Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.

PubMed Central

Gleeson, D; Smith, N D; Boyer, J L

1989-01-01

Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pHi) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pHi was higher (7.28 +/- 09) in +HCO3- than in -HCO3- (7.16 +/- 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pHi in +HCO3- but caused reversible 0.1-0.2-U acidification in -HCO3- or in +HCO3- after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na+ replacement in +HCO3- alos caused acidification which was amiloride independent but DIDS inhibitible. The recovery of pHi from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ effluxmax by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pHi recovery in +HCO3- was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3- exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3- dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3- transport. We conclude that Na+-HCO3- cotransport, in addition to Na+/H+ exchange, is an important regulator of pHi in rat hepatocytes. PMID:2544626

cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells.

PubMed

Bachmann, Oliver; Franke, Kristin; Yu, Haoyang; Riederer, Brigitte; Li, Hong C; Soleimani, Manoocher; Manns, Michael P; Seidler, Ursula

2008-12-22

The renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. Na+- and HCO3-dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10(-5) M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10(-4) M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na+/HCO3- cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.

Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16.

PubMed

Saggu, Miguel; Zebger, Ingo; Ludwig, Marcus; Lenz, Oliver; Friedrich, Bärbel; Hildebrandt, Peter; Lendzian, Friedhelm

2009-06-12

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

[NiFe] hydrogenases: a common active site for hydrogen metabolism under diverse conditions.

PubMed

Shafaat, Hannah S; Rüdiger, Olaf; Ogata, Hideaki; Lubitz, Wolfgang

2013-01-01

Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these "nonstandard" [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term "oxygen-tolerance". This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Fidelity of metal insertion into hydrogenases.

PubMed

Magalon, A; Blokesch, M; Zehelein, E; Böck, A

2001-06-15

The fidelity of metal incorporation into the active center of hydrogenase 3 from Escherichia coli was studied by analyzing the inhibition of the maturation pathway by zinc and other transition metals. Hydrogenase maturation of wild-type cells was significantly affected only by concentrations of zinc or cadmium higher than 200 microM, whereas a mutant with a lesion in the nickel uptake system displayed a total blockade of the proteolytic processing of the precursor form into the mature form of the large subunit after growth in the presence of 10 microM Zn(2+). The precursor could not be processed in vitro by the maturation endopeptidase even in the presence of an excess of nickel ions. Evidence is presented that zinc does not interfere with the incorporation of iron into the metal center. Precursor of the large subunit accumulated in nickel proficient cells formed a transient substrate complex with the cognate endoprotease HycI whereas that of zinc-supplemented cells did not. The results show that zinc can intrude the nickel-dependent maturation pathway only when nickel uptake is blocked. Under this condition zinc appears to be incorporated at the nickel site of the large subunit and delivers a precursor not amenable to proteolytic processing since the interaction with the endoprotease is blocked.

Ab Initio Electronic Structure Calculation of [4Fe-3S] Cluster of Hydrogenase as Dihydrogen Dissociation/Production Catalyst

NASA Astrophysics Data System (ADS)

Kim, Jaehyun; Kang, Jiyoung; Nishigami, Hiroshi; Kino, Hiori; Tateno, Masaru

2018-03-01

Hydrogenases catalyze both the dissociation and production of dihydrogen (H2). Most hydrogenases are inactivated rapidly and reactivated slowly (in vitro), in the presence of dioxygen (O2) and H2, respectively. However, membrane-bound [NiFe] hydrogenases (MBHs) sustain their activity even together with O2, which is termed "O2 tolerance". In previous experimental analyses, an MBH was shown to include a hydroxyl ion (OH-) bound to an Fe of the super-oxidized [4Fe-3S]5+ cluster in the proximity of the [NiFe] catalytic cluster. In this study, the functional role of the OH- in the O2 tolerance was investigated by ab initio electronic structure calculation of the [4Fe-3S] proximal cluster. The analysis revealed that the OH- significantly altered the electronic structure, thereby inducing the delocalization of the lowest unoccupied molecular orbital (LUMO) toward the [NiFe] catalytic cluster, which may intermediate the electron transfer between the catalytic and proximal clusters. This can promote the O2-tolerant catalytic cycle in the hydrogenase reaction.

Resonant inelastic X-ray scattering on synthetic nickel compounds and Ni-Fe hydrogenase protein

NASA Astrophysics Data System (ADS)

Sanganas, Oliver; Löscher, Simone; Pfirrmann, Stefan; Marinos, Nicolas; Glatzel, Pieter; Weng, Tsu-Chien; Limberg, Christian; Driess, Matthias; Dau, Holger; Haumann, Michael

2009-11-01

Ni-Fe hydrogenases are proteins catalyzing the oxidative cleavage of dihydrogen (H2) and proton reduction to H2 at high turnover rates. Their active site is a heterobimetallic center comprising one Ni and one Fe atom. To understand the function of the site, well resolved structural and electronic information is required. Such information is expected to become accessible by high resolution X-ray absorption and emission techniques, which are rapidly developing at third generation synchrotron radiation sources. We studied a number of synthetic Ni compounds, which mimic relevant features of the Ni site in hydrogenases, and the Ni site in the soluble, NAD-reducing hydrogenase (SH) from the bacterium Ralstonia eutropha by resonant inelastic X-ray scattering (RIXS) using a Rowland-type spectrometer at the ESRF. The SH is particularly interesting because its H2-cleavage reaction is highly resistant against inhibition by O2. Kα-fluorescence detected RIXS planes in the 1s→3d region of the X-ray absorption spectrum were recorded on the protein which allow to extract L3-edge type spectra Spectral features of the protein are compared to those of the model compounds.

Reversible [4Fe-3S] cluster morphing in an O(2)-tolerant [NiFe] hydrogenase.

PubMed

Frielingsdorf, Stefan; Fritsch, Johannes; Schmidt, Andrea; Hammer, Mathias; Löwenstein, Julia; Siebert, Elisabeth; Pelmenschikov, Vladimir; Jaenicke, Tina; Kalms, Jacqueline; Rippers, Yvonne; Lendzian, Friedhelm; Zebger, Ingo; Teutloff, Christian; Kaupp, Martin; Bittl, Robert; Hildebrandt, Peter; Friedrich, Bärbel; Lenz, Oliver; Scheerer, Patrick

2014-05-01

Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H(2) oxidation at high O(2). We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range.

Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approach.

PubMed

Baltazar, Carla S A; Teixeira, Vitor H; Soares, Cláudio M

2012-04-01

Hydrogenases are metalloenzymes that catalyze the reversible reaction H(2)<->2H(+) + 2e(-), being potentially useful in H(2) production or oxidation. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O(2) inhibition and produce more H(2) than standard [NiFe] hydrogenases. However, the molecular determinants responsible for these properties remain unknown. Hydrophobic pathways for H(2) diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H(2) and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a standard [NiFe] hydrogenase. We performed molecular dynamics simulations of H(2) diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas (Teixeira et al. in Biophys J 91:2035-2045, 2006). The comparison showed that H(2) density near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H(2) to the active site. We have also determined a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas (Teixeira et al. in Proteins 70:1010-1022, 2008). The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H(2) and proton pathways. © SBIC 2012

The effect of plasma pre-treatment on NaHCO3 desizing of blended sizes on cotton fabrics

NASA Astrophysics Data System (ADS)

Li, Xuming; Qiu, Yiping

2012-03-01

The influence of the He/O2 atmospheric pressure plasma jet pre-treatment on subsequent NaHCO3 desizing of blends of starch phosphate and poly(vinyl alcohol) on cotton fabrics is investigated. Atomic force microscopy and scanning electron microscopy analysis indicate that the surface topography of the samples has significantly changed and the surface roughness increases with an increase in plasma exposure time. X-ray photoelectron spectroscopy analysis shows that a larger number of oxygen-containing polar groups are formed on the sized fabric surface after the plasma treatment. The results of the percent desizing ratio (PDR) indicate that the plasma pretreatment facilitated the blended sizes removal from the cotton fabrics in subsequent NaHCO3 treatment and the PDR increases with prolonging plasma treatment time. The plasma technology is a promising pretreatment for desizing of blended sizes due to dramatically reduced desizing time.

Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae

PubMed Central

Berghofer, Y.; Klein, A.

1995-01-01

Methanococcus voltae, which contains four different gene groups that encode [NiFe]-hydrogenases, was transformed with integration vectors to achieve polar inactivation of two of the four hydrogenase operons that encode the selenium-free enzymes Vhc and Frc. Transformants which were selected by their acquired puromycin resistance showed site-specific insertions in either the vhc or frc operon by single crossover events. Southern hybridization revealed tandem integrations of whole vectors in the vhc operon, whereas only one vector copy was found in the frc operon. Northern (RNA) hybridizations showed a pac transcript of defined size, indicating strong termination in front of the hydrogenase genes downstream. In spite of the apparent abolition of expression of selenium-free hydrogenases through these polar insertions, they were not lethal to cells upon growth in selenium-deprived minimal medium, which we had previously shown to strongly induce transcription of the respective operons in M. voltae. Instead, like wild-type control cultures, transformants responded to selenium deprivation only with a reduction in growth rate. We conclude that loss of the potential to express a selenium-free hydrogenase can nevertheless be balanced by very small amounts of selenium hydrogenases under laboratory conditions in which the hydrogen supply is not likely to be a limiting growth factor. PMID:16535019

Ingestion of Sodium Bicarbonate (NaHCO3) Following a Fatiguing Bout of Exercise Accelerates Postexercise Acid-Base Balance Recovery and Improves Subsequent High-Intensity Cycling Time to Exhaustion.

PubMed

Gough, Lewis A; Rimmer, Steven; Osler, Callum J; Higgins, Matthew F

2017-10-01

This study evaluated the ingestion of sodium bicarbonate (NaHCO 3 ) on postexercise acid-base balance recovery kinetics and subsequent high-intensity cycling time to exhaustion. In a counterbalanced, crossover design, nine healthy and active males (age: 23 ± 2 years, height: 179 ± 5 cm, body mass: 74 ± 9 kg, peak mean minute power (W peak ) 256 ± 45 W, peak oxygen uptake (V̇O 2peak ) 46 ± 8 ml.kg -1 .min -1 ) performed a graded incremental exercise test, two familiarization and two experimental trials. Experimental trials consisted of cycling to volitional exhaustion (T LIM1 ) at 100% W PEAK on two occasions (T LIM1 and T LIM2 ) interspersed by a 90 min passive recovery period. Using a double-blind approach, 30 min into a 90 min recovery period participants ingested either 0.3 g.kg -1 body mass sodium bicarbonate (NaHCO 3 ) or a placebo (PLA) containing 0.1 g.kg -1 body mass sodium chloride (NaCl) mixed with 4 ml.kg -1 tap water and 1 ml.kg -1 orange squash. The mean differences between T LIM2 and T LIM1 was larger for PLA compared with NaHCO 3 (-53 ± 53 vs. -20 ± 48 s; p = .008, d = 0.7, CI =-0.3, 1.6), indicating superior subsequent exercise time to exhaustion following NaHCO 3 . Blood lactate [Bla - ] was similar between treatments post T LIM1 , but greater for NaHCO 3 post T LIM2 and 5 min post T LIM2 . Ingestion of NaHCO 3 induced marked increases (p < .01) in both blood pH (+0.07 ± 0.02, d = 2.6, CI = 1.2, 3.7) and bicarbonate ion concentration [HCO 3 - ] (+6.8 ± 1.6 mmo.l -1 , d = 3.4, CI = 1.8, 4.7) compared with the PLA treatment, before T LIM2 . It is likely both the acceleration of recovery, and the marked increases of acid-base after T LIM1 contributed to greater T LIM2 performance compared with the PLA condition.

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS.

PubMed

Tengölics, Roland; Mészáros, Lívia; Győri, E; Doffkay, Zsolt; Kovács, Kornél L; Rákhely, Gábor

2014-10-01

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled. Copyright © 2014 Elsevier B.V. All rights reserved.

hypD as a Marker for [NiFe]-Hydrogenases in Microbial Communities of Surface Waters

PubMed Central

Beimgraben, Christian; Gutekunst, Kirstin; Opitz, Friederike

2014-01-01

Hydrogen is an important trace gas in the atmosphere. Soil microorganisms are known to be an important part of the biogeochemical H2 cycle, contributing 80 to 90% of the annual hydrogen uptake. Different aquatic ecosystems act as either sources or sinks of hydrogen, but the contribution of their microbial communities is unknown. [NiFe]-hydrogenases are the best candidates for hydrogen turnover in these environments since they are able to cope with oxygen. As they lack sufficiently conserved sequence motifs, reliable markers for these enzymes are missing, and consequently, little is known about their environmental distribution. We analyzed the essential maturation genes of [NiFe]-hydrogenases, including their frequency of horizontal gene transfer, and found hypD to be an applicable marker for the detection of the different known hydrogenase groups. Investigation of two freshwater lakes showed that [NiFe]-hydrogenases occur in many prokaryotic orders. We found that the respective hypD genes cooccur with oxygen-tolerant [NiFe]-hydrogenases (groups 1 and 5) mainly of Actinobacteria, Acidobacteria, and Burkholderiales; cyanobacterial uptake hydrogenases (group 2a) of cyanobacteria; H2-sensing hydrogenases (group 2b) of Burkholderiales, Rhizobiales, and Rhodobacterales; and two groups of multimeric soluble hydrogenases (groups 3b and 3d) of Legionellales and cyanobacteria. These findings support and expand a previous analysis of metagenomic data (M. Barz et al., PLoS One 5:e13846, 2010, http://dx.doi.org/10.1371/journal.pone.0013846) and further identify [NiFe]-hydrogenases that could be involved in hydrogen cycling in aquatic surface waters. PMID:24727276

A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases

PubMed Central

Cracknell, James A.; Wait, Annemarie F.; Lenz, Oliver; Friedrich, Bärbel; Armstrong, Fraser A.

2009-01-01

In biology, rapid oxidation and evolution of H2 is catalyzed by metalloenzymes known as hydrogenases. These enzymes have unusual active sites, consisting of iron complexed by carbonyl, cyanide, and thiolate ligands, often together with nickel, and are typically inhibited or irreversibly damaged by O2. The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H2 as an energy source with O2 as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an “O2-tolerant” hydrogenase. The mechanism of O2 tolerance of Re MBH has been probed by measuring H2 oxidation activity in the presence of O2 over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O2 and subsequent reactivation of the active site. Most significantly, O2 tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H2 affinity or the kinetics of O2 attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O2 tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases. PMID:19934053

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis

PubMed Central

Lohner, Svenja T; Deutzmann, Jörg S; Logan, Bruce E; Leigh, John; Spormann, Alfred M

2014-01-01

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism. PMID:24844759

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis.

PubMed

Lohner, Svenja T; Deutzmann, Jörg S; Logan, Bruce E; Leigh, John; Spormann, Alfred M

2014-08-01

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<-414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.

Isotopic fractionation associated with [NiFe]- and [FeFe]-hydrogenases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Hui; Gandhi, Hasand; Cornish, Adam J.

2016-01-30

Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. In this manuscript, we used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in theirmore » active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27-0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41-0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing us with a more complete picture of the three reactions catalyzed by hydrogenases. The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.« less

Energy-converting [NiFe] hydrogenases: more than just H2 activation.

PubMed

Hedderich, Reiner; Forzi, Lucia

2005-01-01

The well-characterized [NiFe] hydrogenases have a key function in the H2 metabolism of various microorganisms. A subfamily of the [NiFe] hydrogenases with unique properties has recently been identified. The six conserved subunits that build the core of these membrane-bound hydrogenases share sequence similarity with subunits that form the catalytic core of energy-conserving NADH:quinone oxidoreductases (complex I). The physiological role of some of these hydrogenases is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or polyferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase subfamily mainly function in providing the cell with reduced ferredoxin using H2 as electron donor in a reaction driven by reverse electron transport. These hydrogenases have therefore been designated as energy-converting [NiFe] hydrogenases. Copyright 2005 S. Karger AG, Basel.

Oxygen-resistant hydrogenases and methods for designing and making same

DOEpatents

King, Paul [Golden, CO; Ghirardi, Maria L [Lakewood, CO; Seibert, Michael [Lakewood, CO

2009-03-10

The invention provides oxygen- resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H.sub.2 in a light catalyzed reaction having water as the reactant.

Oxygen-resistant hydrogenases and methods for designing and making same

DOEpatents

King, Paul; Ghirardi, Maria Lucia; Seibert, Michael

2014-03-04

The invention provides oxygen-resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H.sub.2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H.sub.2 in a light catalyzed reaction having water as the reactant.

Hydrogenase polypeptide and methods of use

DOEpatents

Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

2016-02-02

Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution.

PubMed

Yonemoto, Isaac T; Matteri, Christopher W; Nguyen, Thao Amy; Smith, Hamilton O; Weyman, Philip D

2013-07-02

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [Ni

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

PubMed Central

2013-01-01

Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe

Process and genes for expression and overexpression of active [FeFe] hydrogenases

DOEpatents

Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

2014-09-16

A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

Three different [NiFe] hydrogenases confer metabolic flexibility in the obligate aerobe Mycobacterium smegmatis.

PubMed

Berney, Michael; Greening, Chris; Hards, Kiel; Collins, Desmond; Cook, Gregory M

2014-01-01

Mycobacterium smegmatis is an obligate aerobe that harbours three predicted [NiFe] hydrogenases, Hyd1 (MSMEG_2262–2263), Hyd2 (MSMEG_2720-2719) and Hyd3 (MSMEG_3931-3928). We show here that these three enzymes differ in their phylogeny, regulation and catalytic activity. Phylogenetic analysis revealed that Hyd1 groups with hydrogenases that oxidize H2 produced by metabolic processes, and Hyd2 is homologous to a novel group of putative high-affinity hydrogenases. Hyd1 and Hyd2 respond to carbon and oxygen limitation, and, in the case of Hyd1, hydrogen supplementation. Hydrogen consumption measurements confirmed that both enzymes can oxidize hydrogen. In contrast, the phylogenetic analysis and activity measurements of Hyd3 are consistent with the enzyme evolving hydrogen. Hyd3 is controlled by DosR, a regulator that responds to hypoxic conditions. The strict dependence of hydrogen oxidation of Hyd1 and Hyd2 on oxygen suggests that the enzymes are oxygen tolerant and linked to the respiratory chain. This unique combination of hydrogenases allows M. smegmatis to oxidize hydrogen at high (Hyd1) and potentially tropospheric (Hyd2) concentrations, as well as recycle reduced equivalents by evolving hydrogen (Hyd3). The distribution of these hydrogenases throughout numerous soil and marine species of actinomycetes suggests that oxic hydrogen metabolism provides metabolic flexibility in environments with changing nutrient fluxes.

An S-Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O2 -Tolerant Hydrogenase.

PubMed

Lindenmaier, Nils J; Wahlefeld, Stefan; Bill, Eckhard; Szilvási, Tibor; Eberle, Christopher; Yao, Shenglai; Hildebrandt, Peter; Horch, Marius; Zebger, Ingo; Driess, Matthias

2017-02-13

To understand the molecular details of O 2 -tolerant hydrogen cycling by a soluble NAD + -reducing [NiFe] hydrogenase, we herein present the first bioinspired heterobimetallic S-oxygenated [NiFe] complex as a structural and vibrational spectroscopic model for the oxygen-inhibited [NiFe] active site. This compound and its non-S-oxygenated congener were fully characterized, and their electronic structures were elucidated in a combined experimental and theoretical study with emphasis on the bridging sulfenato moiety. Based on the vibrational spectroscopic properties of these complexes, we also propose novel strategies for exploring S-oxygenated intermediates in hydrogenases and similar enzymes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of the bidirectional hydrogenase in cyanobacteria.

PubMed

Carrieri, Damian; Wawrousek, Karen; Eckert, Carrie; Yu, Jianping; Maness, Pin-Ching

2011-09-01

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni-Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed. Copyright © 2011 Elsevier Ltd. All rights reserved.

A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

PubMed

Ng, Gordon; Tom, Curtis G S; Park, Angela S; Zenad, Lounis; Ludwig, Robert A

2009-01-01

Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such

Effects of chronic NaHCO3 ingestion during interval training on changes to muscle buffer capacity, metabolism, and short-term endurance performance.

PubMed

Edge, Johann; Bishop, David; Goodman, Carmel

2006-09-01

This study determined the effects of altering the H(+) concentration during interval training, by ingesting NaHCO(3) (Alk-T) or a placebo (Pla-T), on changes in muscle buffer capacity (beta m), endurance performance, and muscle metabolites. Pre- and posttraining peak O(2) uptake (V(O2 peak)), lactate threshold (LT), and time to fatigue at 100% pretraining V(O2 peak) intensity were assessed in 16 recreationally active women. Subjects were matched on the LT, were randomly placed into the Alk-T (n = 8) or Pla-T (n = 8) groups, and performed 8 wk (3 days/wk) of six to twelve 2-min cycle intervals at 140-170% of their LT, ingesting NaHCO(3) or a placebo before each training session (work matched between groups). Both groups had improvements in beta m (19 vs. 9%; P < 0.05) and V(O2 peak) (22 vs. 17%; P < 0.05) after the training period, with no differences between groups. There was a significant correlation between pretraining beta m and percent change in beta m (r = -0.70, P < 0.05). There were greater improvements in both the LT (26 vs. 15%; P = 0.05) and time to fatigue (164 vs. 123%; P = 0.05) after Alk-T, compared with Pla-T. There were no changes to pre- or postexercise ATP, phosphocreatine, creatine, and intracellular lactate concentrations, or pH(i) after training. Our findings suggest that training intensity, rather than the accumulation of H(+) during training, may be more important to improvements in beta m. The group ingesting NaHCO(3) before each training session had larger improvements in the LT and endurance performance, possibly because of a reduced metabolic acidosis during training and a greater improvement in muscle oxidative capacity.

Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.

PubMed

Adam, Nicole; Perner, Mirjam

2017-01-01

Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

PubMed Central

Tamagnini, Paula; Axelsson, Rikard; Lindberg, Pia; Oxelfelt, Fredrik; Wünschiers, Röbbe; Lindblad, Peter

2002-01-01

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included. PMID:11875125

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

PubMed Central

Watrous, Mary M.; Clark, Sandra; Kutty, Razia; Huang, Shouqin; Rudolph, Frederick B.; Hughes, Joseph B.; Bennett, George N.

2003-01-01

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a Km of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R2 = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities. PMID:12620841

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module.

PubMed

Long, Minnan; Liu, Jingjing; Chen, Zhifeng; Bleijlevens, Boris; Roseboom, Winfried; Albracht, Simon P J

2007-01-01

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M (r) = 52 kDa) and a small (M (r) = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe-S clusters, three [2Fe-2S] clusters and five [4Fe-4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe-S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H(2)-dependent reduction of NAD(+). Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni-Fe active site and a [4Fe-4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni(a)-C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.

Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I.

PubMed

Hedderich, Reiner

2004-02-01

[NiFe] hydrogenases are well-characterized enzymes that have a key function in the H2 metabolism of various microorganisms. In the recent years a subfamily of [NiFe] hydrogenases with unique properties has been identified. The members of this family form multisubunit membrane-bound enzyme complexes composed of at least four hydrophilic and two integral membrane proteins. These six conserved subunits, which built the core of these hydrogenases, have closely related counterparts in energy-conserving NADH:quinone oxidoreductases (complex I). However, the reaction catalyzed by these hydrogenases differs significantly from the reaction catalyzed by complex I. For some of these hydrogenases the physiological role is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or poly-ferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase family mainly function to provide the cell with reduced ferredoxin with H2 as electron donor in a reaction driven by reverse electron transport. As complex I these hydrogenases function as ion pumps and have therefore been designated as energy-converting [NiFe] hydrogenases.

PSD-95 interacts with NBCn1 and enhances channel-like activity without affecting Na/HCO(3) cotransport.

PubMed

Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R Kyle; Smith, Yoland; Yun, C Chris; Choi, Inyeong

2012-01-01

The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO(3)(-) movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO(3) cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO(3) cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. Copyright © 2012 S. Karger AG, Basel.

PSD-95 Interacts with NBCn1 and Enhances Channel-like Activity without Affecting Na/HCO3 Cotransport

PubMed Central

Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R. Kyle; Smith, Yoland; Yun, C. Chris; Choi, Inyeong

2013-01-01

Background/Aims The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. PMID:23183381

Quantum chemical approaches to [NiFe] hydrogenase.

PubMed

Vaissier, Valerie; Van Voorhis, Troy

2017-05-09

The mechanism by which [NiFe] hydrogenase catalyses the oxidation of molecular hydrogen is a significant yet challenging topic in bioinorganic chemistry. With far-reaching applications in renewable energy and carbon mitigation, significant effort has been invested in the study of these complexes. In particular, computational approaches offer a unique perspective on how this enzyme functions at an electronic and atomistic level. In this article, we discuss state-of-the art quantum chemical methods and how they have helped deepen our comprehension of [NiFe] hydrogenase. We outline the key strategies that can be used to compute the (i) geometry, (ii) electronic structure, (iii) thermodynamics and (iv) kinetic properties associated with the enzymatic activity of [NiFe] hydrogenase and other bioinorganic complexes. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

[NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics.

PubMed

Brugna-Guiral, Marianne; Tron, Pascale; Nitschke, Wolfgang; Stetter, Karl-Otto; Burlat, Benedicte; Guigliarelli, Bruno; Bruschi, Mireille; Giudici-Orticoni, Marie Thérèse

2003-04-01

Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

The [NiFe]-Hydrogenase of Pyrococcus furiosus Exhibits a New Type of Oxygen Tolerance.

PubMed

Kwan, Patrick; McIntosh, Chelsea L; Jennings, David P; Hopkins, R Chris; Chandrayan, Sanjeev K; Wu, Chang-Hao; Adams, Michael W W; Jones, Anne K

2015-10-28

We report the first direct electrochemical characterization of the impact of oxygen on the hydrogen oxidation activity of an oxygen-tolerant, group 3, soluble [NiFe]-hydrogenase: hydrogenase I from Pyrococcus furiosus (PfSHI), which grows optimally near 100 °C. Chronoamperometric experiments were used to probe the sensitivity of PfSHI hydrogen oxidation activity to both brief and prolonged exposure to oxygen. For experiments between 15 and 80 °C, following short (<200 s) exposure to 14 μM O2 under oxidizing conditions, PfSHI always maintains some fraction of its initial hydrogen oxidation activity; i.e., it is oxygen-tolerant. Reactivation experiments show that two inactive states are formed by interaction with oxygen and both can be quickly (<150 s) reactivated. Analogous experiments, in which the interval of oxygen exposure is extended to 900 s, reveal that the response is highly temperature-dependent. At 25 °C, under sustained 1% O2/ 99% H2 exposure, the H2oxidation activity drops nearly to zero. However, at 80 °C, up to 32% of the enzyme's oxidation activity is retained. Reactivation of PfSHI following sustained exposure to oxygen occurs on a much longer time scale (tens of minutes), suggesting that a third inactive species predominates under these conditions. These results stand in contrast to the properties of oxygen-tolerant, group 1 [NiFe]-hydrogenases, which form a single state upon reaction with oxygen, and we propose that this new type of hydrogenase should be referred to as oxygen-resilient. Furthermore, PfSHI, like other group 3 [NiFe]-hydrogenases, does not possess the proximal [4Fe3S] cluster associated with the oxygen tolerance of some group 1 enzymes. Thus, a new mechanism is necessary to explain the observed oxygen tolerance in soluble, group 3 [NiFe]-hydrogenases, and we present a model integrating both electrochemical and spectroscopic results to define the relationships of these inactive states.

H2 conversion in the presence of O2 as performed by the membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha.

PubMed

Lenz, Oliver; Ludwig, Marcus; Schubert, Torsten; Bürstel, Ingmar; Ganskow, Stefanie; Goris, Tobias; Schwarze, Alexander; Friedrich, Bärbel

2010-04-26

[NiFe]-hydrogenases catalyze the oxidation of H(2) to protons and electrons. This reversible reaction is based on a complex interplay of metal cofactors including the Ni-Fe active site and several [Fe-S] clusters. H(2) catalysis of most [NiFe]-hydrogenases is sensitive to dioxygen. However, some bacteria contain hydrogenases that activate H(2) even in the presence of O(2). There is now compelling evidence that O(2) affects hydrogenase on three levels: 1) H(2) catalysis, 2) hydrogenase maturation, and 3) H(2)-mediated signal transduction. Herein, we summarize the genetic, biochemical, electrochemical, and spectroscopic properties related to the O(2) tolerance of hydrogenases resident in the facultative chemolithoautotroph Ralstonia eutropha H16. A focus is given to the membrane-bound [NiFe]-hydogenase, which currently represents the best-characterized member of O(2)-tolerant hydrogenases.

Electronic structure of the unique [4Fe-3S] cluster in O2-tolerant hydrogenases characterized by 57Fe Mössbauer and EPR spectroscopy

PubMed Central

Pandelia, Maria-Eirini; Bykov, Dmytro; Izsak, Robert; Infossi, Pascale; Giudici-Orticoni, Marie-Thérèse; Bill, Eckhard; Neese, Frank; Lubitz, Wolfgang

2013-01-01

Iron–sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H2 catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O2-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent 57Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron–sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron–sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher. PMID:23267108

Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

PubMed Central

Robey, Ian F.; Baggett, Brenda K.; Kirkpatrick, Nathaniel D.; Roe, Denise J.; Dosescu, Julie; Sloane, Bonnie F.; Hashim, Arig Ibrahim; Morse, David L.; Raghunand, Natarajan; Gatenby, Robert A.; Gillies, Robert J.

2010-01-01

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO3 selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by 31P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO3 therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO3 therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease. PMID:19276390

O₂migration rates in [NiFe] hydrogenases. A joint approach combining free-energy calculations and kinetic modeling.

PubMed

Topin, Jérémie; Diharce, Julien; Fiorucci, Sébastien; Antonczak, Serge; Golebiowski, Jérôme

2014-01-23

Hydrogenases are promising candidates for the catalytic production of green energy by means of biological ways. The major impediment to such a production is rooted in their inhibition under aerobic conditions. In this work, we model dioxygen migration rates in mutants of a hydrogenase of Desulfovibrio fructusovorans. The approach relies on the calculation of the whole potential of mean force for O2 migration within the wild-type as well as in V74M, V74F, and V74Q mutant channels. The three free-energy barriers along the entire migration pathway are converted into chemical rates through modeling based on Transition State Theory. The use of such a model recovers the trend of O2 migration rates among the series.

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

PubMed Central

2009-01-01

Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co

Mechanism of Hydrogen Production in [Fe-Fe]-Hydrogenase: A Density Functional Theory Study (Preprint)

DTIC Science & Technology

2007-03-01

of NiFe hydrogenases. Dalton Transactions 2003,4030-4038. (9) Armstrong, F. A., Hydrogenases: active site puzzles and progress. Current Opinion in...DFT Investigation of Structural, Electronic, and Catalytic Properties of Diiron Complexes Related to the [2Fe]H Subcluster of Fe-Only Hydrogenases...Hydrogenases: Effects of Redox State and Ligand Characteristics on Structural, Electronic, and Reactivity Properties of Complexes Related to the [2Fe]H

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.

PubMed

Rousset, M; Montet, Y; Guigliarelli, B; Forget, N; Asso, M; Bertrand, P; Fontecilla-Camps, J C; Hatchikian, E C

1998-09-29

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

PubMed Central

Rousset, Marc; Montet, Yael; Guigliarelli, Bruno; Forget, Nicole; Asso, Marcel; Bertrand, Patrick; Fontecilla-Camps, Juan C.; Hatchikian, E. Claude

1998-01-01

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed. PMID:9751716

Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH

PubMed Central

Losey, Nathaniel A.; Mus, Florence; Peters, John W.; Le, Huynh M.

2017-01-01

ABSTRACT Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the

Solar powered biohydrogen production requires specific localization of the hydrogenase

DOE PAGES

Burroughs, Nigel J.; Boehm, Marko; Eckert, Carrie; ...

2014-09-04

Cyanobacteria contain a bidirectional [NiFe] hydrogenase which transiently produces hydrogen upon exposure of anoxic cells to light, potentially acting as a “valve” releasing excess electrons from the electron transport chain. However, its interaction with the photosynthetic electron transport chain remains unclear. By GFP-tagging the HoxF diaphorase subunit we show that the hydrogenase is thylakoid associated, comprising a population dispersed uniformly through the thylakoids and a subpopulation localized to discrete puncta in the distal thylakoid. Thylakoid localisation of both the HoxH and HoxY hydrogenase subunits is confirmed by immunogold electron microscopy. The diaphorase HoxE subunit is essential for recruitment to themore » dispersed thylakoid population, potentially anchoring the hydrogenase to the membrane, but aggregation to puncta occurs through a distinct HoxE-independent mechanism. Membrane association does not require NDH-1. Localization is dynamic on a scale of minutes, with anoxia and high light inducing a significant redistribution between these populations in favour of puncta. Lastly, since HoxE is essential for access to its electron donor, electron supply to the hydrogenase depends on a physiologically controlled localization, potentially offering a new avenue to enhance photosynthetic hydrogen production by exploiting localization/aggregation signals.« less

Turning Cellulose Waste Into Electricity: Hydrogen Conversion by a Hydrogenase Electrode

PubMed Central

Abramov, Sergey M.; Sadraddinova, Elmira R.; Shestakov, Andrey I.; Voronin, Oleg G.; Karyakin, Arkadiy A.; Zorin, Nikolay A.; Netrusov, Alexander I.

2013-01-01

Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L-1 h-1 and 1 mM L-1 h-1, respectively. An enzymatic fuel cell (EFC) with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h) to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value. PMID:24312437

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H/sub 2/ metabolism in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sankar, P.; Lee, J.H.; Shanmugam, K.T.

1985-04-01

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2more » (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.« less

Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.

PubMed

Avilan, Luisana; Roumezi, Baptiste; Risoul, Véronique; Bernard, Christophe Sébastien; Kpebe, Arlette; Belhadjhassine, Mayssène; Rousset, Marc; Brugna, Myriam; Latifi, Amel

2018-04-24

The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H 2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H 2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H 2 . As a result, their H 2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H 2 production activities, but their sensitivity to O 2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O 2 . The diazotrophic filamentous cyanobacteria protect their nitrogenases from O 2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O 2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 μmol-H 2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H 2 production by an O 2 -sensitive hydrogenase.

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

PubMed

Plummer, Scott M; Plummer, Mark A; Merkel, Patricia A; Hagen, Moira; Biddle, Jennifer F; Waidner, Lisa A

2016-11-01

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production. Copyright © 2016 Elsevier Inc. All rights reserved.

Improving cyanobacterail O 2-tolerance using CBS hydrogenase for hydrogen production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maness, Pin-Ching; Eckert, Carrie; Wawrousek, Karen

2016-11-11

Cyanobacterial H 2 production is a viable path to renewable H 2 with water serving as the electron donor and sunlight the energy source. A grand challenge is the sensitivity of the underlying hydrogenase to O 2, the latter an inherent byproduct of oxygenic photosynthesis. This challenge has been identified as a technical barrier in the Fuel Cell Technologies Office (FCTO) Multi-year Research, Development and Deployment Plan. One solution is to express in cyanobacterium an O 2-tolerant hydrogenase to circumvent this barrier. We have uncovered an O 2-tolerant hydrogenase from a photosynthetic bacterium Rubrivivax gelatinosus CBS (Casa Bonita Strain; hereaftermore » “CBS”) with a half-life near 21 h when exposed to ambient O 2. We sequenced the CBS genome and identified two sets of maturation machineries hyp1 and hyp2. Transcripts expression analysis and mutagenesis revealed that hyp1 is responsible for the assembly of the O 2-tolerant CO-oxidation (Coo) hydrogenase and hyp2 is involved in the maturation of a H 2-uptake hydrogenase. The structural genes encoding the O 2-tolerant hydrogenase (cooLXUH) and maturation genes hyp1FABCDE were therefore cloned and expressed in the model cyanobacterium Synechocystis sp. PCC 6803. We obtained several recombinants displaying hydrogenase activity in a Synechocystis host lacking background activity, suggesting that the CBS hydrogenase is active in Synechocystis. Yet the activity is extremely low. To ensure balanced protein expression, we systematically optimized heterologous expression of 10 CBS genes by using stronger promoters and better ribosome binding site. Moreover we attempted the expression of cooM and cooK genes, verified to be important in CBS to afford activity. CooM is a very large protein and both CooM and CooK are membrane-associated. These properties limited our success in expressing both genes in Synechocystis, although they were both expressed in E. coli yet with no activity. This project was terminated in

[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation.

PubMed

Peters, John W; Schut, Gerrit J; Boyd, Eric S; Mulder, David W; Shepard, Eric M; Broderick, Joan B; King, Paul W; Adams, Michael W W

2015-06-01

The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Evolutionary and Biotechnological Implications of Robust Hydrogenase Activity in Halophilic Strains of Tetraselmis

PubMed Central

D'Adamo, Sarah; Jinkerson, Robert E.; Boyd, Eric S.; Brown, Susan L.; Baxter, Bonnie K.; Peters, John W.; Posewitz, Matthew C.

2014-01-01

Although significant advances in H2 photoproduction have recently been realized in fresh water algae (e.g. Chlamydomonas reinhardtii), relatively few studies have focused on H2 production and hydrogenase adaptations in marine or halophilic algae. Salt water organisms likely offer several advantages for biotechnological H2 production due to the global abundance of salt water, decreased H2 and O2 solubility in saline and hypersaline systems, and the ability of extracellular NaCl levels to influence metabolism. We screened unialgal isolates obtained from hypersaline ecosystems in the southwest United States and identified two distinct halophilic strains of the genus Tetraselmis (GSL1 and QNM1) that exhibit both robust fermentative and photo H2-production activities. The influence of salinity (3.5%, 5.5% and 7.0% w/v NaCl) on H2 production was examined during anoxic acclimation, with the greatest in vivo H2-production rates observed at 7.0% NaCl. These Tetraselmis strains maintain robust hydrogenase activity even after 24 h of anoxic acclimation and show increased hydrogenase activity relative to C. reinhardtii after extended anoxia. Transcriptional analysis of Tetraselmis GSL1 enabled sequencing of the cDNA encoding the FeFe-hydrogenase structural enzyme (HYDA) and its maturation proteins (HYDE, HYDEF and HYDG). In contrast to freshwater Chlorophyceae, the halophilic Tetraselmis GSL1 strain likely encodes a single HYDA and two copies of HYDE, one of which is fused to HYDF. Phylogenetic analyses of HYDA and concatenated HYDA, HYDE, HYDF and HYDG in Tetraselmis GSL1 fill existing knowledge gaps in the evolution of algal hydrogenases and indicate that the algal hydrogenases sequenced to date are derived from a common ancestor. This is consistent with recent hypotheses that suggest fermentative metabolism in the majority of eukaryotes is derived from a common base set of enzymes that emerged early in eukaryotic evolution with subsequent losses in some organisms. PMID

Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro

PubMed Central

Yacoby, Iftach; Pochekailov, Sergii; Toporik, Hila; Ghirardi, Maria L.; King, Paul W.; Zhang, Shuguang

2011-01-01

Photosynthetic water splitting, coupled to hydrogenase-catalyzed hydrogen production, is considered a promising clean, renewable source of energy. It is widely accepted that the oxygen sensitivity of hydrogen production, combined with competition between hydrogenases and NADPH-dependent carbon dioxide fixation are the main limitations for its commercialization. Here we provide evidence that, under the anaerobic conditions that support hydrogen production, there is a significant loss of photosynthetic electrons toward NADPH production in vitro. To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP+-oxidoreductase (FNR), and NADP+. Replacing the hydrogenase with a ferredoxin-hydrogenase fusion switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction. These results suggest a new direction for improvement of biohydrogen production and a means to further resolve the mechanisms that control partitioning of photosynthetic electron transport. PMID:21606330

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

PubMed Central

Greening, Chris; Biswas, Ambarish; Carere, Carlo R; Jackson, Colin J; Taylor, Matthew C; Stott, Matthew B; Cook, Gregory M; Morales, Sergio E

2016-01-01

Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H2) is a niche process. To gain a broader insight into the importance of microbial H2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H2. The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H2-based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H2-sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content (pO2) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H2

Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter.

PubMed

Loh, Shih-Hurng; Chen, Wei-Hwa; Chiang, Cheng-Hsien; Tsai, Chien-Sung; Lee, Guo-Chen; Jin, Jong-Shiaw; Cheng, Tzu-Hurng; Chen, Jin-Jer

2002-01-01

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes. Copyright 2002 National Science Council, ROC and S. Karger AG, Basel

Mechanism of Selective Nickel Transfer from HypB to HypA, Escherichia coli [NiFe]-Hydrogenase Accessory Proteins.

PubMed

Lacasse, Michael J; Douglas, Colin D; Zamble, Deborah B

2016-12-13

[NiFe]-hydrogenase enzymes catalyze the reversible reduction of protons to molecular hydrogen and serve as a vital component of the metabolism of many pathogens. The synthesis of the bimetallic catalytic center requires a suite of accessory proteins, and the penultimate step, nickel insertion, is facilitated by the metallochaperones HypA and HypB. In Escherichia coli, nickel moves from a site in the GTPase domain of HypB to HypA in a process accelerated by GDP. To determine how the transfer of nickel is controlled, the impacts of HypA and nucleotides on the properties of HypB were examined. Integral to this work was His2Gln HypA, a mutant with attenuated nickel affinity that does not support hydrogenase production in E. coli. This mutation inhibits the translocation of nickel from HypB. H2Q-HypA does not modulate the apparent metal affinity of HypB, but the stoichiometry and stability of the HypB-nickel complex are modulated by the nucleotide. Furthermore, the HypA-HypB interaction was detected by gel filtration chromatography if HypB was loaded with GDP, but not a GTP analogue, and the protein complex dissociated upon binding of nickel to His2 of HypA. In contrast, a nucleotide does not modulate the binding of zinc to HypB, and loading zinc into the GTPase domain of HypB inhibits formation of the complex with HypA. These results demonstrate that GTP hydrolysis controls both metal binding and protein-protein interactions, conferring selective and directional nickel transfer during [NiFe]-hydrogenase biosynthesis.

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

PubMed

Chandrayan, Sanjeev K; McTernan, Patrick M; Hopkins, R Christopher; Sun, Junsong; Jenney, Francis E; Adams, Michael W W

2012-01-27

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

PubMed Central

Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

2012-01-01

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

New FeFe-hydrogenase genes identified in a metagenomic fosmid library from a municipal wastewater treatment plant as revealed by high-throughput sequencing.

PubMed

Tomazetto, Geizecler; Wibberg, Daniel; Schlüter, Andreas; Oliveira, Valéria M

2015-01-01

A fosmid metagenomic library was constructed with total community DNA obtained from a municipal wastewater treatment plant (MWWTP), with the aim of identifying new FeFe-hydrogenase genes encoding the enzymes most important for hydrogen metabolism. The dataset generated by pyrosequencing of a fosmid library was mined to identify environmental gene tags (EGTs) assigned to FeFe-hydrogenase. The majority of EGTs representing FeFe-hydrogenase genes were affiliated with the class Clostridia, suggesting that this group is the main hydrogen producer in the MWWTP analyzed. Based on assembled sequences, three FeFe-hydrogenase genes were predicted based on detection of the L2 motif (MPCxxKxxE) in the encoded gene product, confirming true FeFe-hydrogenase sequences. These sequences were used to design specific primers to detect fosmids encoding FeFe-hydrogenase genes predicted from the dataset. Three identified fosmids were completely sequenced. The cloned genomic fragments within these fosmids are closely related to members of the Spirochaetaceae, Bacteroidales and Firmicutes, and their FeFe-hydrogenase sequences are characterized by the structure type M3, which is common to clostridial enzymes. FeFe-hydrogenase sequences found in this study represent hitherto undetected sequences, indicating the high genetic diversity regarding these enzymes in MWWTP. Results suggest that MWWTP have to be considered as reservoirs for new FeFe-hydrogenase genes. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

[FeFe]-Hydrogenases: recent developments and future perspectives.

PubMed

Wittkamp, F; Senger, M; Stripp, S T; Apfel, U-P

2018-06-08

[FeFe]-Hydrogenases are the most efficient enzymes for catalytic hydrogen turnover. Their H2 production efficiency is hitherto unrivalled. However, functional details of the catalytic machinery and possible modes of application are discussed controversially. The incorporation of synthetically modified cofactors and utilization of semi-artificial enzymes only recently allowed us to shed light on key steps of the catalytic cycle. Herein, we summarize the essential findings regarding the redox chemistry of [FeFe]-hydrogenases and discuss their catalytic hydrogen turnover. We furthermore will give an outlook on potential research activities and exploit the utilization of synthetic cofactor mimics.

Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1

PubMed Central

Taketa, Midori; Nakagawa, Hanae; Habukawa, Mao; Osuka, Hisao; Kihira, Kiyohito; Komori, Hirofumi; Shibata, Naoki; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi; Yoon, Ki-Seok; Ogo, Seiji; Shomura, Yasuhito; Higuchi, Yoshiki

2015-01-01

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space group C2, with unit-cell parameters a = 131.43, b = 189.71, c = 124.59 Å, β = 109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, V M was calculated to be 2.2 Å3  Da−1, which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms. PMID:25615977

Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase.

PubMed

Ogata, Hideaki; Nishikawa, Koji; Lubitz, Wolfgang

2015-04-23

The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H(-)) bridging the Ni and Fe and a proton (H(+)) attached to the sulphur of a cysteine ligand. The Ni-H(-) and Fe-H(-) bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN(-) ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.

Optimizing cyanobacteria growth conditions in a sealed environment to enable chemical inhibition tests with volatile chemicals.

PubMed

Johnson, Tylor J; Zahler, Jacob D; Baldwin, Emily L; Zhou, Ruanbao; Gibbons, William R

2016-07-01

Cyanobacteria are currently being engineered to photosynthetically produce next-generation biofuels and high-value chemicals. Many of these chemicals are highly toxic to cyanobacteria, thus strains with increased tolerance need to be developed. The volatility of these chemicals may necessitate that experiments be conducted in a sealed environment to maintain chemical concentrations. Therefore, carbon sources such as NaHCO3 must be used for supporting cyanobacterial growth instead of CO2 sparging. The primary goal of this study was to determine the optimal initial concentration of NaHCO3 for use in growth trials, as well as if daily supplementation of NaHCO3 would allow for increased growth. The secondary goal was to determine the most accurate method to assess growth of Anabaena sp. PCC 7120 in a sealed environment with low biomass titers and small sample volumes. An initial concentration of 0.5g/L NaHCO3 was found to be optimal for cyanobacteria growth, and fed-batch additions of NaHCO3 marginally improved growth. A separate study determined that a sealed test tube environment is necessary to maintain stable titers of volatile chemicals in solution. This study also showed that a SYTO® 9 fluorescence-based assay for cell viability was superior for monitoring filamentous cyanobacterial growth compared to absorbance, chlorophyll α (chl a) content, and biomass content due to its accuracy, small sampling size (100μL), and high throughput capabilities. Therefore, in future chemical inhibition trials, it is recommended that 0.5g/L NaHCO3 is used as the carbon source, and that culture viability is monitored via the SYTO® 9 fluorescence-based assay that requires minimum sample size. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of Hydrogenase and Mixed Sulfate-Reducing Bacterial Populations on the Corrosion of Steel

PubMed Central

Bryant, Richard D.; Jansen, Wayne; Boivin, Joe; Laishley, Edward J.; Costerton, J. William

1991-01-01

The importance of hydrogenase activity to corrosion of steel was assessed by using mixed populations of sulfate-reducing bacteria isolated from corroded and noncorroded oil pipelines. Biofilms which developed on the steel studs contained detectable numbers of sulfate-reducing bacteria (104 increasing to 107/0.5 cm2). However, the biofilm with active hydrogenase activity (i.e., corrosion pipeline organisms), as measured by a semiquantitative commercial kit, was associated with a significantly higher corrosion rate (7.79 mm/year) relative to noncorrosive biofilm (0.48 mm/year) with 105 sulfate-reducing bacteria per 0.5 cm2 but no measurable hydrogenase activity. The importance of hydrogenase and the microbial sulfate-reducing bacterial population making up the biofilm are discussed relative to biocorrosion. Images PMID:16348560

Heterologous Expression of the Desulfovibrio gigas [NiFe] Hydrogenase in Desulfovibrio fructosovorans MR400

PubMed Central

Rousset, Marc; Magro, Valérie; Forget, Nicole; Guigliarelli, Bruno; Belaich, Jean-Pierre; Hatchikian, E. Claude

1998-01-01

The ability of Desulfovibrio fructosovorans MR400 ΔhynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon. PMID:9733707

Heterologous expression of the Desulfovibrio gigas [NiFe] hydrogenase in Desulfovibrio fructosovorans MR400.

PubMed

Rousset, M; Magro, V; Forget, N; Guigliarelli, B; Belaich, J P; Hatchikian, E C

1998-09-01

The ability of Desulfovibrio fructosovorans MR400 DeltahynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

PubMed Central

Bowman, Lisa; Flanagan, Lindsey; Fyfe, Paul K.; Parkin, Alison; Hunter, William N.; Sargent, Frank

2014-01-01

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. PMID:24428762

Effect of NaHCO3 treatments on the activity of cell wall-degrading enzymes produced by Penicillium digitatum during the pathogenesis process on grapefruit.

PubMed

Venditti, Tullio; D'hallewin, Guy; Ladu, Gianfranca; Petretto, Giacomo L; Pintore, Giorgio; Labavitch, John M

2018-03-25

The present study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with NaHCO 3 , at two different pH (5 and 8.3), on cell wall-degrading enzymes activity, over a period of 72 h. The treatment with NaHCO 3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, if compared to the control or the same treatment at pH 5. On the contrary, the pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5 treated plugs up to 72 h. The rapid increase in pectin methyl esterase activity, under alkaline conditions, is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. This article is protected by copyright. All rights reserved.

Protein Interactions and Localization of the Escherichia coli Accessory Protein HypA during Nickel Insertion to [NiFe] Hydrogenase*

PubMed Central

Chan Chung, Kim C.; Zamble, Deborah B.

2011-01-01

Nickel delivery during maturation of Escherichia coli [NiFe] hydrogenase 3 includes the accessory proteins HypA, HypB, and SlyD. Although the isolated proteins have been characterized, little is known about how they interact with each other and the hydrogenase 3 large subunit, HycE. In this study the complexes of HypA and HycE were investigated after modification with the Strep-tag II. Multiprotein complexes containing HypA, HypB, SlyD, and HycE were observed, consistent with the assembly of a single nickel insertion cluster. An interaction between HypA and HycE did not require the other nickel insertion proteins, but HypB was not found with the large subunit in the absence of HypA. The HypA-HycE complex was not detected in the absence of the HypC or HypD proteins, involved in the preceding iron insertion step, and this interaction is enhanced by nickel brought into the cell by the NikABCDE membrane transporter. Furthermore, without the hydrogenase 1, 2, and 3 large subunits, complexes between HypA, HypB, and SlyD were observed. These results support the hypothesis that HypA acts as a scaffold for assembly of the nickel insertion proteins with the hydrogenase precursor protein after delivery of the iron center. At different stages of the hydrogenase maturation process, HypA was observed at or near the cell membrane by using fluorescence confocal microscopy, as was HycE, suggesting membrane localization of the nickel insertion event. PMID:22016389

Hydrogenase in N/sub 2/-fixing cyanobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tel-or, E.; Luijk, L.W.; Packer, L.

Hydrogenase has been examined in two species of aerobically grown cyanobacteria (blue-green algae), Nostoc muscorum and Anabaena cylindrica, with respect to H/sub 2/ production and consumption. These activities are found both in heterocysts and in vegetative cell preparations, but the characteristics of the enzyme in the two cell types differ. H/sub 2/ production requires an artificial electron mediator such as methylviologen, and it can be driven by artificial electron donors with and without light and by a wide variety of organic substrates for which enzymes exist for NADP and methylviologen reduction. This activity is similar in heterocysts and vegetative cellsmore » of both species and is mainly found in the soluble rather than membrane fraction. H/sub 2/ consumption, however, occurs without added mediators or acceptors at 10-fold higher rates than H/sub 2/ production and 10-fold greater activity in heterocysts. H/sub 2/ consumption activity is membrane bound, has a high affinity for H/sub 2/ (K/sub m/ = 50 ..mu..M), and is augmented by light and low concentrations of oxygen. This activity of hydrogenase is mainly found in heterocysts and is poised unidirectionally toward H/sub 2/ consumption. Since nitrogenase activity is localized in heterocysts, it suggests that H/sub 2/ leaked by nitrogenase during N/sub 2/ fixation can be recycled by hydrogenase.« less

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

PubMed Central

Chatelus, C; Carrier, P; Saignes, P; Libert, M F; Berlier, Y; Lespinat, P A; Fauque, G; Legall, J

1987-01-01

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena. PMID:3310883

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.

PubMed Central

Fu, C; Javedan, S; Moshiri, F; Maier, R J

1994-01-01

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product. PMID:8197192

Kinetics and thermodynamics of gas diffusion in a NiFe hydrogenase.

PubMed

Topin, Jérémie; Rousset, Marc; Antonczak, Serge; Golebiowski, Jérôme

2012-03-01

We have investigated O₂ and H₂ transport across a NiFe hydrogenase at the atomic scale by means of computational methods. The Wild Type protein has been compared with the V74Q mutant. Two distinct methodologies have been applied to study the gas access to the active site. Temperature locally enhanced sampling simulations have emphasized the importance of protein dynamics on gas diffusion. The O₂ diffusion free energy profiles, obtained by umbrella sampling, are in agreement with the known kinetic data and show that in the V74Q mutant, the inhibition process is lowered from both a kinetic and a thermodynamic point of view. Copyright © 2011 Wiley Periodicals, Inc.

Radical S-Adenosyl-l-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors*

PubMed Central

Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; Broderick, Joan B.

2015-01-01

Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. The FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-l-methionine enzymes are vital for the assembly of all three of these diverse cofactors. This minireview presents and discusses the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors. PMID:25477518

Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

PubMed

Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

2016-09-01

The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori

PubMed Central

Servetas, Stephanie L.; Benoit, Stéphane L.; Maier, Robert J.; Maroney, Michael J.

2017-01-01

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance. PMID:28809946

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori.

PubMed

Blum, Faith C; Hu, Heidi Q; Servetas, Stephanie L; Benoit, Stéphane L; Maier, Robert J; Maroney, Michael J; Merrell, D Scott

2017-01-01

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

Brownian Dynamics and Molecular Dynamics Study of the Association between Hydrogenase and Ferredoxin from Chlamydomonas reinhardtii

PubMed Central

Long, Hai; Chang, Christopher H.; King, Paul W.; Ghirardi, Maria L.; Kim, Kwiseon

2008-01-01

The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer. PMID:18621810

Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Byer, Amanda S.; Shepard, Eric M.; Peters, John W.

Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe 7S 9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesismore » of these remarkable metal cofactors.« less

Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

DOE PAGES

Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; ...

2014-12-04

Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe 7S 9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesismore » of these remarkable metal cofactors.« less

Arsenic treatment and power generation with a dual-chambered fuel cell with anionic and cationic membranes using NaHCO3 anolyte and HCl or NaCl catholyte.

PubMed

Maitlo, Hubdar Ali; Kim, Jung Hwan; Park, Joo Yang

2017-04-01

Dual-chambered fuel cells with an iron anode and an air-carbon cathode separated by an ion exchange membranes have been used to treat arsenate during power production. To select an effective catholyte, the dual-chambered fuel cell consisted 90 mL of 0.1 M HCl or 0.5 M NaCl as the catholyte and 1 L of 0.1 M NaHCO 3 as the anolyte at an initial pH 5. The 0.1 M HCl was an effective catholyte, with which 1 ppm arsenate in 1 L of anolyte was reduced to 5 ppb in 1 h, and the maximum power density was about 6.3 w/m 2 with an anion exchange membrane fuel cell (AEM_FC) and 4.4 w/m 2 with a cation exchange membrane fuel cell (CEM_FC). Therefore, 90 mL of 0.1 M HCl was used as a catholyte to treat 20 L of 0.1 M NaHCO 3 anolyte containing 1 ppm arsenate at an initial pH of 5 or 7. The arsenate level at pH 5 decreased to less than 5 ppb in 4 h, and the maximum power density was 5.9 W/m 2 and 4.7 W/m 2 with AEM_FC and CEM_FC, respectively. When using 0.01 M NaHCO 3 as the anolyte at pH 5, arsenate was reduced to less than 5 ppb in 8 and 24 h for AEC_FC and CEM_FC, respectively. However, when using an anolyte at pH 7, arsenate could not be effectively removed in 24 h. Therefore, when using carbonate as an anolyte, the solution should be adjusted to a weakly acidic pH in order to remove arsenate. Copyright © 2017 Elsevier Ltd. All rights reserved.

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies.

PubMed

Livingston, D J; Fox, J A; Orme-Johnson, W H; Walsh, C T

1987-07-14

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

PubMed Central

2017-01-01

Catalysis of H2 production and oxidation reactions is critical in renewable energy systems based around H2 as a clean fuel, but the present reliance on platinum-based catalysts is not sustainable. In nature, H2 is oxidized at minimal overpotential and high turnover frequencies at [NiFe] catalytic sites in hydrogenase enzymes. Although an outline mechanism has been established for the [NiFe] hydrogenases involving heterolytic cleavage of H2 followed by a first and then second transfer of a proton and electron away from the active site, details remain vague concerning how the proton transfers are facilitated by the protein environment close to the active site. Furthermore, although [NiFe] hydrogenases from different organisms or cellular environments share a common active site, they exhibit a broad range of catalytic characteristics indicating the importance of subtle changes in the surrounding protein in controlling their behavior. Here we review recent time-resolved infrared (IR) spectroscopic studies and IR spectroelectrochemical studies carried out in situ during electrocatalytic turnover. Additionally, we re-evaluate the significant body of IR spectroscopic data on hydrogenase active site states determined through more conventional solution studies, in order to highlight mechanistic steps that seem to apply generally across the [NiFe] hydrogenases, as well as steps which so far seem limited to specific groups of these enzymes. This analysis is intended to help focus attention on the key open questions where further work is needed to assess important aspects of proton and electron transfer in the mechanism of [NiFe] hydrogenases. PMID:28413691

Molecular Dynamics Studies of Proton Transport in Hydrogenase and Hydrogenase Mimics.

PubMed

Ginovska, B; Raugei, S; Shaw, W J

2016-01-01

There is extensive interest in hydrogenases based on their ability to rapidly and efficiently interconvert H2 with protons and electrons, and their (typically) superior function relative to molecular mimics. Understanding the function of enzymes is one approach to implementing design features to make better catalysts and is an approach we have implemented in our work. In this review, we will discuss our efforts to develop design principles from enzymes, with specific focus on proton transport. We will also present computational studies of the mimics we have investigated with similar methodologies. We will discuss the mechanisms used by small scaffolds on molecular mimics which in many cases are surprisingly similar to those used by nature, while in other cases, computational analysis allowed us to reveal an unexpected role. Computational methods provide one of the best ways, and in some cases, the only way, to gain insight into the mechanistic details of enzymes. In this review, we illustrate the general computational strategy we used to study the proton pathway of [FeFe]-hydrogenase, and the similar strategy to investigate small molecules. We present the main results we obtained and how our computational work stimulated or worked in concert with experimental investigations. We also focus on estimation of errors and convergence of properties in the simulations. These studies demonstrate the powerful results that can be obtained by the close pairing of experimental and theoretical approaches. Copyright © 2016 Elsevier Inc. All rights reserved. Battelle, operator of PNNL, under Contract No: DE-AC05-76RL01830 with US DoE.

Synthesis and reactivity of mononuclear iron models of [Fe]-hydrogenase that contain an acylmethylpyridinol ligand.

PubMed

Hu, Bowen; Chen, Dafa; Hu, Xile

2014-02-03

[Fe]-hydrogenase has a single iron-containing active site that features an acylmethylpyridinol ligand. This unique ligand environment had yet to be reproduced in synthetic models; however the synthesis and reactivity of a new class of small molecule mimics of [Fe]-hydrogenase in which a mono-iron center is ligated by an acylmethylpyridinol ligand has now been achieved. Key to the preparation of these model compounds is the successful C-O cleavage of an alkyl ether moiety to form the desired pyridinol ligand. Reaction of solvated complex [(2-CH2CO-6-HOC5H3N)Fe(CO)2(CH3CN)2](+)(BF4)(-) with thiols or thiophenols in the presence of NEt3 yielded 5-coordinate iron thiolate complexes. Further derivation produced complexes [(2-CH2CO-6-HOC5H3N)Fe(CO)2(SCH2CH2OH)] and [(2-CH2CO-6-HOC5H3N)Fe(CO)2(CH3COO)], which can be regarded as models of FeGP cofactors of [Fe]-hydrogenase extracted by 2-mercaptoethanol and acetic acid, respectively. When the derivative complexes were treated with HBF4 ⋅Et2O, the solvated complex was regenerated by protonation of the thiolate ligands. The reactivity of several models with CO, isocyanide, cyanide, and H2 was also investigated. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H 2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H 2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H 2. The resulting strain photoproduces H 2 and self-reports its own H 2 production through fluorescence. Furthermore, this model system represents amore » unique method of developing hydrogenase-based H 2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H 2.« less

Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production

DOE PAGES

Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; ...

2016-08-17

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H 2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H 2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H 2. The resulting strain photoproduces H 2 and self-reports its own H 2 production through fluorescence. Furthermore, this model system represents amore » unique method of developing hydrogenase-based H 2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H 2.« less

Metal insertion into NiFe-hydrogenases.

PubMed

Blokesch, M; Paschos, A; Theodoratou, E; Bauer, A; Hube, M; Huth, S; Böck, A

2002-08-01

The synthesis and the insertion of the metallocentre of NiFe-hydrogenases is a complex process, in which seven maturation enzymes plus ATP, GTP and carbamoyl phosphate are involved. The review summarizes what is known about the properties and activities of these auxiliary proteins, and postulates a pathway along which maturation may take place.

Essential role of the electroneutral Na+-HCO3- cotransporter NBCn1 in murine duodenal acid-base balance and colonic mucus layer build-up in vivo.

PubMed

Singh, Anurag Kumar; Xia, Weiliang; Riederer, Brigitte; Juric, Marina; Li, Junhua; Zheng, Wen; Cinar, Ayhan; Xiao, Fang; Bachmann, Oliver; Song, Penghong; Praetorius, Jeppe; Aalkjaer, Christian; Seidler, Ursula

2013-04-15

Duodenal epithelial cells need efficient defence strategies during gastric acidification of the lumen, while colonic mucosa counteracts damage by pathogens by building up a bacteria-free adherent mucus layer. Transport of HCO3(-) is considered crucial for duodenal defence against acid as well as for mucus release and expansion, but the transport pathways involved are incompletely understood. This study investigated the significance of the electroneutral Na(+)-HCO3(-) cotransporter NBCn1 for duodenal defence against acid and colonic mucus release. NBCn1 was localized to the basolateral membrane of duodenal villous enterocytes and of colonic crypt cells, with predominant expression in goblet cells. Duodenal villous enterocyte intracellular pH was studied before and during a luminal acid load by two-photon microscopy in exteriorized, vascularly perfused, indicator (SNARF-1 AM)-loaded duodenum of isoflurane-anaesthetized, systemic acid-base-controlled mice. Acid-induced HCO3(-) secretion was measured in vivo by single-pass perfusion and pH-stat titration. After a luminal acid load, NBCn1-deficient duodenocytes were unable to recover rapidly from intracellular acidification and could not respond adequately with protective HCO3(-) secretion. In the colon, build-up of the mucus layer was delayed, and a decreased thickness of the adherent mucus layer was observed, suggesting that basolateral HCO3(-) uptake is essential for optimal release of mucus. The electroneutral Na(+)-HCO3(-) cotransporter NBCn1 displays a differential cellular distribution in the murine intestine and is essential for HCO3(-)-dependent mucosal protective functions, such as recovery of intracellular pH and HCO3(-) secretion in the duodenum and secretion of mucus in the colon.

A [NiFe]hydrogenase model that catalyses the release of hydrogen from formic acid.

PubMed

Nguyen, Nga T; Mori, Yuki; Matsumoto, Takahiro; Yatabe, Takeshi; Kabe, Ryota; Nakai, Hidetaka; Yoon, Ki-Seok; Ogo, Seiji

2014-11-11

We report the decomposition of formic acid to hydrogen and carbon dioxide, catalysed by a NiRu complex originally developed as a [NiFe]hydrogenase model. This is the first example of H2 evolution, catalysed by a [NiFe]hydrogenase model, which does not require additional energy.

Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase

PubMed Central

Kalms, Jacqueline; Schmidt, Andrea; Utesch, Tillmann; von Stetten, David; van der Linden, Peter; Royant, Antoine; Mroginski, Maria Andrea; Carpentier, Philippe; Scheerer, Patrick

2018-01-01

[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a “soak-and-freeze” derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme. PMID:29463722

Impact of amino acid substitutions near the catalytic site on the spectral properties of an O2-tolerant membrane-bound [NiFe] hydrogenase.

PubMed

Saggu, Miguel; Ludwig, Marcus; Friedrich, Bärbel; Hildebrandt, Peter; Bittl, Robert; Lendzian, Friedhelm; Lenz, Oliver; Zebger, Ingo

2010-04-26

[NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O(2) tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O(2)-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni(r)-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Ni(u)-A state, an irreversible inactive redox state, and the light-induced Ni(a)-L state even at room temperature.

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure.

PubMed

Wulff, Philip; Thomas, Claudia; Sargent, Frank; Armstrong, Fraser A

2016-03-01

'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

Approaches to efficient molecular catalyst systems for photochemical H2 production using [FeFe]-hydrogenase active site mimics.

PubMed

Wang, Mei; Chen, Lin; Li, Xueqiang; Sun, Licheng

2011-12-28

The research on structural and functional biomimics of the active site of [FeFe]-hydrogenases is in an attempt to elucidate the mechanisms of H(2)-evolution and uptake at the [FeFe]-hydrogenase active site, and to learn from Nature how to create highly efficient H(2)-production catalyst systems. Undoubtedly, it is a challenging, arduous, and long-term work. In this perspective, the progresses in approaches to photochemical H(2) production using mimics of the [FeFe]-hydrogenase active site as catalysts in the last three years are reviewed, with emphasis on adjustment of the redox potentials and hydrophilicity of the [FeFe]-hydrogenase active site mimics to make them efficient catalysts for H(2) production. With gradually increasing understanding of the chemistry of the [FeFe]-hydrogenases and their mimics, more bio-inspired proton reduction catalysts with significantly improved efficiency of H(2) production will be realized in the future. This journal is © The Royal Society of Chemistry 2011

Theoretical investigation of aerobic and anaerobic oxidative inactivation of the [NiFe]-hydrogenase active site.

PubMed

Breglia, Raffaella; Greco, Claudio; Fantucci, Piercarlo; De Gioia, Luca; Bruschi, Maurizio

2018-01-17

The extraordinary capability of [NiFe]-hydrogenases to catalyse the reversible interconversion of protons and electrons into dihydrogen (H 2 ) has stimulated numerous experimental and theoretical studies addressing the direct utilization of these enzymes in H 2 production processes. Unfortunately, the introduction of these natural H 2 -catalysts in biotechnological applications is limited by their inhibition under oxidising (aerobic and anaerobic) conditions. With the aim of contributing to overcome this limitation, we studied the oxidative inactivation mechanism of [NiFe]-hydrogenases by performing Density Functional Theory (DFT) calculations on a very large model of their active site in which all the amino acids forming the first and second coordination spheres of the NiFe cluster have been explicitly included. We identified an O 2 molecule and two H 2 O molecules as sources of the two oxygen atoms that are inserted at the active site of the inactive forms of the enzyme (Ni-A and Ni-B) under aerobic and anaerobic conditions, respectively. Furthermore, our results support the experimental evidence that the Ni-A-to-Ni-B ratio strongly depends on the number of reducing equivalents available for the process and on the oxidizing conditions under which the reaction takes place.

Expression of a clostridial [FeFe]-hydrogenase in Chlamydomonas reinhardtii prolongs photo-production of hydrogen from water splitting

DOE PAGES

Noone, Seth; Ratcliff, Kathleen; Davis, ReAnna; ...

2016-12-24

The high oxygen (O 2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H 2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O 2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD ( hydA1– hydA2–) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H 2 photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized andmore » matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O 2 inactivation of CaI led to more sustained H 2 photoproduction when cultures were challenged with O 2 or kept under prolonged illumination at solar intensities. Lastly, these results provide new insights into the requisites for attaining photobiological H 2 production from water splitting using a more O 2-tolerant hydrogenase.« less

Guiding Principles of Hydrogenase Catalysis Instigated and Clarified by Protein Film Electrochemistry.

PubMed

Armstrong, Fraser A; Evans, Rhiannon M; Hexter, Suzannah V; Murphy, Bonnie J; Roessler, Maxie M; Wulff, Philip

2016-05-17

is also a substrate, and implicit in the description of some hydrogenases as "H2/O2 oxidoreductases" is the hypothesis that fast and efficient multielectron transfer is a key to O2 tolerance because it promotes complete reduction of O2 to harmless water. Not only is a novel [4Fe-3S] cluster (able to transfer two electrons consecutively) an important component, but connections to additional electron sources (other Fe-S clusters, an electrode, another quaternary structure unit, or the physiological membrane itself) ensure that H2 oxidation can be sustained in the presence of O2, as demonstrated with enzyme fuel cells able to operate on a H2/air mixture. Manipulating the H-H bond in the active site is the simplest proton-coupled electron-transfer reaction to be catalyzed by an enzyme. Unlike small molecular catalysts or the surfaces of materials, metalloenzymes are far better suited to engineering the all-important outer-coordination shell. Hence, recent successful site-directed mutagenesis of the conserved outer-shell "canopy" residues in a [NiFe]-hydrogenase opens up new opportunities for understanding the mechanism of H2 activation beyond the role of the inner coordination shell.

Dithiolato-bridged nickel-iron complexes as models for the active site of [NiFe]-hydrogenases.

PubMed

Song, Li-Cheng; Yang, Xi-Yue; Cao, Meng; Gao, Xiu-Yun; Liu, Bei-Bei; Zhu, Liang; Jiang, Feng

2017-03-30

The structural and functional modeling of the active site of [NiFe]-hydrogenases has been proved to be challenging to a great extent. Herein, we report the synthesis, structures, and some properties of the NiFe-based dicarbonyl, terminal hydride, and μ-hydroxo models for the active site of [NiFe]-hydrogenases.

Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton Transfer Pathway in [NiFe] Hydrogenases

PubMed Central

Szőri-Dorogházi, Emma; Maróti, Gergely; Szőri, Milán; Nyilasi, Andrea; Rákhely, Gábor; Kovács, Kornél L.

2012-01-01

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis. PMID:22511957

Concerto catalysis--harmonising [NiFe]hydrogenase and NiRu model catalysts.

PubMed

Ichikawa, Koji; Nonaka, Kyoshiro; Matsumoto, Takahiro; Kure, Bunsho; Yoon, Ki-Seok; Higuchi, Yoshiki; Yagi, Tatsuhiko; Ogo, Seiji

2010-03-28

This communication reports the successful merging of the chemical properties of a natural [NiFe]hydrogenase (Desulfovibrio vulgaris Miyazaki F) and our previously reported [NiRu] hydrogenase-mimic. The catalytic activity of both the natural enzyme and the mimic is almost identical, with the exception of working pH ranges, and this allows us to use them simultaneously in the same reaction flask. In such a manner, isotope exchange between D(2) and H(2)O could be conducted over an extended pH range (about 2-10) in one pot under mild conditions at ambient temperature and pressure.

Novel [NiFe]- and [FeFe]-Hydrogenase Gene Transcripts Indicative of Active Facultative Aerobes and Obligate Anaerobes in Earthworm Gut Contents▿†

PubMed Central

Schmidt, Oliver; Wüst, Pia K.; Hellmuth, Susanne; Borst, Katharina; Horn, Marcus A.; Drake, Harold L.

2011-01-01

The concomitant occurrence of molecular hydrogen (H2) and organic acids along the alimentary canal of the earthworm is indicative of ongoing fermentation during gut passage. Fermentative H2 production is catalyzed by [FeFe]-hydrogenases and group 4 [NiFe]-hydrogenases in obligate anaerobes (e.g., Clostridiales) and facultative aerobes (e.g., Enterobacteriaceae), respectively, functional groups that might respond differently to contrasting redox conditions. Thus, the objectives of this study were to assess the redox potentials of the alimentary canal of Lumbricus terrestris and analyze the hydrogenase transcript diversities of H2 producers in glucose-supplemented gut content microcosms. Although redox potentials in the core of the alimentary canal were variable on an individual worm basis, average redox potentials were similar. The lowest redox potentials occurred in the foregut and midgut regions, averaging 40 and 110 mV, respectively. Correlation plots between hydrogenase amino acid sequences and 16S rRNA gene sequences indicated that closely related hydrogenases belonged to closely related taxa, whereas distantly related hydrogenases did not necessarily belong to distantly related taxa. Of 178 [FeFe]-hydrogenase gene transcripts, 177 clustered in 12 Clostridiales-affiliated operational taxonomic units, the majority of which were indicative of heretofore unknown hydrogenases. Of 86 group 4 [NiFe]-hydrogenase gene transcripts, 79% and 21% were affiliated with organisms in the Enterobacteriaceae and Aeromonadaceae, respectively. The collective results (i) suggest that fermenters must cope with variable and moderately oxidative redox conditions along the alimentary canal, (ii) demonstrate that heretofore undetected hydrogenases are present in the earthworm gut, and (iii) corroborate previous findings implicating Clostridiaceae and Enterobacteriaceae as active fermentative taxa in earthworm gut content. PMID:21784904

Structural Characterization of the Novel and Thermal Stable Hydrogenases from the Purple Sulfur Bacteria Thiocapsa Roseopersicina and Lamprobacter Modestohalophilus

DTIC Science & Technology

2011-08-01

production activity achieved for hydrogenase encapsulated in sol–gel material doped with carbon nanotubes . J. Mater. Chem., 20, 1065 (2010). Abstract...Doping hydrogenase-containing sol-gel materials with multi-walled carbon nanotubes , polyethylene glycol and methyl viologen results in a greater than...homogeneous T. roseopersicina hydrogenase and of multi-walled carbon nanotubes in silica gel was studied. It is shown that in such material between the

Synthetic Models for Nickel-Iron Hydrogenase Featuring Redox-Active Ligands.

PubMed

Schilter, David; Gray, Danielle L; Fuller, Amy L; Rauchfuss, Thomas B

2017-05-01

The nickel-iron hydrogenase enzymes efficiently and reversibly interconvert protons, electrons, and dihydrogen. These redox proteins feature iron-sulfur clusters that relay electrons to and from their active sites. Reported here are synthetic models for nickel-iron hydrogenase featuring redox-active auxiliaries that mimic the iron-sulfur cofactors. The complexes prepared are Ni II (μ-H)Fe II Fe II species of formula [(diphosphine)Ni(dithiolate)(μ-H)Fe(CO) 2 (ferrocenylphosphine)] + or Ni II Fe I Fe II complexes [(diphosphine)Ni(dithiolate)Fe(CO) 2 (ferrocenylphosphine)] + (diphosphine = Ph 2 P(CH 2 ) 2 PPh 2 or Cy 2 P(CH 2 ) 2 PCy 2 ; dithiolate = - S(CH 2 ) 3 S - ; ferrocenylphosphine = diphenylphosphinoferrocene, diphenylphosphinomethyl(nonamethylferrocene) or 1,1'-bis(diphenylphosphino)ferrocene). The hydride species is a catalyst for hydrogen evolution, while the latter hydride-free complexes can exist in four redox states - a feature made possible by the incorporation of the ferrocenyl groups. Mixed-valent complexes of 1,1'-bis(diphenylphosphino)ferrocene have one of the phosphine groups unbound, with these species representing advanced structural models with both a redox-active moiety (the ferrocene group) and a potential proton relay (the free phosphine) proximal to a nickel-iron dithiolate.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of

The ERK pathway regulates Na(+)-HCO(3)(-) cotransport activity in adult rat cardiomyocytes.

PubMed

Baetz, Delphine; Haworth, Robert S; Avkiran, Metin; Feuvray, Danielle

2002-11-01

The sarcolemmal Na(+)-HCO cotransporter (NBC) is stimulated by intracellular acidification and acts as an acid extruder. We examined the role of the ERK pathway of the MAPK cascade as a potential mediator of NBC activation by intracellular acidification in the presence and absence of angiotensin II (ANG II) in adult rat ventricular myocytes. Intracellular pH (pH(i)) was recorded with the use of seminaphthorhodafluor-1. The NH method was used to induce an intracellular acid load. NBC activation was significantly decreased with the ERK inhibitors PD-98059 and U-0126. NBC activity after acidification was increased in the presence of ANG II (pH(i) range of 6.75-7.00). ANG II plus PD-123319 (AT(2) antagonist) still increased NBC activity, whereas ANG II plus losartan (AT(1) antagonist) did not affect it. ERK phosphorylation (measured by immunoblot analysis) during intracellular acidification was increased by ANG II, an effect that was abolished by losartan and U-0126. In conclusion, the MAPK(ERK)-dependent pathway facilitates the rate of pH(i) recovery from acid load through NBC activity and is involved in the AT(1) receptor-mediated stimulation of such activity by ANG II.

Reaction fronts of the autocatalytic hydrogenase reaction

NASA Astrophysics Data System (ADS)

Gyevi-Nagy, László; Lantos, Emese; Gehér-Herczegh, Tünde; Tóth, Ágota; Bagyinka, Csaba; Horváth, Dezső

2018-04-01

We have built a model to describe the hydrogenase catalyzed, autocatalytic, reversible hydrogen oxidation reaction where one of the enzyme forms is the autocatalyst. The model not only reproduces the experimentally observed front properties, but also explains the found hydrogen ion dependence. Furthermore, by linear stability analysis, two different front types are found in good agreement with the experiments.

Fractionation of Sulfur Isotopes by Desulfovibrio vulgaris Mutants Lacking Periplasmic Hydrogenases or the Type I Tetraheme Cytochrome c3

NASA Astrophysics Data System (ADS)

Sim, M.; Ono, S.; Bosak, T.

2012-12-01

A large fraction of anaerobic mineralization of organic compounds relies on microbial sulfate reduction. Sulfur isotope fractionation by these microbes has been widely used to trace the biogeochemical cycling of sulfur and carbon, but intracellular mechanisms behind the wide range of fractionations observed in nature and cultures are not fully understood. In this study, we investigated the influence of electron transport chain components on the fractionation of sulfur isotopes by culturing Desulfovibrio vulgaris Hildenborough mutants lacking hydrogenases or type I tetraheme cytochrome c3 (Tp1-c3). The mutants were grown both in batch and continuous cultures. All tested mutants grew on lactate or pyruvate as the sole carbon and energy sources, generating sulfide. Mutants lacking cytoplasmic and periplasmic hydrogenases exhibited similar growth physiologies and sulfur isotope fractionations to their parent strains. On the other hand, a mutant lacking Tp1-c3 (ΔcycA) fractionated the 34S/32S ratio more than the wild type, evolving H2 in the headspace and exhibiting a lower specific respiration rate. In the presence of high concentrations of pyruvate, the growth of ΔcycA relied largely on fermentation rather than sulfate reduction, even when sulfate was abundant, producing the largest sulfur isotope effect observed in this study. Differences between sulfur isotope fractionation by ΔcycA and the wild type highlight the effect of electron transfer chains on the magnitude of sulfur isotope fractionation. Because Tp1-c3 is known to exclusively shuttle electrons from periplasmic hydrogenases to transmembrane complexes, electron transfers in the absence of Tp1-c3 should bypass the periplasmic hydrogen cycling, and the loss of reducing equivalents in the form of H2 can impair the flow of electrons from organic acids to sulfur, increasing isotope fractionation. Larger fractionation by ΔcycA can inform interpretations of sulfur isotope data at an environmental scale as well

Cyanobacterial Hydrogenases and Hydrogen Metabolism Revisited: Recent Progress and Future Prospects

PubMed Central

Khanna, Namita; Lindblad, Peter

2015-01-01

Cyanobacteria have garnered interest as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms can utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical processes. Our limited understanding of the cellular hydrogen production pathway is a primary setback in the potential scale-up of this process. In this regard, the present review discusses the recent insight around ferredoxin/flavodoxin as the likely electron donor to the bidirectional Hox hydrogenase instead of the generally accepted NAD(P)H. This may have far reaching implications in powering solar driven hydrogen production. However, it is evident that a successful hydrogen-producing candidate would likely integrate enzymatic traits from different species. Engineering the [NiFe] hydrogenases for optimal catalytic efficiency or expression of a high turnover [FeFe] hydrogenase in these photo-autotrophs may facilitate the development of strains to reach target levels of biohydrogen production in cyanobacteria. The fundamental advancements achieved in these fields are also summarized in this review. PMID:26006225

Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases

NASA Astrophysics Data System (ADS)

Winkler, Martin; Senger, Moritz; Duan, Jifu; Esselborn, Julian; Wittkamp, Florian; Hofmann, Eckhard; Apfel, Ulf-Peter; Stripp, Sven Timo; Happe, Thomas

2017-07-01

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier's principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

Mechanism of Hydrogen Production in [Fe-Fe]-Hydrogenase: A Density Functional Theory Study (Postprint)

DTIC Science & Technology

2007-03-01

Chem. Soc. 2001, 123, 1596-1601. (8) Volbeda, A.; Fontecilla-Camps, J. C. The Active Site and Catalytic Mechanism of NiFe Hydrogenases. Dalton Trans... Properties of Diiron Complexes Related to the [2Fe]H Subcluster of Fe-Only Hydrogenases. Inorg. Chem. 2002, 41, 1421-1429. (16) Bruschi, M.; Fantucci, P...Structural, Electronic, and Reactivity Properties of Complexes Related to the [2Fe]H Subcluster. Inorg. Chem. 2003, 42, 4773-4781. (17) Bruschi, M.; Fantucci

Roles of HynAB and Ech, the Only Two Hydrogenases Found in the Model Sulfate Reducer Desulfovibrio gigas

PubMed Central

Morais-Silva, Fabio O.; Santos, Catia I.; Rodrigues, Rute

2013-01-01

Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism. PMID:23974026

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

PubMed

Howlett, Robert M; Hughes, Bethan M; Hitchcock, Andrew; Kelly, David J

2012-06-01

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

Expression of Shewanella oneidensis MR-1 [FeFe]-Hydrogenase Genes in Anabaena sp. Strain PCC 7120

PubMed Central

Gärtner, Katrin; Lechno-Yossef, Sigal; Cornish, Adam J.; Wolk, C. Peter

2012-01-01

H2 generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H2 from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O2 sensitive. Certain filamentous cyanobacteria protect nitrogenase from O2 by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter PhetN. Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O2. PMID:23023750

The structurally unique photosynthetic Chlorella variabilis NC64A hydrogenase does not interact with plant-type ferredoxins.

PubMed

Engelbrecht, Vera; Rodríguez-Maciá, Patricia; Esselborn, Julian; Sawyer, Anne; Hemschemeier, Anja; Rüdiger, Olaf; Lubitz, Wolfgang; Winkler, Martin; Happe, Thomas

2017-09-01

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H 2 ) production and H 2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H 2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity). Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, S E; Hopkins, R C; Blanchette, C

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

PubMed Central

Hexter, Suzannah V.; Grey, Felix; Happe, Thomas; Climent, Victor; Armstrong, Fraser A.

2012-01-01

The extraordinary ability of Fe- and Ni-containing enzymes to catalyze rapid and efficient H+/H2 interconversion—a property otherwise exclusive to platinum metals—has been investigated in a series of experiments combining variable-temperature protein film voltammetry with mathematical modeling. The results highlight important differences between the catalytic performance of [FeFe]-hydrogenases and [NiFe]-hydrogenases and justify a simple model for reversible catalytic electron flow in enzymes and electrocatalysts that should be widely applicable in fields as diverse as electrochemistry, catalysis, and bioenergetics. The active site of [FeFe]-hydrogenases, an intricate Fe-carbonyl complex known as the “H cluster,” emerges as a supreme catalyst. PMID:22802675

Impact of the chemicals, essential for the purification process of strict Fe-hydrogenase, on the corrosion of mild steel.

PubMed

Rouvre, Ingrid; Gauquelin, Charles; Meynial-Salles, Isabelle; Basseguy, Régine

2016-06-01

The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.1 M pH7 Tris-HCl medium with or without pure hydrogenase. The results showed that hydrogenase and the additional molecules were in competition, and the electrochemical response could not be attributed solely to hydrogenase. Then, solutions with additional chemicals of different compositions were studied electrochemically. DTT polluted the electrochemical signal by increasing the Eoc by 35 mV 24 h after the injection of 300 μL of control solutions with DTT, whereas it drastically decreased the corrosion rate by increasing the charge transfer resistance (Rct 10 times the initial value). Thus, DTT was shown to have a strong antagonistic effect on corrosion and was removed from the purification process. An optimal composition of the medium was selected (0.5 mM dithionite, 7.5 mM desthiobiotin) that simultaneously allowed a high activity of hydrogenase and a lower impact on the electrochemical response for corrosion tests. Copyright © 2016 Elsevier B.V. All rights reserved.

Secretagogue stimulation enhances NBCe1 (electrogenic Na(+)/HCO(3)(-) cotransporter) surface expression in murine colonic crypts.

PubMed

Yu, Haoyang; Riederer, Brigitte; Stieger, Nicole; Boron, Walter F; Shull, Gary E; Manns, Michael P; Seidler, Ursula E; Bachmann, Oliver

2009-12-01

A Na(+)/HCO(3)(-) cotransporter (NBC) is located in the basolateral membrane of the gastrointestinal epithelium, where it imports HCO(3)(-) during stimulated anion secretion. Having previously demonstrated secretagogue activation of NBC in murine colonic crypts, we now asked whether vesicle traffic and exocytosis are involved in this process. Electrogenic NBCe1-B was expressed at significantly higher levels than electroneutral NBCn1 in colonic crypts as determined by QRT-PCR. In cell surface biotinylation experiments, a time-dependent increase in biotinylated NBCe1 was observed, which occurred with a peak of +54.8% after 20 min with forskolin (P < 0.05) and more rapidly with a peak of +59.8% after 10 min with carbachol (P < 0.05) and which corresponded well with the time course of secretagogue-stimulated colonic bicarbonate secretion in Ussing chamber experiments. Accordingly, in isolated colonic crypts pretreated with forskolin and carbachol for 10 min, respectively, and subjected to immunohistochemistry, the NBCe1 signal showed a markedly stronger colocalization with the E-cadherin signal, which was used as a membrane marker, compared with the untreated control. Cytochalasin D did not change the observed increase in membrane abundance, whereas colchicine alone enhanced NBCe1 membrane expression without an additional increase after carbachol or forskolin, and LY294002 had a marked inhibitory effect. Taken together, our results demonstrate a secretagogue-induced increase of NBCe1 membrane expression. Vesicle traffic and exocytosis might thus represent a novel mechanism of intestinal NBC activation by secretagogues.

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

PubMed Central

Flanagan, Lindsey A.; Parkin, Alison

2016-01-01

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

DOE PAGES

Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; ...

2015-08-10

The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique ‘wagging’ mode involving H- motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate amore » low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H- binding Ni more tightly than Fe. Lastly, the present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.« less

Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy

PubMed Central

Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin; Schilter, David; Pelmenschikov, Vladimir; van Gastel, Maurice; Neese, Frank; Rauchfuss, Thomas B.; Gee, Leland B.; Scott, Aubrey D.; Yoda, Yoshitaka; Tanaka, Yoshihito; Lubitz, Wolfgang; Cramer, Stephen P.

2015-01-01

The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the 57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique ‘wagging' mode involving H− motion perpendicular to the Ni(μ-H)57Fe plane was studied using 57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)57Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)57Fe(CO)3]+ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H− binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts. PMID:26259066

Synthetic Models for Nickel–Iron Hydrogenase Featuring Redox-Active Ligands*

PubMed Central

Schilter, David; Gray, Danielle L.; Fuller, Amy L.; Rauchfuss, Thomas B.

2017-01-01

The nickel–iron hydrogenase enzymes efficiently and reversibly interconvert protons, electrons, and dihydrogen. These redox proteins feature iron–sulfur clusters that relay electrons to and from their active sites. Reported here are synthetic models for nickel–iron hydrogenase featuring redox-active auxiliaries that mimic the iron–sulfur cofactors. The complexes prepared are NiII(μ-H)FeIIFeII species of formula [(diphosphine)Ni(dithiolate)(μ-H)Fe(CO)2(ferrocenylphosphine)]+ or NiIIFeIFeII complexes [(diphosphine)Ni(dithiolate)Fe(CO)2(ferrocenylphosphine)]+ (diphosphine = Ph2P(CH2)2PPh2 or Cy2P(CH2)2PCy2; dithiolate = −S(CH2)3S−; ferrocenylphosphine = diphenylphosphinoferrocene, diphenylphosphinomethyl(nonamethylferrocene) or 1,1′-bis(diphenylphosphino)ferrocene). The hydride species is a catalyst for hydrogen evolution, while the latter hydride-free complexes can exist in four redox states – a feature made possible by the incorporation of the ferrocenyl groups. Mixed-valent complexes of 1,1′-bis(diphenylphosphino)ferrocene have one of the phosphine groups unbound, with these species representing advanced structural models with both a redox-active moiety (the ferrocene group) and a potential proton relay (the free phosphine) proximal to a nickel–iron dithiolate. PMID:28819328

Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism.

PubMed

Cha, Minseok; Chung, Daehwan; Westpheling, Janet

2016-02-01

The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.

Thermodynamic description of Tc(iv) solubility and carbonate complexation in alkaline NaHCO3-Na2CO3-NaCl systems.

PubMed

Baumann, A; Yalçıntaş, E; Gaona, X; Polly, R; Dardenne, K; Prüßmann, T; Rothe, J; Altmaier, M; Geckeis, H

2018-03-28

The solubility of 99 Tc(iv) was investigated in dilute to concentrated carbonate solutions (0.01 M ≤ C tot ≤ 1.0 M, with C tot = [HCO 3 - ] + [CO 3 2- ]) under systematic variation of ionic strength (I = 0.3-5.0 M NaHCO 3 -Na 2 CO 3 -NaCl-NaOH) and pH m (-log[H + ] = 8.5-14.5). Strongly reducing conditions (pe + pH m ≈ 2) were set with Sn(ii). Carbonate enhances the solubility of Tc(iv) in alkaline conditions by up to 3.5 log 10 -units compared to carbonate-free systems. Solvent extraction and XANES confirmed that Tc was kept as +IV during the timeframe of the experiments (≤ 650 days). Solid phase characterization performed by XAFS, XRD, SEM-EDS, chemical analysis and TG-DTA confirmed that TcO 2 ·0.6H 2 O(am) controls the solubility of Tc(iv) under the conditions investigated. Slope analysis of the solubility data in combination with solid/aqueous phase characterization and DFT calculations indicate the predominance of the species Tc(OH) 3 CO 3 - at pH m ≤ 11 and C tot ≥ 0.01 M, for which thermodynamic and activity models are derived. Solubility data obtained above pH m ≈ 11 indicates the formation of previously unreported Tc(iv)-carbonate species, possibly Tc(OH) 4 CO 3 2- , although the likely formation of additional complexes prevents deriving a thermodynamic model valid for this pH m -region. This work provides the most comprehensive thermodynamic dataset available for the system Tc 4+ -Na + -Cl - -OH - -HCO 3 - -CO 3 2- -H 2 O(l) valid under a range of conditions relevant for nuclear waste disposal.

Force Field Development and Molecular Dynamics of [NiFe] Hydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle MA; Xiong, Yijia; Straatsma, TP

2012-05-09

Classical molecular force-field parameters describing the structure and motion of metal clusters in [NiFe] hydrogenase enzymes can be used to compare the dynamics and thermodynamics of [NiFe] under different oxidation, protonation, and ligation circumstances. Using density functional theory (DFT) calculations of small model clusters representative of the active site and the proximal, medial, and distal Fe/S metal centers and their attached protein side chains, we have calculated classical force-field parameters for [NiFe] in reduced and oxidized states, including internal coordinates, force constants, and atom-centered charges. Derived force constants revealed that cysteinate ligands bound to the metal ions are more flexiblemore » in the Ni-B active site, which has a bridging hydroxide ligand, than in the Ni-C active site, which has a bridging hydride. Ten nanosecond all-atom, explicit-solvent MD simulations of [NiFe] hydrogenase in oxidized and reduced catalytic states established the stability of the derived force-field parameters in terms of C{alpha} and metal cluster fluctuations. Average active site structures from the protein MD simulations are consistent with [NiFe] structures from the Protein Data Bank, suggesting that the derived force-field parameters are transferrable to other hydrogenases beyond the structure used for testing. A comparison of experimental H{sub 2}-production rates demonstrated a relationship between cysteinate side chain rotation and activity, justifying the use of a fully dynamic model of [NiFe] metal cluster motion.« less

Solution structure and backbone dynamics of an endopeptidase HycI from Escherichia coli: implications for mechanism of the [NiFe] hydrogenase maturation.

PubMed

Yang, Fan; Hu, Wei; Xu, Huimin; Li, Congmin; Xia, Bin; Jin, Changwen

2007-02-09

[NiFe] hydrogenases are metalloenzymes involved in many biological processes concerning the metabolism of hydrogen. The maturation of the large subunit of these hydrogenases requires the cleavage of a peptide at the C terminus by an endopeptidase before the final formation of the [NiFe] metallocenter. HycI is an endopeptidase of the M52 family and responsible for the C-terminal cleavage of the large subunit of hydrogenase 3 in Escherichia coli. Although extensive studies were performed, the molecular mechanism of recognition and cleavage of hydrogenase 3 remains elusive. Herein, we report the solution structure of E. coli HycI determined by high resolution nuclear magnetic resonance spectroscopy. This is the first solution structure of the apo form of endopeptidase of the M52 family reported thus far. The overall structure is similar to the crystal structure of holo-HybD in the same family. However, significant diversity was observed between the two structures. Especially, HycI shows an open conformation at the putative nickel-binding site, whereas HybD adopts a closed conformation. In addition, we performed backbone dynamic studies to probe the motional properties of the apo form of HycI. Furthermore, the metal ion titration experiments provide insightful information on the substrate recognition and cleavage processes. Taken together, our current structural, biochemical, and dynamic studies extend the knowledge of the M52 family proteins and provide novel insights into the biological function of HycI.

HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

PubMed Central

Lindberg, Pia; Devine, Ellenor; Stensjö, Karin

2012-01-01

The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium. PMID:22020512

Evidence for the role of a Na(+)/HCO(3)(-) cotransporter in trout hepatocyte pHi regulation.

PubMed

Furimsky, M; Moon, T W; Perry, S F

2000-07-01

The mechanisms of intracellular pH (pHi) regulation were examined in hepatocytes of the rainbow trout Oncorhynchus mykiss. pHi was monitored using the pH-sensitive fluorescent dye BCECF, and the effects of various media and pharmacological agents were examined for their influence on baseline pHi and recovery rates from acid and base loading. Rates of Na(+) uptake were measured using (22)Na, and changes in membrane potential were examined using the potentiometric fluorescent dye Oxonol VI. The rate of proton extrusion following acid loading was diminished by the blockade of either Na(+)/H(+) exchange (using amiloride) or anion transport (using DIDS). The removal of external HCO(3)(-) and the abolition of outward K(+) diffusion by the channel blocker Ba(2+) also decreased the rate of proton extrusion following acid load. Depolarization of the cell membrane with 50 mmol l(-)(1) K(+), however, did not affect pHi. The rate of recovery from base loading was significantly diminished by the blockade of anion transport, removal of external HCO(3)(-) and, to a lesser extent, by blocking Na(+)/H(+) exchange. The blockade of K(+) conductance had no effect. The decrease in Na(+) uptake rate observed in the presence of the anion transport blocker DIDS and the DIDS-sensitive hyperpolarization of membrane potential during recovery from acid loading suggest that a Na(+)-dependent electrogenic transport system is involved in the restoration of pHi after intracellular acidification. The effects on baseline pHi indicate that the different membrane exchangers are tonically active in the maintenance of steady-state pHi. This study confirms the roles of a Na(+)/H(+) exchanger and a Cl(-)/HCO(3)(-) exchanger in the regulation of trout hepatocyte pHi and provides new evidence that a Na(+)/HCO(3)(-) cotransporter contributes to pHi regulation.

Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase.

PubMed

Shomura, Yasuhito; Yoon, Ki-Seok; Nishihara, Hirofumi; Higuchi, Yoshiki

2011-10-16

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2) → 2H(+) + 2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme.

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase

PubMed Central

Bürstel, Ingmar; Siebert, Elisabeth; Zebger, Ingo; Friedrich, Bärbel

2016-01-01

Hydrogenases are nature’s key catalysts involved in both microbial consumption and production of molecular hydrogen. H2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H2, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry—that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H2 oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O2, employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N10-formyl-tetrahydrofolate (N10-CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N10-CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates. PMID:27930319

Atomic Resolution Modeling of the Ferredoxin:[FeFe] Hydrogenase Complex from Chlamydomonas reinhardtii

PubMed Central

Chang, Christopher H.; King, Paul W.; Ghirardi, Maria L.; Kim, Kwiseon

2007-01-01

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H2 from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H2 evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2. PMID:17660315

Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii.

PubMed

Chang, Christopher H; King, Paul W; Ghirardi, Maria L; Kim, Kwiseon

2007-11-01

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.

Properties of the [NiFe]-hydrogenase maturation protein HypD.

PubMed

Blokesch, Melanie; Böck, August

2006-07-24

A mutational screen of amino acid residues of hydrogenase maturation protein HypD from Escherichia coli disclosed that seven conserved cysteine residues located in three different motifs in HypD are essential. Evidence is presented for potential functions of these motifs in the maturation process.

Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei.

PubMed

Worm, Petra; Stams, Alfons J M; Cheng, Xu; Plugge, Caroline M

2011-01-01

Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO(2) to formylmethanofuran. The best candidates for F(420)-dependent N(5),N(10)-methyl-H(4) MPT and N(5),N(10),-methylene-H(4)MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases.

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases.

PubMed

Wulff, Philip; Day, Christopher C; Sargent, Frank; Armstrong, Fraser A

2014-05-06

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H2 + O2 = 2 H2O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H2O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H2 and 10% O2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H2 oxidation. The oxidase activity is too slow to be useful in removing O2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation.

Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

PubMed Central

Wu, Chang-Hao; McTernan, Patrick M.; Walter, Mary E.; Adams, Michael W. W.

2015-01-01

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed. PMID:26543406

Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

DOE PAGES

Wu, Chang-Hao; McTernan, Patrick M.; Walter, Mary E.; ...

2015-01-01

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus , amore » member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.« less

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

2012-04-01

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus,more » led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.« less

Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12.

PubMed Central

Stoker, K; Reijnders, W N; Oltmann, L F; Stouthamer, A H

1989-01-01

To isolate genes from Escherichia coli which regulate the labile hydrogenase activity, a plasmid library was used to transform hydL mutants lacking the labile hydrogenase. A single type of gene, designated hydG, was isolated. This gene also partially restored the hydrogenase activity in hydF mutants (which are defective in all hydrogenase isoenzymes), although the low hydrogenase 1 and 2 levels were not induced. Therefore, hydG apparently regulates, specifically, the labile hydrogenase activity. Restoration of this latter activity in hydF mutants was accompanied by a proportional increase of the H2 uptake activity, suggesting a functional relationship. H2:fumarate oxidoreductase activity was not restored in complemented hydL mutants. These latter strains may therefore lack, in addition to the labile hydrogenase, a second component (provisionally designated component R), possibly an electron carrier coupling H2 oxidation to the anerobic respiratory chain. Sequence analysis showed an open reading frame of 1,314 base pairs for hydG. It was preceded by a ribosome-binding site but apparently lacked a promoter. Minicell experiments revealed a single polypeptide of approximately 50 kilodaltons. Comparison of the predicted amino acid sequence with a protein sequence data base revealed strong homology to NtrC from Klebsiella pneumoniae, a DNA-binding transcriptional activator. The 411 base pairs upstream from pHG40 contained a second open reading frame overlapping hydG by four bases. The deduced amino acid sequence showed considerable homology with the C-terminal part of NtrB. This sequence was therefore assumed to be part of a second gene, encoding the NtrB-like component, and was designated hydH. The labile hydrogenase activity in E. coli is apparently regulated by a multicomponent system analogous to the NtrB-NtrC system. This conclusion is in agreement with the results of Birkmann et al. (A. Birkmann, R. G. Sawers, and A. Böck, Mol. Gen. Genet. 210:535-542, 1987), who

Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum

DOE PAGES

Biswas, Ranjita; Zheng, Tianyong; Olson, Daniel G.; ...

2015-02-12

The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl-CoA reduction to ethanol. C. thermocellum encodes four hydrogenases and rather than delete each individually, we targeted a hydrogenase maturase gene (hydG), involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe]more » hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in hydG ech was undetectable and ethanol yield increased nearly 2-fold compared to wild type. Interestingly, mutant growth improved upon the addition of acetate, which led to increased expression of genes related to sulfate metabolism, suggesting these mutants may use sulfate as a terminal electron acceptor to balance redox reactions. Genomic analysis of hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation is found in ethanol tolerant C. thermocellum strain E50C, hydG and hydG ech are not more ethanol tolerant than wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. The dramatic increase in ethanol production here suggests that targeting protein post-translational modification is a promising new approach for inactivation of multiple enzymes simultaneously for metabolic engineering.« less

Delivery of Iron-Sulfur Clusters to the Hydrogen-Oxidizing [NiFe]-Hydrogenases in Escherichia coli Requires the A-Type Carrier Proteins ErpA and IscA

PubMed Central

Pinske, Constanze; Sawers, R. Gary

2012-01-01

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the

NRVS and EPR Spectroscopy of 57Fe-enriched [FeFe] Hydrogenase Indicate Stepwise Assembly of the H-cluster†

PubMed Central

Kuchenreuther, Jon M.; Guo, Yisong; Wang, Hongxin; Myers, William K.; George, Simon J.; Boyke, Christine A.; Yoda, Yoshitaka; Alp, E. Ercan; Zhao, Jiyong; Britt, R. David; Swartz, James R.; Cramer, Stephen P.

2013-01-01

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe–S clusters that facilitate electron transfer to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H2. Here, we have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe–4S] ferredoxin as well as a model complex resembling the [2Fe]H catalytic domain of the H-cluster. In order to enrich the hydrogenase with 57Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with 56Fe-labeled Fe–S clusters was activated in vitro using 57Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe]H subcluster with 57Fe, which NRVS confirms by detecting 57Fe–CO and 57Fe–CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second 57Fe–S cluster. EPR spectroscopy indicates that this 57Fe-enriched metal center is not the [4Fe– 4S]H subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an 56Fe-enriched [4Fe–4S]H cluster during in vitro maturation, providing unambiguous evidence for stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases. PMID:23249091

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

PubMed Central

Petrenko, Alexander; Stein, Matthias

2017-01-01

Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron–sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from Density Functional Theory (DFT) calculations. The distal cubane FeS-cluster has a three-cysteine and one-histidine coordination [Fe4S4](His)(Cys)3 first ligation sphere. The reorganization energy (inner- and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and an all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe4S4](Cys)4 case. The fast and efficient electron transfer from the distal iron–sulfur cluster is realized by a fine-tuned protein environment, which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering. PMID:28067774

Identification of a catalytic iron-hydride at the H-cluster of [FeFe]-hydrogenase

DOE PAGES

Mulder, David W.; Guo, Yisong; Ratzloff, Michael W.; ...

2016-12-14

Hydrogenases couple electrochemical potential to the reversible chemical transformation of H 2 and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (H hyd) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S] H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe] H) with CO and CN- ligands. Mossbauer analysis and density functional theory (DFT) calculations show that H hyd consists of a reduced [4Fe-4S] H + coupled to a diferrous [2Fe] Hmore » with a terminally bound Fe-hydride. The existence of the Fe-hydride in Hhyd was demonstrated by an unusually low Mossbauer isomer shift of the distal Fe of the [2Fe] H subcluster. As a result, a DFT model of H hyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.« less

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase.

PubMed

Goris, Tobias; Wait, Annemarie F; Saggu, Miguel; Fritsch, Johannes; Heidary, Nina; Stein, Matthias; Zebger, Ingo; Lendzian, Friedhelm; Armstrong, Fraser A; Friedrich, Bärbel; Lenz, Oliver

2011-05-01

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

Hydrogenase activity in the thermophile mastigocladus laminosus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benemann, J.R.; Miyamoto, K.; Hallenbeck, P.C.

Hydrogenase activity in the thermophilic cyanobacterium, Mastigocladus laminosus was studied both in vivo and in vitro. In vivo hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H/sub 2/-supported acetylene reduction in reductant-limited cultures was a light-dependent, but O/sub 2/-independent reaction. In vitro hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for Anabaena cylindrica.

Catalysts for hydrogen evolution from the [NiFe] hydrogenase to the Ni2P(001) surface: the importance of ensemble effect.

PubMed

Liu, Ping; Rodriguez, José A

2005-10-26

Density functional theory (DFT) was employed to investigate the behavior of a series of catalysts used in the hydrogen evolution reaction (HER, 2H(+) + 2e(-) --> H(2)). The kinetics of the HER was studied on the [NiFe] hydrogenase, the [Ni(PS3*)(CO)](1)(-) and [Ni(PNP)(2)](2+) complexes, and surfaces such as Ni(111), Pt(111), or Ni(2)P(001). Our results show that the [NiFe] hydrogenase exhibits the highest activity toward the HER, followed by [Ni(PNP)(2)](2+) > Ni(2)P > [Ni(PS3*)(CO)](1)(-) > Pt > Ni in a decreasing sequence. The slow kinetics of the HER on the surfaces is due to the fact that the metal hollow sites bond hydrogen too strongly to allow the facile removal of H(2). In fact, the strong H-Ni interaction on Ni(2)P(001) can lead to poisoning of the highly active sites of the surface, which enhances the rate of the HER and makes it comparable to that of the [NiFe] hydrogenase. In contrast, the promotional effect of H-poisoning on the HER on Pt and Ni surfaces is relatively small. Our calculations suggest that among all of the systems investigated, Ni(2)P should be the best practical catalyst for the HER, combining the high thermostability of the surfaces and high catalytic activity of the [NiFe] hydrogenase. The good behavior of Ni(2)P(001) toward the HER is found to be associated with an ensemble effect, where the number of active Ni sites is decreased due to presence of P, which leads to moderate bonding of the intermediates and products with the surface. In addition, the P sites are not simple spectators and directly participate in the HER.

Effective identification of (NH4)2CO3 and NH4HCO3 concentrations in NaHCO3 regeneration process from desulfurized waste.

PubMed

Govindan, Muthuraman; Karunakaran, Kannan; Nallasamy, Palanisami; Moon, Il Shik

2015-01-01

This work describes the quantitative analysis of (NH4)2CO3 and NH4HCO3 using a simple solution phase titration method. Back titration results at various (NH4)2CO3-NH4HCO3 ratios demonstrated that 6:4 ratio caused a 3% error in their differentiation, but very high errors were found at other ratios. A similar trend was observed for the double indicator method, especially when strong acid HCl was used as a titrant, where still less errors (2.5%) at a middle ratio of (NH4)2CO3-NH4HCO3 was found. Remaining ratios with low (NH4)2CO3 (2:8, 4:6) show high +ve error (found concentration is less) and high (NH4)2CO3 (7:3, 8:2, and 9:1) show high -ve error (found concentration is higher) and vice versa for NH4HCO3. In replacement titration using Na2SO4, at both higher end ratios of (NH4)2CO3-NH4HCO3 (2:8 and 9:1), both -ve and +ve errors were minimized to 75% by partial equilibrium arrest between (NH4)2CO3 and NH2COONH4, instead of more than 100% observed in back titration and only double indicator methods. In the presence of (NH4)2SO4 both -ve and +ve error% are completely reduced to 3±1 at ratios 2:8, 4:6, and 6:4 of (NH4)2CO3-NH4HCO3, which demonstrates that the equilibrium transformation between NH2COONH4 and (NH4)2CO3 is completely controlled. The titration conducted at lower temperature (5 °C) in the presence of (NH4)2SO4 at higher ratios of (NH4)2CO3-NH4HCO3 (7:3, 8:2,and 9:1) shows complete minimization of both -ve and +ve errors to 2±1%, which explains the complete arresting of equilibrium transformation. Finally, the developed method shows 2±1% error in differentiation of CO3(2-) and HCO3(-) in the regeneration process of NaHCO3 from crude desulfurized sample. The developed method is more promising to differentiate CO3(2-) and HCO3(-) in industrial applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Key hydride vibrational modes in [NiFe] hydrogenase model compounds studied by resonance Raman spectroscopy and density functional calculations.

PubMed

Shafaat, Hannah S; Weber, Katharina; Petrenko, Taras; Neese, Frank; Lubitz, Wolfgang

2012-11-05

Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. While many enzymatic states of the [NiFe] hydrogenase have been studied extensively, there are multiple catalytically relevant EPR-silent states that remain poorly characterized. Analysis of model compounds using new spectroscopic techniques can provide a framework for the study of these elusive states within the protein. We obtained optical absorption and resonance Raman (RR) spectra of (dppe)Ni(μ-pdt)Fe(CO)(3) and [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)(3)][BF(4)], which are structural and functional model compounds for the EPR-silent Ni-SI and Ni-R states of the [NiFe] hydrogenase active site. The studies presented here use RR spectroscopy to probe vibrational modes of the active site, including metal-hydride stretching vibrations along with bridging ligand-metal and Fe-CO bending vibrations, with isotopic substitution used to identify key metal-hydride modes. The metal-hydride vibrations are essentially uncoupled and represent isolated, localized stretching modes; the iron-hydride vibration occurs at 1530 cm(-1), while the nickel-hydride vibration is observed at 945 cm(-1). The significant discrepancy between the metal-hydride vibrational frequencies reflects the slight asymmetry in the metal-hydride bond lengths. Additionally, time-dependent density functional theory (TD-DFT) calculations were carried out to obtain theoretical RR spectra of these compounds. On the basis of the detailed comparison of theory and experiment, the dominant electronic transitions and significant normal modes probed in the RR experiments were assigned; the primary transitions in the visible wavelengths represent metal-to-metal and metal-to-ligand charge transfer bands. Inherent properties of metal-hydride vibrational modes in resonance Raman spectra and DFT calculations are discussed together with the prospects of observing such vibrational modes in metal-hydride-containing proteins. Such a

The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production.

PubMed

McIntosh, Chelsea L; Germer, Frauke; Schulz, Rüdiger; Appel, Jens; Jones, Anne K

2011-07-27

Protein film electrochemistry (PFE) was utilized to characterize the catalytic activity and oxidative inactivation of a bidirectional [NiFe]-hydrogenase (HoxEFUYH) from the cyanobacterium Synechocystis sp. PCC 6803. PFE provides precise control of the redox potential of the adsorbed enzyme so that its activity can be monitored under changing experimental conditions as current. The properties of HoxEFUYH are different from those of both the standard uptake and the "oxygen-tolerant" [NiFe]-hydrogenases. First, HoxEFUYH is biased toward proton reduction as opposed to hydrogen oxidation. Second, despite being expressed under aerobic conditions in vivo, HoxEFUYH is clearly not oxygen-tolerant. Aerobic inactivation of catalytic hydrogen oxidation by HoxEFUYH is total and nearly instantaneous, producing two inactive states. However, unlike the Ni-A and Ni-B inactive states of standard [NiFe]-hydrogenases, both of these states are quickly (<90 s) reactivated by removal of oxygen and exposure to reducing conditions. Third, proton reduction continues at 25-50% of the maximal rate in the presence of 1% oxygen. Whereas most previously characterized [NiFe]-hydrogenases seem to be preferential hydrogen oxidizing catalysts, the cyanobacterial enzyme works effectively in both directions. This unusual catalytic bias as well as the ability to be quickly reactivated may be essential to fulfilling the physiological role in cyanobacteria, organisms expected to experience swings in cellular reduction potential as they switch between aerobic conditions in the light and dark anaerobic conditions. Our results suggest that the uptake [NiFe]-hydrogenases alone are not representative of the catalytic diversity of [NiFe]-hydrogenases, and the bidirectional heteromultimeric enzymes may serve as valuable models to understand the diverse mechanisms of tuning the reactivity of the hydrogen activating site.

SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells.

PubMed

Pinske, Constanze; Sargent, Frank; Sawers, R Gary

2015-04-01

Fermentatively growing Escherichia coli cells have three active [NiFe]-hydrogenases (Hyd), two of which, Hyd-1 and Hyd-2, contribute to H2 oxidation while Hyd-3 couples formate oxidation to H2 evolution. Biosynthesis of all Hyd involves the insertion of a Fe(CN)2CO group and a subsequent insertion of nickel ions through the HypA/HybF, HypB and SlyD proteins. With high nickel concentrations the presence of none of these proteins is required, but under normal growth conditions and during late stationary growth SlyD is important for hydrogenase activities. The slyD mutation reduced H2 production during exponential phase growth by about 50%. Assaying stationary phase grown cells for the coupling of Hyd activity to the respiratory chain or formate-dependent H2 evolution showed that SlyD is essential for both H2 evolution and H2 oxidation. Although introduction of plasmid-coded slyD resulted in an overall decrease of Hyd-2 polypeptides in slyD and hypA slyD mutants, processing and dye-reducing activity of the Hyd-2 enzyme was nevertheless restored. Similarly, introduction of the slyD plasmid restored only some H2 evolution in the slyD mutant while Hyd-3 polypeptides and dye-reducing activity were fully restored. Taken together, these results indicate an essential role for SlyD in the generation of the fully cofactor-equipped hydrogenase large subunits in the stationary phase where the level of each Hyd enzyme is finely tuned by SlyD for optimal enzyme activity.

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases

PubMed Central

Wulff, Philip; Day, Christopher C.; Sargent, Frank; Armstrong, Fraser A.

2014-01-01

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H2 + O2 = 2 H2O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H2O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H2 and 10% O2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H2 oxidation. The oxidase activity is too slow to be useful in removing O2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation. PMID:24715724

Mechanism of O2 diffusion and reduction in FeFe hydrogenases

NASA Astrophysics Data System (ADS)

Kubas, Adam; Orain, Christophe; de Sancho, David; Saujet, Laure; Sensi, Matteo; Gauquelin, Charles; Meynial-Salles, Isabelle; Soucaille, Philippe; Bottin, Hervé; Baffert, Carole; Fourmond, Vincent; Best, Robert B.; Blumberger, Jochen; Léger, Christophe

2017-01-01

FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre.

PubMed

Fritsch, Johannes; Scheerer, Patrick; Frielingsdorf, Stefan; Kroschinsky, Sebastian; Friedrich, Bärbel; Lenz, Oliver; Spahn, Christian M T

2011-10-16

Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(2)-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H(2) oxidation and as an electron-delivering device upon O(2) attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H(2)-converting catalysts that are capable of cycling H(2) in air.

Molecular Dynamics Studies of Proton Transport in Hydrogenase and Hydrogenase Mimics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ginovska-Pangovska, Bojana; Raugei, Simone; Shaw, Wendy J.

2016-08-02

Protons are used throughout the biological world for a number of functions, from charge balance to energy carriers. Metalloenzymes use protons as energy carriers and control proton movement both temporally and spatially. Despite the interest and need for controlled proton movement in other systems, the scientific community has not been able to develop extensive general rules for developing synthetic proton pathways. In part this is due to the challenging nature of studying these large and complex molecules experimentally, although experiments have gleaned extensive critical insight. While computational methods are also challenging because of the large size of the molecules, theymore » have been critical in advancing our knowledge of proton movement through pathways, but even further, they have advanced our knowledge in how protonation and proton movement is correlated with large and small scale molecular motions and electron movement. These studies often complement experimental studies but provide insight and depth simply not possible in many cases in the absence of theory. In this chapter, we will discuss advances and methods used in understanding proton movement in hydrogenases.« less

Structure of a [NiFe] hydrogenase maturation protease HycI provides insights into its substrate selectivity.

PubMed

Kwon, Sunghark; Nishitani, Yuichi; Hirao, Yoshinori; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

2018-04-15

The immature large subunit of [NiFe] hydrogenases undergoes C-terminal cleavage by a specific protease in the final step of the post-translational process before assembly with other subunits. It has been reported that the [NiFe] hydrogenase maturation protease HycI from Thermococcus kodakarensis (TkHycI) has the catalytic ability to target the membrane-bound hydrogenase large subunit MbhL from T. kodakarensis. However, the detailed mechanism of its substrate recognition remains elusive. We determined the crystal structure of TkHycI at 1.59 Å resolution to clarify how TkHycI recognizes its own substrate MbhL. Although the overall structure of TkHycI is similar to that of its homologous protease TkHybD, TkHycI adopts a larger loop than TkHybD, thereby creating a broad and deep cleft. We analyzed the structural properties of the TkHycI cleft probably involved in its substrate recognition. Our findings provide novel and profound insights into the substrate selectivity of TkHycI. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus.

PubMed

Pandelia, Maria-Eirini; Nitschke, Wolfgang; Infossi, Pascale; Giudici-Orticoni, Marie-Thérèse; Bill, Eckhard; Lubitz, Wolfgang

2011-04-12

Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygen-tolerant Hydrogenase I from Aquifex aeolicus such electron "wires" form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O(2) to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a [3Fe4S] and two [4Fe4S], but also a unique high-potential (HP) state was found. On the basis of (57)Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the [4Fe4S](3+) state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3+, 2+, and 1+ oxidation states within a very small potential range. We suggest that the (1 + /2+) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress.

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii.

PubMed

Godaux, Damien; Bailleul, Benjamin; Berne, Nicolas; Cardol, Pierre

2015-06-01

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments. © 2015 American Society of Plant Biologists. All Rights Reserved.

Nickel containing CO dehydrogenases and hydrogenases.

PubMed

Ragsdale, S W

2000-01-01

The two redox catalysts described here can generate very low potential electrons in one direction and perform chemically difficult reductions in the other. The chemical transformations occur at unusual metal clusters. Spectroscopic, crystallographic, and kinetic analyses are converging on answers to how the metals in these clusters are arranged and how they are involved in the chemical and redox steps. The first structure of CO dehydrogenase, which will appear in the next year, will help define a firm chemical basis for future mechanistic studies. In the immediate future, we hope to learn whether the hydride intermediate in hydrogenase or the carbonyl intermediate in CO dehydrogenase bind to the Ni or Fe subsites in these heterometallic clusters. Or perhaps could they be bridged to two metals? Inter- and intramolecular wires have been proposed that connect the catalytic redox machine to proximal redox centers leading eventually to the ultimate redox partners. Elucidating the pathways of electron flow is a priority for the future. There is evidence for molecular channels delivering substrates to the active sites of these enzymes. In the next few years, these channels will be better defined. The products of CO2 and proton reduction are passed to the active sites of other enzymes and, in the case of H2, even passed from one organism to another. In the future, the mechanism of gas transfer will be uncovered. General principles of how these redox reactions are catalyzed are becoming lucid as the reactions are modeled theoretically and experimentally. Proton and CO2 reduction and the generation of C-C bonds from simple precursors are important reactions in industry. H2 could be the clean fuel of the future. Hopefully, the knowledge gained from studies of hydrogenase, CO dehydrogenase, and acetyl-CoA synthase can be used to improve life on earth.

The Physiological Functions and Structural Determinants of Catalytic Bias in the [FeFe]-Hydrogenases CpI and CpII of Clostridium pasteurianum Strain W5.

PubMed

Therien, Jesse B; Artz, Jacob H; Poudel, Saroj; Hamilton, Trinity L; Liu, Zhenfeng; Noone, Seth M; Adams, Michael W W; King, Paul W; Bryant, Donald A; Boyd, Eric S; Peters, John W

2017-01-01

The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro , with CpII capturing

Potentials of using mixed culture bacteria incorporated with sodium bicarbonate for hydrogen production from water hyacinth.

PubMed

Wazeri, Alaa; Elsamadony, Mohamed; Roux, Sophie Le; Peu, Pascal; Tawfik, Ahmed

2018-05-08

The aim of this study is to assess the potentials of using mixed culture bacteria incorporated with different concentrations of NaHCO 3 for hydrogen production from water hyacinth (WH). The lowest hydrogen yield (HY) of 30.4 ± 1.9 mL/g TVS , H 2 content (HC) of 19.5 ± 1.5% and hydrogenase enzyme (HE) activity of 0.06 ± 0.01 mgM.B reduced /min were registered for the cultures without supplementation of NaHCO 3 . The HY, HC, and HE activity were maximized at levels of 69.2 ± 4.3 mL/g TVS, 58.4 ± 3.6% and 0.18 ± 0.01 mgM.B reduced /min. respectively for the anaerobes supplied with 3.0 g NaHCO 3 /L. Furthermore, cellulose, hemicellulose, and lignin destruction efficiencies were 37.2 ± 2.3, 30.0 ± 1.9 and 20.9 ± 1.3% respectively due to the increase of cellulase and xylanase activities up to 2.73 ± 0.17 and 1.87 ± 0.12 U/mL, respectively. Moreover, the abundance of Firmicutes was substantially increased and accounted for 71% of the total OTU's. Microbes belonging to the order Clostridiales and OPB54 were particularly enriched in the medium supplemented with NaHCO 3 . Copyright © 2018 Elsevier Ltd. All rights reserved.

Isolation, purification and characterization of the hydrogen evolution promoting factor of hydrogenase of Spirulina platensis

NASA Astrophysics Data System (ADS)

Gu, Tian-Qing; Zhang, Hui-Miao; Sun, Shi-Hua

1996-03-01

A component (s-factor) with obvious promoting effect on hydrogen evolution of hydrogenase has been isolated and extracted from a cell-free preparation of Spirulina platensis. The effect of the s-factor in the reaction system is similar to that of Na2S2O4, but is coupled with light. The s-factor has the maximum absorption peak at 620 nm in the oxidized state, at 590 nm in the reduced state. The partially purified s-factor showed two bands by SDS-PAGE and is distinctly different from phycocyanin, which has no change of oxidized state and reduced state absorption spectra, and also has no promoting effect on hydrogenase of Spirulina platensis under the light.

Enzyme electrokinetics: hydrogen evolution and oxidation by Allochromatium vinosum [NiFe]-hydrogenase.

PubMed

Léger, Christophe; Jones, Anne K; Roseboom, Winfried; Albracht, Simon P J; Armstrong, Fraser A

2002-12-31

The mechanism of catalytic hydrogen evolution and oxidation by Allochromatium vinosum [NiFe]-hydrogenase has been studied by protein film voltammetry (PFV) with the enzyme adsorbed at a pyrolytic graphite edge electrode. By analyzing the entire shapes of catalytic voltammograms, the energetics of the catalytic cycles (reduction potentials and acidity constants of the active states), including the detailed profiles of activity against pH and the sequences of proton and electron transfers, have been determined, and these are discussed with respect to the mechanism. PFV, which probes rates as a continuous function of the electrochemical potential (i.e., in the "potential domain"), is proven to be an invaluable tool for determining the redox properties of an active site in the presence of its substrate, at room temperature, and during turnover. This is especially relevant in the case of the active states of hydrogenase, since one of its substrates (the proton) is always present at significant levels in the titration medium at physiological pH values.

Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states.

PubMed

Volbeda, Anne; Martin, Lydie; Barbier, Elodie; Gutiérrez-Sanz, Oscar; De Lacey, Antonio L; Liebgott, Pierre-Pol; Dementin, Sébastien; Rousset, Marc; Fontecilla-Camps, Juan C

2015-01-01

Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.

Hydrogenase activity of mineral-associated and suspended populations of Desulfovibrio Desulfuricans Essex 6

USDA-ARS?s Scientific Manuscript database

The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate...

Structure prediction and molecular simulation of gases diffusion pathways in hydrogenase.

PubMed

Sundaram, Shanthy; Tripathi, Ashutosh; Gupta, Vipul

2010-10-06

Although hydrogen is considered to be one of the most promising future energy sources and the technical aspects involved in using it have advanced considerably, the future supply of hydrogen from renewable sources is still unsolved. The [Fe]- hydrogenase enzymes are highly efficient H(2) catalysts found in ecologically and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. While these enzymes can occur in several forms, H(2) catalysis takes place at a unique [FeS] prosthetic group or H-cluster, located at the active site. 3D structure of the protein hydA1 hydrogenase from Chlamydomonas reinhardtti was predicted using the MODELER 8v2 software. Conserved region was depicted from the NCBI CDD Search. Template selection was done on the basis NCBI BLAST results. For single template 1FEH was used and for multiple templates 1FEH and 1HFE were used. The result of the Homology modeling was verified by uploading the file to SAVS server. On the basis of the SAVS result 3D structure predicted using single template was chosen for performing molecular simulation. For performing molecular simulation three strategies were used. First the molecular simulation of the protein was performed in solvated box containing bulk water. Then 100 H(2) molecules were randomly inserted in the solvated box and two simulations of 50 and 100 ps were performed. Similarly 100 O(2) molecules were randomly placed in the solvated box and again 50 and 100 ps simulation were performed. Energy minimization was performed before each simulation was performed. Conformations were saved after each simulation. Analysis of the gas diffusion was done on the basis of RMSD, Radius of Gyration and no. of gas molecule/ps plot.

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

PubMed Central

Pandelia, Maria-Eirini; Nitschke, Wolfgang; Infossi, Pascale; Giudici-Orticoni, Marie-Thérèse; Bill, Eckhard; Lubitz, Wolfgang

2011-01-01

Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygen-tolerant Hydrogenase I from Aquifex aeolicus such electron “wires” form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O2 to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a [3Fe4S] and two [4Fe4S], but also a unique high-potential (HP) state was found. On the basis of 57Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the [4Fe4S]3+ state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3+, 2+, and 1+ oxidation states within a very small potential range. We suggest that the (1 + /2+) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress. PMID:21444783

The Physiological Functions and Structural Determinants of Catalytic Bias in the [FeFe]-Hydrogenases CpI and CpII of Clostridium pasteurianum Strain W5

DOE PAGES

Therien, Jesse B.; Artz, Jacob H.; Poudel, Saroj; ...

2017-07-12

Here, the first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogenmore » production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with Cp

The Physiological Functions and Structural Determinants of Catalytic Bias in the [FeFe]-Hydrogenases CpI and CpII of Clostridium pasteurianum Strain W5

PubMed Central

Therien, Jesse B.; Artz, Jacob H.; Poudel, Saroj; Hamilton, Trinity L.; Liu, Zhenfeng; Noone, Seth M.; Adams, Michael W. W.; King, Paul W.; Bryant, Donald A.; Boyd, Eric S.; Peters, John W.

2017-01-01

The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess

Flexible plastic bioreactors for photobiological hydrogen production by hydrogenase-deficient cyanobacteria.

PubMed

Kitashima, Masaharu; Masukawa, Hajime; Sakurai, Hidehiro; Inoue, Kazuhito

2012-01-01

Uptake hydrogenase mutant cells of the cyanobacterium Nostoc sp. PCC 7422 photobiologically produced H(2) catalyzed by nitrogenase for several days in H(2)-barrier transparent plastic bags, and accumulated H(2) in the presence of O(2) evolved by photosynthesis. Their H(2) production activity was higher in the sealed flexible bags than in stoppered serum bottles of fixed gas volume.

The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron-sulphur clusters.

PubMed

Kurkin, Sergei; Meuer, Jörn; Koch, Jürgen; Hedderich, Reiner; Albracht, Simon P J

2002-12-01

The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.

Multiscale simulations give insight into the hydrogen in and out pathways of [NiFe]-hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans.

PubMed

Oteri, Francesco; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

2014-12-04

[NiFe]-hydrogenases catalyze the cleavage of molecular hydrogen into protons and electrons and represent promising tools for H2-based technologies such as biofuel cells. However, many aspects of these enzymes remain to be understood, in particular how the catalytic center can be protected from irreversible inactivation by O2. In this work, we combined homology modeling, all-atom molecular dynamics, and coarse-grain Brownian dynamics simulations to investigate and compare the dynamic and mechanical properties of two [NiFe]-hydrogenases: the soluble O2-sensitive enzyme from Desulfovibrio fructosovorans, and the O2-tolerant membrane-bound hydrogenase from Aquifex aeolicus. We investigated the diffusion pathways of H2 from the enzyme surface to the central [NiFe] active site, and the possible proton pathways that are used to evacuate hydrogen after the oxidation reaction. Our results highlight common features of the two enzymes, such as a Val/Leu/Arg triad of key residues that controls ligand migration and substrate access in the vicinity of the active site, or the key role played by a Glu residue for proton transfer after hydrogen oxidation. We show specificities of each hydrogenase regarding the enzymes internal tunnel network or the proton transport pathways.

Nickel-centred proton reduction catalysis in a model of [NiFe] hydrogenase

NASA Astrophysics Data System (ADS)

Brazzolotto, Deborah; Gennari, Marcello; Queyriaux, Nicolas; Simmons, Trevor R.; Pécaut, Jacques; Demeshko, Serhiy; Meyer, Franc; Orio, Maylis; Artero, Vincent; Duboc, Carole

2016-11-01

Hydrogen production through water splitting is one of the most promising solutions for the storage of renewable energy. [NiFe] hydrogenases are organometallic enzymes containing nickel and iron centres that catalyse hydrogen evolution with performances that rival those of platinum. These enzymes provide inspiration for the design of new molecular catalysts that do not require precious metals. However, all heterodinuclear NiFe models reported so far do not reproduce the Ni-centred reactivity found at the active site of [NiFe] hydrogenases. Here, we report a structural and functional NiFe mimic that displays reactivity at the Ni site. This is shown by the detection of two catalytic intermediates that reproduce structural and electronic features of the Ni-L and Ni-R states of the enzyme during catalytic turnover. Under electrocatalytic conditions, this mimic displays high rates for H2 evolution (second-order rate constant of 2.5 × 104 M-1 s-1 turnover frequency of 250 s-1 at 10 mM H+ concentration) from mildly acidic solutions.

Nickel centred H+ reduction catalysis in a model of [NiFe] Hydrogenase

PubMed Central

Brazzolotto, Deborah; Gennari, Marcello; Queyriaux, Nicolas; Simmons, Trevor R.; Pécaut, Jacques; Demeshko, Serhiy; Meyer, Franc; Orio, Maylis; Artero, Vincent; Duboc, Carole

2017-01-01

Hydrogen production through water splitting is one of the most promising solutions for the storage of renewable energy. [NiFe] hydrogenases are organometallic enzymes containing nickel and iron centers that catalyze hydrogen evolution with performances that rival those of platinum. These enzymes provide inspiration for the design of new molecular catalysts that do not require precious metals. However, all heterodinuclear NiFe models reported so far do not reproduce the Ni-centered reactivity found at the active site of [NiFe] hydrogenases. Here we report a structural and functional NiFe mimic that displays reactivity at the Ni site. This is shown by the detection of two catalytic intermediates that reproduce structural and electronic features of the Ni-L and Ni-R states of the enzyme during catalytic turnover. Under electrocatalytic conditions, this mimic displays high rates for H2 evolution (second order rate constant of 2.5 104 M-1s-1; turnover frequency of 225 s-1 at 10 mM H+ concentration) from mildly acidic solutions. PMID:27768098

The chronic toxicity of sodium bicarbonate, a major component of coal bed natural gas produced waters

USGS Publications Warehouse

Farag, Aïda M.; Harper, David D.

2014-01-01

Sodium bicarbonate (NaHCO3) is the principal salt in coal bed natural gas produced water from the Powder River Structural Basin, Wyoming, USA, and concentrations of up to 3000 mg NaHCO3/L have been documented at some locations. No adequate studies have been performed to assess the chronic effects of NaHCO3 exposure. The present study was initiated to investigate the chronic toxicity and define sublethal effects at the individual organism level to explain the mechanisms of NaHCO3 toxicity. Three chronic experiments were completed with fathead minnows (Pimephales promelas), 1 with white suckers (Catostomus commersoni), 1 with Ceriodaphnia dubia, and 1 with a freshwater mussel, (Lampsilis siliquoidea). The data demonstrated that approximately 500 mg NaHCO3/L to 1000 mg NaHCO3/L affected all species of experimental aquatic animals in chronic exposure conditions. Freshwater mussels were the least sensitive to NaHCO3 exposure, with a 10-d inhibition concentration that affects 20% of the sample population (IC20) of 952 mg NaHCO3/L. The IC20 for C. dubia was the smallest, at 359 mg NaHCO3/L. A significant decrease in sodium–potassium adenosine triphosphatase (Na+/K+ ATPase) together with the lack of growth effects suggests that Na+/K+ ATPase activity was shut down before the onset of death. Several histological anomalies, including increased incidence of necrotic cells, suggested that fish were adversely affected as a result of exposure to >450 mg NaHCO3/L.

Low concentration of sodium bicarbonate improves the bioactive compound levels and antioxidant and α-glucosidase inhibitory activities of tartary buckwheat sprouts.

PubMed

Qin, Peiyou; Wei, Aichun; Zhao, Degang; Yao, Yang; Yang, Xiushi; Dun, Baoqing; Ren, Guixing

2017-06-01

This study aimed to investigate the effects of different concentrations of sodium bicarbonate (NaHCO 3 ) on the accumulation of flavonoids, total phenolics and d-chiro-inositol (DCI), as well as the antioxidant and α-glucosidase inhibitory activities, in tartary buckwheat sprouts. Treatment with low concentrations of NaHCO 3 (0.05, 0.1, and 0.2%) resulted in an increase in flavonoids, total phenolic compounds and DCI concentrations, and improved DPPH radical-scavenging and α-glucosidase inhibition activities compared with the control (0%). The highest levels of total flavonoids (26.69mg/g DW), individual flavonoids (rutin, isoquercitrin, quercetin, and kaempferol), total phenolic compounds (29.31mg/g DW), DCI (12.56mg/g DW), as well as antioxidant and α-glucosidase inhibition activities, were observed in tartary buckwheat sprouts treated with 0.05% NaHCO 3 for 96h. These results indicated that appropriate treatment with NaHCO 3 could improve the healthy benefits of tartary buckwheat sprouts. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of bicarbonate on menadione-induced redox cycling and cytotoxicity: potential involvement of the carbonate radical.

PubMed

Aljuhani, Naif; Michail, Karim; Karapetyan, Zubeida; Siraki, Arno G

2013-10-01

We have investigated the effect of NaHCO3 on menadione redox cycling and cytotoxicity. A cell-free system utilized menadione and ascorbic acid to catalyze a redox cycle, and we utilized murine hepatoma (Hepa 1c1c7) cells for in vitro experiments. Experiments were performed using low (2 mmol/L) and physiological (25 mmol/L) levels of NaHCO3 in buffer equilibrated to physiological pH. Using oximetry, ascorbic acid oxidation, and ascorbyl radical detection, we found that menadione redox cycling was enhanced by NaHCO3. Furthermore, Hepa 1c1c7 cells treated with menadione demonstrated cytotoxicity that was significantly increased with physiological concentrations of NaHCO3 in the media, compared with low levels of NaHCO3. Interestingly, the inhibition of superoxide dismutase (SOD) with 2 different metal chelators was associated with a protective effect against menadione cytotoxicity. Using isolated protein, we found a significant increase in protein carbonyls with menadione-ascorbate-SOD with physiological NaHCO3 levels; low NaHCO3 or SOD-free reactions produced lower levels of protein carbonyls. In conclusion, these findings suggest that the hydrogen peroxide generated by menadione redox cycling together with NaHCO3-CO2 are potential substrates for SOD peroxidase activity that can lead to carbonate-radical-enhanced cytotoxicity. These findings demonstrate the importance of NaHCO3 in menadione redox cycling and cytotoxicity.

Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment1[OPEN

PubMed Central

Liran, Oded; Milrad, Yuval; Eilenberg, Haviva; Weiner, Iddo

2016-01-01

Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, 18O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells. PMID:27443604

Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

PubMed

Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia R; Balbach, Jochen; Zamble, Deborah B; Haupt, Caroline

2012-03-16

SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

PubMed

Oughli, Alaa A; Vélez, Marisela; Birrell, James A; Schuhmann, Wolfgang; Lubitz, Wolfgang; Plumeré, Nicolas; Rüdiger, Olaf

2018-06-08

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

Oxygen-tolerant [NiFe]-hydrogenases: the individual and collective importance of supernumerary cysteines at the proximal Fe-S cluster.

PubMed

Lukey, Michael J; Roessler, Maxie M; Parkin, Alison; Evans, Rhiannon M; Davies, Rosalind A; Lenz, Oliver; Friedrich, Baerbel; Sargent, Frank; Armstrong, Fraser A

2011-10-26

An important clue to the mechanism for O(2) tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron-sulfur cluster that is proximal to the active site. The O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron-sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O(2).

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2.

PubMed

Preissler, Janina; Wahlefeld, Stefan; Lorent, Christian; Teutloff, Christian; Horch, Marius; Lauterbach, Lars; Cramer, Stephen P; Zebger, Ingo; Lenz, Oliver

2018-01-01

Biocatalysts that mediate the H 2 -dependent reduction of NAD + to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD + -reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O 2 , which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD + -reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1 T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H 2 -oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H 2 -mediated NAD + reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O 2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD + -reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H 2 -driven cofactor recycling under oxic conditions at elevated temperatures. Copyright © 2017 Elsevier B.V. All rights reserved.

Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*

PubMed Central

Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

2014-01-01

The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

Structure and function of photosystem I–[FeFe] hydrogenase protein fusions: An all-atom molecular dynamics study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harris, Bradley J.; Cheng, Xiaolin; Frymier, Paul

2015-12-15

All-atom molecular dynamics (MD) simulation was used to study the solution dynamics and protein protein interactions of protein fusions of photosystem I (PSI) from Thermosynechococcus elongatus and an [FeFe]-hydrogenase (FeFe H 2ase) from Clostridium pasteurianum, a unique complex capable of photocatalytic hydrogen production. This study involved fusions of these two proteins via dithiol linkers of different length including decanedithiol, octanedithiol, and hexanedithiol, for which experimental data had previously been obtained. Evaluation of root-mean-squared deviations (RMSDs) relative to the respective crystal structures of PSI and the FeFe H 2ase shows that these fusion complexes approach stable equilibrium conformations during the MDmore » simulations. Investigating protein mobility via root-mean-squared fluctuations (RMSFs) reveals that tethering via the shortest hexanedithiol linker results in increased atomic fluctuations of both PSI and the hydrogenase in these fusion complexes. Furthermore, evaluation of the inter- and intraprotein electron transfer distances in these fusion complexes indicates that the structural changes in the FeFe H 2ase arising from ligation to PSI via the shortest hexanedithiol linker may hinder electron transport in the hydrogenase, thus providing a molecular level explanation for the observation that the medium-length octanedithiol linker gives the highest hydrogen production rate.« less

International Hydrogenase Conference (7th) Held at the University of Reading on August 24th to 29th 2004.

DTIC Science & Technology

2004-08-19

Johannes Hackstein [ PB GIO rNovel Fe-hydrogenases from the rumen ciliate metagenome . :12.50 :114.00 -1 Lunch [ 114.00 1 7.00 1 Poster Session 2...d.r.o’ g’.e n-.a-.s.e..s from the rumnen ciliate metagenome . p36 Severing, E., Boxma, B., van Alen, T.A., Ricard, G., van Hoek, A.H.A.M., Moon-van...hydrogenases from the rumen ciliate metagenome . Severing, E.’, Boxma, B.1, van Alen, T.A.’, Ricard, G.z, van Hoek, A.H.A.M.’, Moon-van der Staay, S.Y

Electrochemistry of Simple Organometallic Models of Iron-Iron Hydrogenases in Organic Solvent and Water.

PubMed

Gloaguen, Frederic

2016-01-19

Synthetic models of the active site of iron-iron hydrogenases are currently the subjects of numerous studies aimed at developing H2-production catalysts based on cheap and abundant materials. In this context, the present report offers an electrochemist's view of the catalysis of proton reduction by simple binuclear iron(I) thiolate complexes. Although these complexes probably do not follow a biocatalytic pathway, we analyze and discuss the interplay between the reduction potential and basicity and how these antagonist properties impact the mechanisms of proton-coupled electron transfer to the metal centers. This question is central to any consideration of the activity at the molecular level of hydrogenases and related enzymes. In a second part, special attention is paid to iron thiolate complexes holding rigid and unsaturated bridging ligands. The complexes that enjoy mild reduction potentials and stabilized reduced forms are promising iron-based catalysts for the photodriven evolution of H2 in organic solvents and, more importantly, in water.

Occurrence of H2-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus1

PubMed Central

Murillo, Jesús; Villa, Ana; Chamber, Manuel; Ruiz-Argüeso, Tomás

1989-01-01

Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H2 uptake by bacteroids and the relative efficiency of N2 fixation (RE = 1 - [H2 evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H2-uptake activity in symbiosis with O. compressus. The inability of these putative Hup+ strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization

Roles of H2 uptake hydrogenases in Shigella flexneri acid tolerance

PubMed Central

McNorton, Mykeshia M.

2012-01-01

Hydrogenases play many roles in bacterial physiology, and use of H2 by the uptake-type enzymes of animal pathogens is of particular interest. Hydrogenases have never been studied in the pathogen Shigella, so targeted mutant strains were individually generated in the two Shigella flexneri H2-uptake enzymes (Hya and Hyb) and in the H2-evolving enzyme (Hyc) to address their roles. Under anaerobic fermentative conditions, a Hya mutant strain (hya) was unable to oxidize H2, while a Hyb mutant strain oxidized H2 like the wild-type. A hyc strain oxidized more exogenously added hydrogen than the parent. Fluorescence ratio imaging with dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) showed that the parent strain generated a membrane potential 15 times greater than hya. The hya mutant was also by far the most acid-sensitive strain, being even more acid-sensitive than a mutant strain in the known acid-combating glutamate-dependent acid-resistance pathway (GDAR pathway). In severe acid-challenge experiments, the addition of glutamate to hya restored survivability, and this ability was attributed in part to the GDAR system (removes intracellular protons) by mutant strain (e.g. hya/gadBC double mutant) analyses. However, mutant strain phenotypes indicated that a larger portion of the glutamate-rescued acid tolerance was independent of GadBC. The acid tolerance of the hya strains was aided by adding chloride ions to the growth medium. The whole-cell Hya enzyme became more active upon acid exposure (20 min), based on assays of hyc. Indeed, the very high rates of Shigella H2 oxidation by Hya in acid can supply each cell with 2.4×108 protons min−1. Electrons generated from Hya-mediated H2 oxidation at the inner membrane likely counteract cytoplasmic positive charge stress, while abundant proton pools deposited periplasmically likely repel proton influx during severe acid stress. PMID:22628482

Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases.

PubMed

Volbeda, Anne; Martin, Lydie; Cavazza, Christine; Matho, Michaël; Faber, Bart W; Roseboom, Winfried; Albracht, Simon P J; Garcin, Elsa; Rousset, Marc; Fontecilla-Camps, Juan C

2005-05-01

[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we present the crystal structure of the ready Ni-B state of Desulfovibrio fructosovorans [NiFe] hydrogenase and show it to have a putative mu-hydroxo Ni-Fe bridging ligand at the active site. On the other hand, a new, improved refinement procedure of the X-ray diffraction data obtained for putative unready Ni-A/Ni-SU states resulted in a more elongated electron density for the bridging ligand, suggesting that it is a diatomic species. The slow activation of the Ni-A state, compared with the rapid activation of the Ni-B state, is therefore proposed to result from the different chemical nature of the ligands in the two oxidized species. Our results along with very recent electrochemical studies suggest that the diatomic ligand could be hydro-peroxide.

Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis ▿ †

PubMed Central

Kanai, Tamotsu; Matsuoka, Ryoji; Beppu, Haruki; Nakajima, Akihito; Okada, Yoshihiro; Atomi, Haruyuki; Imanaka, Tadayuki

2011-01-01

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S0) or H2 generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H2S- and H2-evolving conditions. H2S generation in PHY1 was equivalent to that of the host strain, and H2 generation was higher in PHY1, suggesting that Hyh functions in the direction of H2 uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H2-evolving conditions. This fact, as well as the impaired H2 generation activity in MHD1, indicated that Mbh is mainly responsible for H2 evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H2 transfer (i.e., H2 evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H2S-evolving conditions and exhibited a lower H2S generation activity, indicating the involvement of Mbx in the S0 reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales. PMID:21515783

Requirements for functional models of the iron hydrogenase active site: D2/H2O exchange activity in ((mu-SMe)(mu-pdt)[Fe(CO)2(PMe3)]2+)[BF4-].

PubMed

Georgakaki, Irene P; Miller, Matthew L; Darensbourg, Marcetta Y

2003-04-21

Hydrogen uptake in hydrogenase enzymes can be assayed by H/D exchange reactivity in H(2)/D(2)O or H(2)/D(2)/H(2)O mixtures. Diiron(I) complexes that serve as structural models for the active site of iron hydrogenase are not active in such isotope scrambling but serve as precursors to Fe(II)Fe(II) complexes that are functional models of [Fe]H(2)ase. Using the same experimental protocol as used previously for ((mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-H(+) (Zhao et al. J. Am. Chem. Soc. 2001, 123, 9710), we now report the results of studies of ((mu-SMe)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-SMe(+), toward H/D exchange. The 1-SMe(+) complex can take up H(2) and catalyze the H/D exchange reaction in D(2)/H(2)O mixtures under photolytic, CO-loss conditions. Unlike 1-H(+), it does not catalyze H(2)/D(2) scrambling under anhydrous conditions. The molecular structure of 1-SMe(+) involves an elongated Fe.Fe separation, 3.11 A, relative to 2.58 A in 1-H(+). It is proposed that the strong SMe(-) bridging ligand results in catalytic activity localized on a single Fe(II) center, a scenario that is also a prominent possibility for the enzyme active site. The single requirement is an open site on Fe(II) available for binding of D(2) (or H(2)), followed by deprotonation by the external base H(2)O (or D(2)O).

Protonation/reduction dynamics at the [4Fe-4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases.

PubMed

Senger, Moritz; Mebs, Stefan; Duan, Jifu; Shulenina, Olga; Laun, Konstantin; Kertess, Leonie; Wittkamp, Florian; Apfel, Ulf-Peter; Happe, Thomas; Winkler, Martin; Haumann, Michael; Stripp, Sven T

2018-01-31

The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN - ) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN - vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN - infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

Metabolic pathways for photobiological hydrogen production by nitrogenase- and hydrogenase-containing unicellular cyanobacteria Cyanothece.

PubMed

Skizim, Nicholas J; Ananyev, Gennady M; Krishnan, Anagha; Dismukes, G Charles

2012-01-20

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.

Heterobimetallic [NiFe] Complexes Containing Mixed CO/CN- Ligands: Analogs of the Active Site of the [NiFe] Hydrogenases.

PubMed

Perotto, Carlo U; Sodipo, Charlene L; Jones, Graham J; Tidey, Jeremiah P; Blake, Alexander J; Lewis, William; Davies, E Stephen; McMaster, Jonathan; Schröder, Martin

2018-03-05

The development of synthetic analogs of the active sites of [NiFe] hydrogenases remains challenging, and, in spite of the number of complexes featuring a [NiFe] center, those featuring CO and CN - ligands at the Fe center are under-represented. We report herein the synthesis of three bimetallic [NiFe] complexes [Ni( N 2 S 2 )Fe(CO) 2 (CN) 2 ], [Ni( S 4 )Fe(CO) 2 (CN) 2 ], and [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ] that each contain a Ni center that bridges through two thiolato S donors to a {Fe(CO) 2 (CN) 2 } unit. X-ray crystallographic studies on [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ], supported by DFT calculations, are consistent with a solid-state structure containing distinct molecules in the singlet ( S = 0) and triplet ( S = 1) states. Each cluster exhibits irreversible reduction processes between -1.45 and -1.67 V vs Fc + /Fc and [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ] possesses a reversible oxidation process at 0.17 V vs Fc + /Fc. Spectroelectrochemical infrared (IR) and electron paramagnetic resonance (EPR) studies, supported by density functional theory (DFT) calculations, are consistent with a Ni III Fe II formulation for [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ] + . The singly occupied molecular orbital (SOMO) in [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ] + is based on Ni 3d z 2 and 3p S with the S contributions deriving principally from the apical S-donor. The nature of the SOMO corresponds to that proposed for the Ni-C state of the [NiFe] hydrogenases for which a Ni III Fe II formulation has also been proposed. A comparison of the experimental structures, and the electrochemical and spectroscopic properties of [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ] and its [Ni( N 2 S 3 )] precursor, together with calculations on the oxidized [Ni( N 2 S 3 )Fe(CO) 2 (CN) 2 ] + and [Ni( N 2 S 3 )] + forms suggests that the binding of the {Fe(CO)(CN) 2 } unit to the {Ni(CysS) 4 } center at the active site of the [NiFe] hydrogenases suppresses thiolate-based oxidative chemistry involving the bridging thiolate S donors

Transcription and Regulation of the Bidirectional Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120▿

PubMed Central

Sjöholm, Johannes; Oliveira, Paulo; Lindblad, Peter

2007-01-01

The filamentous, heterocystous cyanobacterium Nostoc sp. strain PCC 7120 (Anabaena sp. strain PCC 7120) possesses an uptake hydrogenase and a bidirectional enzyme, the latter being capable of catalyzing both H2 production and evolution. The completely sequenced genome of Nostoc sp. strain PCC 7120 reveals that the five structural genes encoding the bidirectional hydrogenase (hoxEFUYH) are separated in two clusters at a distance of approximately 8.8 kb. The transcription of the hox genes was examined under nitrogen-fixing conditions, and the results demonstrate that the cluster containing hoxE and hoxF can be transcribed as one polycistronic unit together with the open reading frame alr0750. The second cluster, containing hoxU, hoxY, and hoxH, is transcribed together with alr0763 and alr0765, located between the hox genes. Moreover, alr0760 and alr0761 form an additional larger operon. Nevertheless, Northern blot hybridizations revealed a rather complex transcription pattern in which the different hox genes are expressed differently. Transcriptional start points (TSPs) were identified 66 and 57 bp upstream from the start codon of alr0750 and hoxU, respectively. The transcriptions of the two clusters containing the hox genes are both induced under anaerobic conditions concomitantly with the induction of a higher level of hydrogenase activity. An additional TSP, within the annotated alr0760, 244 bp downstream from the suggested translation start codon, was identified. Electrophoretic mobility shift assays with purified LexA from Nostoc sp. strain PCC 7120 demonstrated specific interactions between the transcriptional regulator and both hox promoter regions. However, when LexA from Synechocystis sp. strain PCC 6803 was used, the purified protein interacted only with the promoter region of the alr0750-hoxE-hoxF operon. A search of the whole Nostoc sp. strain PCC 7120 genome demonstrated the presence of 216 putative LexA binding sites in total, including recA and rec

Ligand versus metal protonation of an iron hydrogenase active site mimic.

PubMed

Eilers, Gerriet; Schwartz, Lennart; Stein, Matthias; Zampella, Giuseppe; de Gioia, Luca; Ott, Sascha; Lomoth, Reiner

2007-01-01

The protonation behavior of the iron hydrogenase active-site mimic [Fe2(mu-adt)(CO)4(PMe3)2] (1; adt=N-benzyl-azadithiolate) has been investigated by spectroscopic, electrochemical, and computational methods. The combination of an adt bridge and electron-donating phosphine ligands allows protonation of either the adt nitrogen to give [Fe2(mu-Hadt)(CO)4(PMe3)2]+ ([1 H]+), the Fe-Fe bond to give [Fe2(mu-adt)(mu-H)(CO)4(PMe3)2]+ ([1 Hy]+), or both sites simultaneously to give [Fe2(mu-Hadt)(mu-H)(CO)4(PMe3)2]2+ ([1 HHy]2 +). Complex 1 and its protonation products have been characterized in acetonitrile solution by IR, (1)H, and (31)P NMR spectroscopy. The solution structures of all protonation states feature a basal/basal orientation of the phosphine ligands, which contrasts with the basal/apical structure of 1 in the solid state. Density functional calculations have been performed on all protonation states and a comparison between calculated and experimental spectra confirms the structural assignments. The ligand protonated complex [1 H]+ (pKa=12) is the initial, metastable protonation product while the hydride [1 Hy]+ (pKa=15) is the thermodynamically stable singly protonated form. Tautomerization of cation [1 H]+ to [1 Hy]+ does not occur spontaneously. However, it can be catalyzed by HCl (k=2.2 m(-1) s(-1)), which results in the selective formation of cation [1 Hy]+. The protonations of the two basic sites have strong mutual effects on their basicities such that the hydride (pK(a)=8) and the ammonium proton (pK(a)=5) of the doubly protonated cationic complex [1 HHy]2+ are considerably more acidic than in the singly protonated analogues. The formation of dication [1 HHy]2+ from cation [1 H]+ is exceptionally slow with perchloric or trifluoromethanesulfonic acid (k=0.15 m(-1) s(-1)), while the dication is formed substantially faster (k>10(2) m(-1) s(-1)) with hydrobromic acid. Electrochemically, 1 undergoes irreversible reduction at -2.2 V versus ferrocene, and this

Control of the transition between Ni-C and Ni-SI(a) states by the redox state of the proximal Fe-S cluster in the catalytic cycle of [NiFe] hydrogenase.

PubMed

Tai, Hulin; Nishikawa, Koji; Suzuki, Masayuki; Higuchi, Yoshiki; Hirota, Shun

2014-12-08

[NiFe] hydrogenase catalyzes the reversible cleavage of H2. The electrons produced by the H2 cleavage pass through three Fe-S clusters in [NiFe] hydrogenase to its redox partner. It has been reported that the Ni-SI(a), Ni-C, and Ni-R states of [NiFe] hydrogenase are involved in the catalytic cycle, although the mechanism and regulation of the transition between the Ni-C and Ni-SI(a) states remain unrevealed. In this study, the FT-IR spectra under light irradiation at 138-198 K show that the Ni-L state of [NiFe] hydrogenase is an intermediate between the transition of the Ni-C and Ni-SI(a) states. The transition of the Ni-C state to the Ni-SI(a) state occurred when the proximal [Fe4S4]p(2+/+) cluster was oxidized, but not when it was reduced. These results show that the catalytic cycle of [NiFe] hydrogenase is controlled by the redox state of its [Fe4S4]p(2+/+) cluster, which may function as a gate for the electron flow from the NiFe active site to the redox partner. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction

NASA Astrophysics Data System (ADS)

Diakonova, A. N.; Khrushchev, S. S.; Kovalenko, I. B.; Riznichenko, G. Yu; Rubin, A. B.

2016-10-01

Ferredoxin (Fd) protein transfers electrons from photosystem I (PSI) to ferredoxin:NADP+-reductase (FNR) in the photosynthetic electron transport chain, as well as other metabolic pathways. In some photosynthetic organisms including cyanobacteria and green unicellular algae under anaerobic conditions Fd transfers electrons not only to FNR but also to hydrogenase—an enzyme which catalyzes reduction of atomic hydrogen to H2. One of the questions posed by this competitive relationship between proteins is which characteristics of thylakoid stroma media allow switching of the electron flow between the linear path PSI-Fd-FNR-NADP+ and the path PSI-Fd-hydrogenase-H2. The study was conducted using direct multiparticle simulation approach. In this method protein molecules are considered as individual objects that experience Brownian motion and electrostatic interaction with the surrounding media and each other. Using the model we studied the effects of pH and ionic strength (I) upon complex formation between ferredoxin and FNR and ferredoxin and hydrogenase. We showed that the rate constant of Fd-FNR complex formation is constant in a wide range of physiologically significant pH values. Therefore it can be argued that regulation of FNR activity doesn’t involve pH changes in stroma. On the other hand, in the model rate constant of Fd-hydrogenase interaction dramatically depends upon pH: in the range 7-9 it increases threefold. It may seem that because hydrogenase reduces protons it should be more active when pH is acidic. Apparently, regulation of hydrogenase’s affinity to both her reaction partners (H+ and Fd) is carried out by changes in its electrostatic properties. In the dark, the protein is inactive and in the light it is activated and starts to interact with both Fd and H+. Therefore, we can conclude that in chloroplasts the rate of hydrogen production is regulated by pH through the changes in the affinity between hydrogenase and ferredoxin.

A TiO₂ nanoparticle system for sacrificial solar H₂ production prepared by rational combination of a hydrogenase with a ruthenium photosensitizer.

PubMed

Reisner, Erwin; Armstrong, Fraser A

2011-01-01

A hybrid system comprising a hydrogenase and a photosensitizer co-attached to a nanoparticle serves as a rational model for fast dihydrogen (H(2)) production using visible light. This chapter describes a stepwise procedure for preparing TiO(2) nanoparticles functionalized with a hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) and a tris(bipyridyl)ruthenium photosensitizer (RuP). Upon irradiation with visible light, these particles produce H(2) from neutral water at room temperature in the presence of a sacrificial electron donor - a test-system for the cathodic half reaction of water splitting. In particular, we describe how a hydrogenase and a photosensitizer with desired properties, including strong adsorption on TiO(2), can be selected by electrochemical methods. The catalyst Db [NiFeSe]-H is selected for its high H(2) production activity even when H(2) and traces of O(2) are present. Adsorption of Db [NiFeSe]-H and RuP on TiO(2) electrodes results in high electrochemical and photocatalytic activities that translate into nanoparticles exhibiting efficient light harvesting, charge separation, and sacrificial H(2) generation.

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

PubMed Central

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P.; Köpke, Michael

2013-01-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP+ with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0′ = −520 mV). PMID:23893107

NADP-specific electron-bifurcating [FeFe]-hydrogenase in a functional complex with formate dehydrogenase in Clostridium autoethanogenum grown on CO.

PubMed

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P; Köpke, Michael; Thauer, Rudolf K

2013-10-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0' = -520 mV).

Requirements for construction of a functional hybrid complex of photosystem I and [NiFe]-hydrogenase.

PubMed

Schwarze, Alexander; Kopczak, Marta J; Rögner, Matthias; Lenz, Oliver

2010-04-01

The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H(2) sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O(2)-tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH(PsaE)) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H(2) production capacity of the purified MBH(PsaE) fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH(PsaE) with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH(PsaE)-PS I complex formation and its light-driven H(2) production capacity by means of spectroelectrochemistry.

Effect of the culture media optimization, pH and temperature on the biohydrogen production and the hydrogenase activities by Klebsiella pneumoniae ECU-15.

PubMed

Xiao, Yan; Zhang, Xu; Zhu, Minglong; Tan, Wensong

2013-06-01

The low yield of the biohydrogen production is the main constraint for its industrialization process. In order to improve its production, medium compositions of the hydrogen fermentation by Klebsiella pneumoniae ECU-15 were optimized through the response surface methodology (RSM). Experimental results showed that the optimum hydrogen production of 5363.8 ml/L was obtained when the concentration of glucose, the ammonium sulfate and the trace elements were 35.62 g/L, 2.78 g/L and 23.15 ml/L at temperature 37.0°C, pH 6.0. H2 evolving hydrogenase was greatly enhanced by the optimization of the medium compositions. The activity of H2 evolving hydrogenase increased with the temperature, and decreased with the pH, while the activity of the uptake hydrogenase increased with the temperature and the pH. So the biohydrogen production process of the K. pneumoniae ECU-15 was the comprehensive results of the evolution hydrogen process and the uptake hydrogen process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Integrated responses of Na+/HCO3- cotransporters and V-type H+-ATPases in the fish gill and kidney during respiratory acidosis.

PubMed

Perry, S F; Furimsky, M; Bayaa, M; Georgalis, T; Shahsavarani, A; Nickerson, J G; Moon, T W

2003-12-30

Using degenerate primers, followed by 3' and 5' RACE and "long" PCR, a continuous 4050-bp cDNA was obtained and sequenced from rainbow trout (Oncorhynchus mykiss) gill. The cDNA included an open reading frame encoding a deduced protein of 1088 amino acids. A BLAST search of the GenBank protein database demonstrated that the trout gene shared high sequence similarity with several vertebrate Na(+)/HCO(3)(-) cotransporters (NBCs) and in particular, NBC1. Protein alignment revealed that the trout NBC is >80% identical to vertebrate NBC1s and phylogenetic analysis provided additional evidence that the trout NBC is indeed a homolog of NBC1. Using the same degenerate primers, a partial cDNA (404 bp) for NBC was obtained from eel (Anguilla rostrata) kidney. Analysis of the tissue distribution of trout NBC, as determined by Northern blot analysis and real-time PCR, indicated high transcript levels in several absorptive/secretory epithelia including gill, kidney and intestine and significant levels in liver. NBC mRNA was undetectable in eel gill by real-time PCR. In trout, the levels of gill NBC1 mRNA were increased markedly during respiratory acidosis induced by exposure to hypercarbia; this response was accompanied by a transient increase in branchial V-type H(+)-ATPase mRNA levels. Assuming that the branchial NBC1 is localised to basolateral membranes of gill cells and operates in the influx mode (HCO(3)(-) and Na(+) entry into the cell), it would appear that in trout, the expression of branchial NBC1 is transcriptionally regulated to match the requirements of gill pHi regulation rather than to match trans-epithelial HCO(3)(-) efflux requirements for systemic acid-base balance. By analogy with mammalian systems, NBC1 in the kidney probably plays a role in the tubular reabsorption of both Na(+) and HCO(3)(-). During periods of respiratory acidosis, levels of renal NBC1 mRNA increased (after a transient reduction) in both trout and eel, presumably to increase HCO(3

Structural Mimics of the [Fe]-Hydrogenase: A Complete Set for Group VIII Metals.

PubMed

Barik, Chandan Kr; Ganguly, Rakesh; Li, Yongxin; Leong, Weng Kee

2018-06-18

A set of structural mimics of the [Fe]-hydrogenase active site comprising all the group VIII metals, viz., [M(2-NHC(O)C 5 H 4 N)(CO) 2 (2-S-C 5 H 4 N)], has been synthesized. They exist as a mixture of isomers in solution, and the relative stability of the isomers depends on the nature of the metal and the substituent at the 6-position of the pyridine ligand.

Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swartz, James

2017-01-25

Final Project Report describing work to elucidate mechanisms for the activation of [FeFe]-hydrogenases and to explore the impact of the polypeptide scaffolding on the function of the Fe-S redox and catalytic centers with emphasis on improving oxygen tolerance.

Electrostatic roles in electron transfer from [NiFe] hydrogenase to cytochrome c3 from Desulfovibrio vulgaris Miyazaki F.

PubMed

Sugimoto, Yu; Kitazumi, Yuki; Shirai, Osamu; Nishikawa, Koji; Higuchi, Yoshiki; Yamamoto, Masahiro; Kano, Kenji

2017-05-01

Electrostatic interactions between proteins are key factors that govern the association and reaction rate. We spectroscopically determine the second-order reaction rate constant (k) of electron transfer from [NiFe] hydrogenase (H 2 ase) to cytochrome (cyt) c 3 at various ionic strengths (I). The k value decreases with I. To analyze the results, we develop a semi-analytical formula for I dependence of k based on the assumptions that molecules are spherical and the reaction proceeds via a transition state. Fitting of the formula to the experimental data reveals that the interaction occurs in limited regions with opposite charges and with radii much smaller than those estimated from crystal structures. This suggests that local charges in H 2 ase and cyt c 3 play important roles in the reaction. Although the crystallographic data indicate a positive electrostatic potential over almost the entire surface of the proteins, there exists a small region with negative potential on H 2 ase at which the electron transfer from H 2 ase to cyt c 3 may occur. This local negative potential region is identical to the hypothetical interaction sphere predicted by the analysis. Furthermore, I dependence of k is predicted by the Adaptive Poisson-Boltzmann Solver considering all charges of the amino acids in the proteins and the configuration of H 2 ase/cyt c 3 complex. The calculation reproduces the experimental results except at extremely low I. These results indicate that the stabilization derived from the local electrostatic interaction in the H 2 ase/cyt c 3 complex overcomes the destabilization derived from the electrostatic repulsion of the overall positive charge of both proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthetic Active Site Model of the [NiFeSe] Hydrogenase

PubMed Central

Wombwell, Claire; Reisner, Erwin

2015-01-01

A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe(‘S2Se2’)(CO)3] (H2‘S2Se2’=1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni(‘S2Se2’)] with [Fe(CO)3bda] (bda=benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe(‘S2Se2’)(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe(‘S2Se2’)(CO)3] with the previously reported thiolate analogue [NiFe(‘S4’)(CO)3] (H2‘S4’=H2xbsms=1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe(‘S2Se2’)(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe(‘S2Se2’)(CO)3] and [NiFe(‘S4’)(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. PMID:25847470

Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kreuzer, Helen W.; Hill, Eric A.; Moran, James J.

2014-03-01

Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the culturesmore » were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data is consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organism, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.« less

Role of Surface-Capping Ligands in Photoexcited Electron Transfer between CdS Nanorods and [FeFe] Hydrogenase and the Subsequent H 2 Generation

DOE PAGES

Wilker, Molly B.; Utterback, James K.; Greene, Sophie; ...

2017-12-08

Complexes of CdS nanorods and [FeFe] hydrogenase from Clostridium acetobutylicum have been shown to photochemically produce H 2. This study examines the role of the ligands that passivate the nanocrystal surfaces in the electron transfer from photoexcited CdS to hydrogenase and the H 2 generation that follows. We functionalized CdS nanorods with a series of mercaptocarboxylate surface-capping ligands of varying lengths and measured their photoexcited electron relaxation by transient absorption (TA) spectroscopy before and after hydrogenase adsorption. Rate constants for electron transfer from the nanocrystals to the enzyme, extracted by modeling of TA kinetics, decrease exponentially with ligand length, suggestingmore » that the ligand layer acts as a barrier to charge transfer and controls the degree of electronic coupling. Relative light-driven H 2 production efficiencies follow the relative quantum efficiencies of electron transfer, revealing the critical role of surface-capping ligands in determining the photochemical activity of these nanocrystal-enzyme complexes. Our results suggest that the H 2 production in this system could be maximized with a choice of a surface-capping ligand that decreases the distance between the nanocrystal surface and the electron injection site of the enzyme.« less

Role of Surface-Capping Ligands in Photoexcited Electron Transfer between CdS Nanorods and [FeFe] Hydrogenase and the Subsequent H 2 Generation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilker, Molly B.; Utterback, James K.; Greene, Sophie

Complexes of CdS nanorods and [FeFe] hydrogenase from Clostridium acetobutylicum have been shown to photochemically produce H 2. This study examines the role of the ligands that passivate the nanocrystal surfaces in the electron transfer from photoexcited CdS to hydrogenase and the H 2 generation that follows. We functionalized CdS nanorods with a series of mercaptocarboxylate surface-capping ligands of varying lengths and measured their photoexcited electron relaxation by transient absorption (TA) spectroscopy before and after hydrogenase adsorption. Rate constants for electron transfer from the nanocrystals to the enzyme, extracted by modeling of TA kinetics, decrease exponentially with ligand length, suggestingmore » that the ligand layer acts as a barrier to charge transfer and controls the degree of electronic coupling. Relative light-driven H 2 production efficiencies follow the relative quantum efficiencies of electron transfer, revealing the critical role of surface-capping ligands in determining the photochemical activity of these nanocrystal-enzyme complexes. Our results suggest that the H 2 production in this system could be maximized with a choice of a surface-capping ligand that decreases the distance between the nanocrystal surface and the electron injection site of the enzyme.« less

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

PubMed

Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2015-07-01

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Isolation and characterization of the small subunit of the uptake hydrogenase from the cyanobacterium Nostoc punctiforme.

PubMed

Raleiras, Patrícia; Kellers, Petra; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2013-06-21

In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ½) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ½) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS.

Enhanced photocatalytic hydrogen production from an MCM-41-immobilized photosensitizer-[Fe-Fe] hydrogenase mimic dyad.

PubMed

Wang, Wen; Yu, Tianjun; Zeng, Yi; Chen, Jinping; Yang, Guoqiang; Li, Yi

2014-11-01

A covalently linked photosensitizer-catalytic center dyad Ps-Hy, consisting of two bis(2-phenylpyridine)(2,2'-bipyridine)iridium(iii) chromophores (Ps) and a diiron hydrogenase mimic (Hy) was constructed by using click reaction. Ps-Hy was incorporated into K(+)-exchanged molecular sieve MCM-41 to form a composite (Ps-Hy@MCM-41), which has been successfully applied to the photochemical production of hydrogen. The catalytic activity of Ps-Hy@MCM-41 is ∼3-fold higher as compared with that of Ps-Hy in the absence of MCM-41. The incorporation of Ps-Hy into MCM-41 stabilizes the catalyst, and consequently, advances the photocatalysis. The present study provides a potential strategy for improving catalytic efficiency of artificial photosynthesis systems using mesoporous molecular sieves.

Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase.

PubMed

Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD + -reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H 2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme. Here all iron-containing cofactors of the SH were investigated by 57 Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD + -reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13 C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

PubMed

Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

2014-02-05

A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

Synthetic Active Site Model of the [NiFeSe] Hydrogenase.

PubMed

Wombwell, Claire; Reisner, Erwin

2015-05-26

A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe('S2Se2')(CO)3] (H2'S2Se2' = 1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni('S2Se2')] with [Fe(CO)3bda] (bda = benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe('S2Se2')(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe('S2Se2')(CO)3] with the previously reported thiolate analogue [NiFe('S4')(CO)3] (H2'S4' = H2xbsms = 1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe('S2Se2')(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe('S2Se2')(CO)3] and [NiFe('S4')(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Role of the Azadithiolate Cofactor in Models for the [FeFe]-Hydrogenase: Novel Structures and Catalytic Implications

PubMed Central

Olsen, Matthew T.; Rauchfuss, Thomas B.; Wilson, Scott R.

2010-01-01

The report summarizes studies on the redox behavior of synthetic models for the [FeFe]-hydrogenases, consisting of diiron dithiolato carbonyl complexes bearing the amine cofactor and its N-benzyl derivative. Of specific interest are the causes of the low reactivity of oxidized models toward H2, which contrasts with the high activity of these enzymes for H2 oxidation. The redox and acid-base properties of the model complexes [Fe2[(SCH2)2NR](CO)3(dppv)(PMe3)]+ ([2]+ for R = H and [2′]+ for R = CH2C6H5, dppv = cis-1,2-bis(diphenylphosphino)ethylene)) indicate that addition of H2 and followed by deprotonation are (i) endothermic for the mixed valence (FeIIFeI) state and (ii) exothermic for the diferrous (FeIIFeII) state. The diferrous state is shown to be unstable with respect to coordination of the amine to Fe, a derivative of which was characterized crystallographically. The redox and acid-base properties for the mixed valence models differ strongly for those containing the amine cofactor versus those derived from propanedithiolate. Protonation of [2′]+ induces disproportionation to a 1:1 mixture of the ammonium-FeIFeI and the dication [2′]2+ (FeIIFeII). This effect is consistent with substantial enhancement of the basicity of the amine in the FeIFeI state vs the FeIIFeI state. The FeIFeI ammonium compounds are rapid and efficient H-atom donors toward the nitroxyl compound TEMPO. The atom transfer is proposed to proceed via the hydride, as indicated by the reaction of [HFe2[(SCH2)2NH](CO)2(dppv)2]+ with TEMPO. Collectively, the results suggest that proton-coupled electron-transfer pathways should be considered for H2 activation by the [FeFe]-hydrogenases. PMID:21114298

Sunlight-Dependent Hydrogen Production by Photosensitizer/Hydrogenase Systems.

PubMed

Adam, David; Bösche, Lisa; Castañeda-Losada, Leonardo; Winkler, Martin; Apfel, Ulf-Peter; Happe, Thomas

2017-03-09

We report a sustainable in vitro system for enzyme-based photohydrogen production. The [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii was tested for photohydrogen production as a proton-reducing catalyst in combination with eight different photosensitizers. Using the organic dye 5-carboxyeosin as a photosensitizer and plant-type ferredoxin PetF as an electron mediator, HydA1 achieves the highest light-driven turnover number (TON HydA1 ) yet reported for an enzyme-based in vitro system (2.9×10 6  mol(H 2 ) mol(cat) -1 ) and a maximum turnover frequency (TOF HydA1 ) of 550 mol(H 2 ) mol(HydA1) -1  s -1 . The system is fueled very effectively by ambient daylight and can be further simplified by using 5-carboxyeosin and HydA1 as a two-component photosensitizer/biocatalyst system without an additional redox mediator. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Artificial hydrogenases based on cobaloximes and heme oxygenase

DOE PAGES

Bacchi, Marine; Veinberg, Elias; Field, Martin J.; ...

2016-06-06

The insertion of cobaloxime catalysts in the heme-binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H 2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO-based biohybrids incorporating a {Co(dmgH) 2} (dmgH 2 = dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respectmore » to the cobaloxime alone or to analogous sperm whale myoglobin adducts. Here, this study thus provides a strong basis for further improvement of such biohybrids, using well-designed modifications of the second and outer coordination spheres, through site-directed mutagenesis of the host protein.« less

Artificial hydrogenases based on cobaloximes and heme oxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacchi, Marine; Veinberg, Elias; Field, Martin J.

The insertion of cobaloxime catalysts in the heme-binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H 2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO-based biohybrids incorporating a {Co(dmgH) 2} (dmgH 2 = dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respectmore » to the cobaloxime alone or to analogous sperm whale myoglobin adducts. Here, this study thus provides a strong basis for further improvement of such biohybrids, using well-designed modifications of the second and outer coordination spheres, through site-directed mutagenesis of the host protein.« less

Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O 2-tolerant NAD +-reducing [NiFe] hydrogenase

DOE PAGES

Lauterbach, Lars; Wang, Hongxin; Horch, Marius; ...

2014-10-30

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H 2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters,more » which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.« less

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

PubMed Central

Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

2011-01-01

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

Photosensitivity of the Ni-A state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F with visible light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osuka, Hisao; Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma-shi, Nara 630-0192; Shomura, Yasuhito

2013-01-04

Highlights: Black-Right-Pointing-Pointer Ni-A state of [NiFe] hydrogenase showed light sensitivity. Black-Right-Pointing-Pointer New FT-IR bands were observed with light irradiation of the Ni-A state. Black-Right-Pointing-Pointer EPR g-values of the Ni-A state shifted upon light irradiation. Black-Right-Pointing-Pointer The light-induced state converted back to the Ni-A state under the dark condition. -- Abstract: [NiFe] hydrogenase catalyzes reversible oxidation of molecular hydrogen. Its active site is constructed of a hetero dinuclear Ni-Fe complex, and the oxidation state of the Ni ion changes according to the redox state of the enzyme. We found that the Ni-A state (an inactive unready, oxidized state) of [NiFe] hydrogenasemore » from Desulfovibrio vulgaris Miyazaki F (DvMF) is light sensitive and forms a new state (Ni-AL) with irradiation of visible light. The Fourier transform infrared (FT-IR) bands at 1956, 2084 and 2094 cm{sup -1} of the Ni-A state shifted to 1971, 2086 and 2098 cm{sup -1} in the Ni-AL state. The g-values of g{sub x} = 2.30, g{sub y} = 2.23 and g{sub z} = 2.01 for the signals in the electron paramagnetic resonance (EPR) spectrum of the Ni-A state at room temperature varied for -0.009, +0.012 and +0.010, respectively, upon light irradiation. The light-induced Ni-AL state converted back immediately to the Ni-A state under dark condition at room temperature. These results show that the coordination structure of the Fe site of the Ni-A state of [NiFe] hydrogenase is perturbed significantly by light irradiation with relatively small coordination change at the Ni site.« less

Cofactor composition and function of a H2-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy† †Electronic supplementary information (ESI) available: Tables with the simulation parameters and details of the Mössbauer, and EPR spectra (Tables S1–S4). additional EPR and Mössbauer spectra in Fig. S1–S9. See DOI: 10.1039/c5sc01560j Click here for additional data file.

PubMed Central

Roncaroli, Federico; Friedrich, Bärbel; Lenz, Oliver

2015-01-01

The regulatory hydrogenase (RH) from Ralstonia eutropha H16 acts as a sensor for the detection of environmental H2 and regulates gene expression related to hydrogenase-mediated cellular metabolism. In marked contrast to prototypical energy-converting [NiFe] hydrogenases, the RH is apparently insensitive to inhibition by O2 and CO. While the physiological function of regulatory hydrogenases is well established, little is known about the redox cycling of the [NiFe] center and the nature of the iron–sulfur (FeS) clusters acting as electron relay. The absence of any FeS cluster signals in EPR had been attributed to their particular nature, whereas the observation of essentially only two active site redox states, namely Ni-SI and Ni-C, invoked a different operant mechanism. In the present work, we employ a combination of Mössbauer, FTIR and EPR spectroscopic techniques to study the RH, and the results are consistent with the presence of three [4Fe–4S] centers in the small subunit. In the as-isolated, oxidized RH all FeS clusters reside in the EPR-silent 2+ state. Incubation with H2 leads to reduction of two of the [4Fe–4S] clusters, whereas only strongly reducing agents lead to reduction of the third cluster, which is ascribed to be the [4Fe–4S] center in ‘proximal’ position to the [NiFe] center. In the two different active site redox states, the low-spin FeII exhibits distinct Mössbauer features attributed to changes in the electronic and geometric structure of the catalytic center. The results are discussed with regard to the spectral characteristics and physiological function of H2-sensing regulatory hydrogenases. PMID:29142700

Impact of the iron-sulfur cluster proximal to the active site on the catalytic function of an O2-tolerant NAD(+)-reducing [NiFe]-hydrogenase.

PubMed

Karstens, Katja; Wahlefeld, Stefan; Horch, Marius; Grunzel, Miriam; Lauterbach, Lars; Lendzian, Friedhelm; Zebger, Ingo; Lenz, Oliver

2015-01-20

The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.

Synthesis, Characterization, and Reactivity of Functionalized Trinuclear Iron–Sulfur Clusters – A New Class of Bioinspired Hydrogenase Models

PubMed Central

Kaiser, Manuel; Knör, Günther

2015-01-01

The air- and moisture-stable iron–sulfur carbonyl clusters Fe3S2(CO)7(dppm) (1) and Fe3S2(CO)7(dppf) (2) carrying the bisphosphine ligands bis(diphenylphosphanyl)methane (dppm) and 1,1′-bis(diphenylphosphanyl)ferrocene (dppf) were prepared and fully characterized. Two alternative synthetic routes based on different thionation reactions of triiron dodecacarbonyl were tested. The molecular structures of the methylene-bridged compound 1 and the ferrocene-functionalized derivative 2 were determined by single-crystal X-ray diffraction. The catalytic reactivity of the trinuclear iron–sulfur cluster core for proton reduction in solution at low overpotential was demonstrated. These deeply colored bisphosphine-bridged sulfur-capped iron carbonyl systems are discussed as promising candidates for the development of new bioinspired model compounds of iron-based hydrogenases. PMID:26512211

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation.

PubMed

Shepard, Eric M; Byer, Amanda S; Aggarwal, Priyanka; Betz, Jeremiah N; Scott, Anna G; Shisler, Krista A; Usselman, Robert J; Eaton, Gareth R; Eaton, Sandra S; Broderick, Joan B

2017-06-27

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H 2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S] 2+/+ and [2Fe-2S] 2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514-3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S] + and [4Fe-4S] + clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S] + and [2Fe-2S] + cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S] + and [4Fe-4S] + clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

PubMed Central

Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady

2016-01-01

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression. PMID:27547572

Inhibition of AMPK catabolic action by GSK3

PubMed Central

Suzuki, Tsukasa; Bridges, Dave; Nakada, Daisuke; Skiniotis, Georgios; Morrison, Sean J.; Lin, Jiandie; Saltiel, Alan R.; Inoki, Ken

2013-01-01

SUMMARY AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK. PMID:23623684

Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl–Group Channeling during [NiFe]–Hydrogenase Cofactor Generation

PubMed Central

Stripp, Sven T.; Lindenstrauss, Ute; Sawers, R. Gary; Soboh, Basem

2015-01-01

[NiFe]–hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so–called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP–dependent condensation reaction, the C–terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]–hydrogenase active site cofactor. We present a FT–IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF–catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro–electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (–N=C=S) rather than thiocyanate (–S–C≡N). This has important implications for cyanyl–group channeling during [NiFe]–hydrogenase cofactor generation. PMID:26186649

Evolutionarily Conserved nodE, nodO, T1SS, and Hydrogenase System in Rhizobia of Astragalus membranaceus and Caragana intermedia.

PubMed

Yan, Hui; Xie, Jian Bo; Ji, Zhao Jun; Yuan, Na; Tian, Chang Fu; Ji, Shou Kun; Wu, Zhong Yu; Zhong, Liang; Chen, Wen Xin; Du, Zheng Lin; Wang, En Tao; Chen, Wen Feng

2017-01-01

Mesorhizobium species are the main microsymbionts associated with the medicinal or sand-fixation plants Astragalus membranaceus and Caragana intermedia (AC) in temperate regions of China, while all the Mesorhizobium strains isolated from each of these plants could nodulate both of them. However, Rhizobium yanglingense strain CCBAU01603 could nodulate AC plants and it's a high efficiency symbiotic and competitive strain with Caragana . Therefore, the common features shared by these symbiotic rhizobia in genera of Mesorhizobium and Rhizobium still remained undiscovered. In order to study the genomic background influencing the host preference of these AC symbiotic strains, the whole genomes of two ( M. silamurunense CCBAU01550, M. silamurunense CCBAU45272) and five representative strains ( M. septentrionale CCBAU01583, M. amorphae CCBAU01570, M. caraganae CCBAU01502, M. temperatum CCBAU01399, and R. yanglingense CCBAU01603) originally isolated from AC plants were sequenced, respectively. As results, type III secretion systems (T3SS) of AC rhizobia evolved in an irregular pattern, while an evolutionarily specific region including nodE, nodO , T1SS, and a hydrogenase system was detected to be conserved in all these AC rhizobia. Moreover, nodO was verified to be prevalently distributed in other AC rhizobia and was presumed as a factor affecting the nodule formation process. In conclusion, this research interpreted the multifactorial features of the AC rhizobia that may be associated with their host specificity at cross-nodulation group, including nodE, nodZ , T1SS as the possible main determinants; and nodO , hydrogenase system, and T3SS as factors regulating the bacteroid formation or nitrogen fixation efficiency.

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism.

PubMed

Biswas, Ranjita; Wilson, Charlotte M; Giannone, Richard J; Klingeman, Dawn M; Rydzak, Thomas; Shah, Manesh B; Hettich, Robert L; Brown, Steven D; Guss, Adam M

2017-01-01

Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H 2 production, including the hydrogenase maturase hydG and NiFe hydrogenase ech , were deleted from the chromosome of C. thermocellum . While ethanol yield increased, the growth rate of Δ hydG decreased substantially compared to wild type. Addition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ∆hydG , whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG , 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated in C. thermocellum ΔhydG in the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG . Clostridium thermocellum ΔhydGΔech had a higher growth rate than ΔhydG in the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate. Sulfur metabolism is perturbed in C. thermocellum ΔhydG strains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.

Alcohol dehydrogenase and hydrogenase transcript fluctuations during a day-night cycle in Chlamydomonas reinhardtii: the role of anoxia.

PubMed

Whitney, Larisa Angela Swirsky; Loreti, Elena; Alpi, Amedeo; Perata, Pierdomenico

2011-04-01

• The unicellular green alga Chlamydomonas reinhardtii contains two iron (Fe)-hydrogenases which are responsible for hydrogen production under anoxia. In the present work the patterns of expression of alcohol dehydrogenase, a typical anaerobic gene in plants, of the hydrogenases genes (HYD1, HYD2) and of the genes responsible for their maturation (HYDEF, HYDG), were analysed. • The expression patterns were analysed by real-time reverse-transcription polymerase chain reaction in Chlamydomonas cultures during the day-night cycle, as well as in response to oxygen availability. • The results indicated that ADH1, HYD1, HYD2, HYDEF and HYDG were expressed following precise day-night fluctuations. ADH1 and HYD2 were modulated by the day-night cycle. Low oxygen plays an important role for the induction of HYD1, HYDEF and HYDG, while ADH1 and HYD2 expression was relatively insensitive to oxygen availability. • The regulation of the anaerobic gene expression in Chlamydomonas is only partly explained by responses to anoxia. The cell cycle and light-dark cycles are equally important elements in the regulatory network modulating the anaerobic response in Chlamydomonas. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

Unification of [FeFe]-hydrogenases into three structural and functional groups

DOE PAGES

Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; ...

2016-05-27

[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H 2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H 2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA,more » and regulatory proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggests that they are post-translationally modified by phosphorylation. In conclusion, these results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.« less

Protozoa inhibition by different salts: Osmotic stress or ionic stress?

PubMed

Li, Changhao; Li, Jingya; Lan, Christopher Q; Liao, Dankui

2017-09-01

Cell density and morphology changes were tested to examine the effects of salts including NaHCO 3 , NaCl, KHCO 3 , and KCl at 160 mM on protozoa. It was demonstrated that ionic stress rather than osmotic stress led to protozoa cell death and NaHCO 3 was shown to be the most effective inhibitor. Deformation of cells and cell shrinkage were observed when protozoan cells were exposed to polyethylene glycol (PEG) or any of the salts. However, while PEG treated cells could fully recover in both number and size, only a small portion of the salt-treated cells survive and cell size was 36-58% smaller than the regular. The disappearance of salt-treated protozoa cells was hypothetically attributed to disruption of the cytoplasmic membrane of these cells. It is further hypothesized that the PEG-treated protozoan cells carried out regulatory volume increase (RVI) after the osmotic shock but the RVI of salt-treated protozoa was hurdled to varied extents. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1418-1424, 2017. © 2017 American Institute of Chemical Engineers.

Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

PubMed

Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

2016-01-01

The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

A dithiolate-bridged (CN)2(CO)Fe-Ni complex reproducing the IR bands of [NiFe] hydrogenase.

PubMed

Tanino, Soichiro; Li, Zilong; Ohki, Yasuhiro; Tatsumi, Kazuyuki

2009-03-16

A dithiolate-bridged dinuclear Fe-Ni complex, which has the desired fac-(CN)(2)(CO) ligand set at iron, has been synthesized. Its CN/CO bands in the IR spectrum reproduce those of the Ni-A, Ni-B, and Ni-SU states, which indicate that these octahedral Fe(II) centers have similar electronic properties. This result verifies the assignment of a (CN)(2)(CO)Fe(II) moiety in the active site of [NiFe] hydrogenase.

The auxiliary protein HypX provides oxygen tolerance to the soluble [NiFe]-hydrogenase of ralstonia eutropha H16 by way of a cyanide ligand to nickel.

PubMed

Bleijlevens, Boris; Buhrke, Thorsten; van der Linden, Eddy; Friedrich, Bärbel; Albracht, Simon P J

2004-11-05

The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium. A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD+ -reducing hydrogenase. The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypX-negative strain were compared with those from an enzyme purified from a HypX-positive strain. In activity assays under anaerobic conditions both enzyme preparations behaved the same. Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H2 oxidation with NAD+ or benzyl viologen as the electron acceptor. Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme. The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide. The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions.

Characterization of Photochemical Processes for H2 Production by CdS Nanorod-[FeFe] Hydrogenase Complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, K. A.; Wilker, M. B.; Boehm, M.

2012-03-28

We have developed complexes of CdS nanorods capped with 3-mercaptopropionic acid (MPA) and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) that photocatalyze reduction of H{sup +} to H{sub 2} at a CaI turnover frequency of 380-900 s{sup -1} and photon conversion efficiencies of up to 20% under illumination at 405 nm. In this paper, we focus on the compositional and mechanistic aspects of CdS:CaI complexes that control the photochemical conversion of solar energy into H{sub 2}. Self-assembly of CdS with CaI was driven by electrostatics, demonstrated as the inhibition of ferredoxin-mediated H{sub 2} evolution by CaI. Production of H{sub 2} by CdS:CaImore » was observed only under illumination and only in the presence of a sacrificial donor. We explored the effects of the CdS:CaI molar ratio, sacrificial donor concentration, and light intensity on photocatalytic H{sub 2} production, which were interpreted on the basis of contributions to electron transfer, hole transfer, or rate of photon absorption, respectively. Each parameter was found to have pronounced effects on the CdS:CaI photocatalytic activity. Specifically, we found that under 405 nm light at an intensity equivalent to total AM 1.5 solar flux, H{sub 2} production was limited by the rate of photon absorption ({approx}1 ms{sup -1}) and not by the turnover of CaI. Complexes were capable of H{sub 2} production for up to 4 h with a total turnover number of 106 before photocatalytic activity was lost. This loss correlated with inactivation of CaI, resulting from the photo-oxidation of the CdS capping ligand MPA.« less

Hydrogen Activation by Biomimetic [NiFe]-Hydrogenase Model Containing Protected Cyanide Cofactors

PubMed Central

Manor, Brian C.; Rauchfuss, Thomas B.

2013-01-01

Described are experiments that allow incorporation of cyanide cofactors and hydride substrate into active site models [NiFe]-hydrogenases (H2ases). Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe), (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BArF3) to give the adducts (CO)2(CNBArF3)2Fe(pdt)Ni(dxpe), (1(BArF3)2, 2(BArF3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives Et4N[(CO)(CNBArF3)2Fe(H)(pdt)Ni(dxpe)], (dxpe = dppe, Et4N[H3(BArF3)2]; dxpe = dcpe, Et4N[H4(BArF3)2]). Crystallographic analysis shows that Et4N[H3(BArF3)2] generally resembles the active site of the enzyme in the reduced, hydride-containing states (Ni-C/R). The Fe-H…Ni center is unsymmetrical with rFe-H = 1.51(3) and rNi-H = 1.71(3) Å. Both crystallographic and 19F NMR analysis show that the CNBArF3− ligands occupy basal and apical sites. Unlike cationic Ni-Fe hydrides, [H3(BArF3)2]− and [H4(BArF3)2]− oxidize at mild potentials, near the Fc+/0 couple. Electrochemical measurements indicate that in the presence of base, [H3(BArF3)2]− catalyzes the oxidation of H2. NMR evidence indicates dihydrogen bonding between these anionic hydrides and ammonium salts, which is relevant to the mechanism of hydrogenogenesis. In the case of Et4N[H3(BArF3)2], strong acids such as HCl induce H2 release to give the chloride Et4N[(CO)(CNBArF3)2Fe(pdt)(Cl)Ni(dppe)]. PMID:23899049

Direct comparison of the performance of a bio-inspired synthetic nickel catalyst and a [NiFe]-hydrogenase, both covalently attached to electrodes.

PubMed

Rodriguez-Maciá, Patricia; Dutta, Arnab; Lubitz, Wolfgang; Shaw, Wendy J; Rüdiger, Olaf

2015-10-12

The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequential application of NaHCO3, CaCl2 and Candida oleophila (isolate 13L) affects significantly Penicillum expansum growth and the infection degree in apples.

PubMed

Molinu, M G; Pani, G; Venditti, T; Dore, A; Ladu, G; D'Hallewin, G

2011-01-01

The employment of biocontrol agents to restrain postharvest pathogens is an encouraging approach, although, efficacy and consistency are still below those of synthetic pesticides. Up to date, the 'integrated control strategy' seems to be the most promising way to overcome this gap. Here, we report the feasibility to control postharvest decay caused by Penicillium expansum in apples by a 2 min, single or sequential, immersion in water with an antagonistic yeast (Candida oleophila, isolate '13L'), 2% NaHCO3 (SBC) or 1% CaCl2. The treatments were carried out, on appels cv 'Miali' either un-wounded, wounded or wound-pathogen inoculated and then stored at 2 degrees C for 30 d followed by a 6 d simulated marketing period at 20 degrees C or alternatively stored only for 7 d at 20 degrees C. As a general role, the best results were attained when CaCl2 was applied with the yeast or when preceded by the SBC treatment. When the wounding and inoculation took place 24 h before the treatment, the latter application sequence of the two salts was three times more effective compared to the treatment with the sole antagonist, and one time when performed 24 h after the treatment. Interestingly, apples immersed in the sole 2% SBC solution had the highest percentage of decay during storage and when inoculated before moving to the simulated marketing period at 20 degrees C.

Unification of [FeFe]-hydrogenases into three structural and functional groups.

PubMed

Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R; Refai, Mohammed; Schut, Gerrit J; King, Paul W; Maness, Pin-Ching; Adams, Michael W W; Peters, John W; Bothner, Brian; Boyd, Eric S

2016-09-01

[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

PubMed Central

2017-01-01

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514–3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway. PMID:28525271

The Mössbauer Parameters of the Proximal Cluster of Membrane-Bound Hydrogenase Revisited: A Density Functional Theory Study.

PubMed

Tabrizi, Shadan Ghassemi; Pelmenschikov, Vladimir; Noodleman, Louis; Kaupp, Martin

2016-01-12

An unprecedented [4Fe-3S] cluster proximal to the regular [NiFe] active site has recently been found to be responsible for the ability of membrane-bound hydrogenases (MBHs) to oxidize dihydrogen in the presence of ambient levels of oxygen. Starting from proximal cluster models of a recent DFT study on the redox-dependent structural transformation of the [4Fe-3S] cluster, (57)Fe Mössbauer parameters (electric field gradients, isomer shifts, and nuclear hyperfine couplings) were calculated using DFT. Our results revise the previously reported correspondence of Mössbauer signals and iron centers in the [4Fe-3S](3+) reduced-state proximal cluster. Similar conflicting assignments are also resolved for the [4Fe-3S](5+) superoxidized state with particular regard to spin-coupling in the broken-symmetry DFT calculations. Calculated (57)Fe hyperfine coupling (HFC) tensors expose discrepancies in the experimental set of HFC tensors and substantiate the need for additional experimental work on the magnetic properties of the MBH proximal cluster in its reduced and superoxidized redox states.

An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure.

PubMed

Pfeiffer, M; Klein, A; Steinert, P; Schomburg, D

The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism.

A density functional theory study on the active center of Fe-only hydrogenase: characterization and electronic structure of the redox states.

PubMed

Liu, Zhi-Pan; Hu, P

2002-05-08

We have carried out extensive density functional theory (DFT) calculations for possible redox states of the active center in Fe-only hydrogenases. The active center is modeled by [(H(CH(3))S)(CO)(CN(-))Fe(p)(mu-DTN)(mu-CO)Fe(d)(CO)(CN(-))(L)](z)() (z is the net charge in the complex; Fe(p)= the proximal Fe, Fe(d) = the distal Fe, DTN = (-SCH(2)NHCH(2)S-), L is the ligand that bonds with the Fe(d) at the trans position to the bridging CO). Structures of possible redox states are optimized, and CO stretching frequencies are calculated. By a detailed comparison of all the calculated structures and the vibrational frequencies with the available experimental data, we find that (i) the fully oxidized, inactive state is an Fe(II)-Fe(II) state with a hydroxyl (OH(-)) group bonded at the Fe(d), (ii) the oxidized, active state is an Fe(II)-Fe(I) complex which is consistent with the assignment of Cao and Hall (J. Am. Chem. Soc. 2001, 123, 3734), and (iii) the fully reduced state is a mixture with the major component being a protonated Fe(I)-Fe(I) complex and the other component being its self-arranged form, Fe(II)-Fe(II) hydride. Our calculations also show that the exogenous CO can strongly bond with the Fe(II)-Fe(I) species, but cannot bond with the Fe(I)-Fe(I) complex. This result is consistent with experiments that CO tends to inhibit the oxidized, active state, but not the fully reduced state. The electronic structures of all the redox states have been analyzed. It is found that a frontier orbital which is a mixing state between the e(g) of Fe and the 2 pi of the bridging CO plays a key role concerning the reactivity of Fe-only hydrogenases: (i) it is unoccupied in the fully oxidized, inactive state, half-occupied in the oxidized, active state, and fully occupied in the fully reduced state; (ii) the e(g)-2 pi orbital is a bonding state, and this is the key reason for stability of the low oxidation states, such as Fe(I)-Fe(I) complexes; and (iii) in the e(g)-2 pi orbital

Hydrogen-induced structural changes at the nickel site of the regulatory [NiFe] hydrogenase from Ralstonia eutropha detected by X-ray absorption spectroscopy.

PubMed

Haumann, Michael; Porthun, Antje; Buhrke, Thorsten; Liebisch, Peter; Meyer-Klaucke, Wolfram; Friedrich, Bärbel; Dau, Holger

2003-09-23

For the first time, the nickel site of the hydrogen sensor of Ralstonia eutropha, the regulatory [NiFe] hydrogenase (RH), was investigated by X-ray absorption spectroscopy (XAS) at the nickel K-edge. The oxidation state and the atomic structure of the Ni site were investigated in the RH in the absence (air-oxidized, RH(ox)) and presence of hydrogen (RH(+H2)). Incubation with hydrogen is found to cause remarkable changes in the spectroscopic properties. The Ni-C EPR signal, indicative of Ni(III), is detectable only in the RH(+H2) state. XANES and EXAFS spectra indicate a coordination of the Ni in the RH(ox) and RH(+H2) that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H(2) are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RH(ox)) to 8341.1 eV (RH(+H2)) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H(2). One likely interpretation of the data is the detachment of one sulfur ligand in RH(+H2) and the binding of additional (O,N) or H ligands. The following Ni oxidation states and coordinations are proposed: five-coordinated Ni(II)(O,N)(2)S(3) for RH(ox) and six-coordinated Ni((III))(O,N)(3)X(1)S(2) [X being either an (O,N) or H ligand] for RH(+H2). Implications of the structural features of the Ni site of the RH in relation to its function, hydrogen sensing, are discussed.

Amiloride Derivatives Inhibit Coxsackievirus B3 RNA Replication▿

PubMed Central

Harrison, David N.; Gazina, Elena V.; Purcell, Damian F.; Anderson, David A.; Petrou, Steven

2008-01-01

Amiloride derivatives are known blockers of the cellular Na+/H+ exchanger and the epithelial Na+ channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds. PMID:18032495

3,3′-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaver, Laura M., E-mail: beaverl@onid.orst.edu; School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331; Yu, Tian-Wei, E-mail: david.yu@oregonstate.edu

2012-09-15

Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast,more » DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis. -- Highlights: ► DIM inhibits HDAC activity and decreases HDAC2 expression in prostate cancer cells. ► DIM is significantly more effective than I3C at inhibiting HDAC activity. ► I3C has no effect on HDAC protein expression. ► Inhibition of HDAC activity by DIM is associated with increased p21 expression. ► HDAC inhibition may be a novel epigenetic mechanism for cancer prevention with DIM.« less

Modeling the active site of [NiFe] hydrogenases and the [NiFeu] subsite of the C-cluster of carbon monoxide dehydrogenases: low-spin iron(II) versus high-spin iron(II).

PubMed

Weber, Katharina; Erdem, Özlen F; Bill, Eckhard; Weyhermüller, Thomas; Lubitz, Wolfgang

2014-06-16

A series of four [S2Ni(μ-S)2FeCp*Cl] compounds with different tetradentate thiolate/thioether ligands bound to the Ni(II) ion is reported (Cp* = C5Me5). The {S2Ni(μ-S)2Fe} core of these compounds resembles structural features of the active site of [NiFe] hydrogenases. Detailed analyses of the electronic structures of these compounds by Mössbauer and electron paramagnetic resonance spectroscopy, magnetic measurements, and density functional theory calculations reveal the oxidation states Ni(II) low spin and Fe(II) high spin for the metal ions. The same electronic configurations have been suggested for the Cred1 state of the C-cluster [NiFeu] subsite in carbon monoxide dehydrogenases (CODH). The Ni-Fe distance of ∼3 Å excludes a metal-metal bond between nickel and iron, which is in agreement with the computational results. Electrochemical experiments show that iron is the redox active site in these complexes, performing a reversible one-electron oxidation. The four complexes are discussed with regard to their similarities and differences both to the [NiFe] hydrogenases and the C-cluster of Ni-containing CODH.

IGFBP-3, hypoxia and TNF-{alpha} inhibit adiponectin transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zappala, Giovanna, E-mail: zappalag@mail.nih.gov; Rechler, Matthew M.; Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD

2009-05-15

The thiazolidinedione rosiglitazone, an agonist ligand for the nuclear receptor PPAR-{gamma}, improves insulin sensitivity in part by stimulating transcription of the insulin-sensitizing adipokine adiponectin. It activates PPAR-{gamma}-RXR-{alpha} heterodimers bound to PPAR-{gamma} response elements in the adiponectin promoter. Rosiglitazone-stimulated adiponectin protein synthesis in 3T3-L1 mouse adipocytes has been shown to be inhibited by IGFBP-3, which can be induced by hypoxia and the proinflammatory cytokine, TNF-{alpha}, two inhibitors of adiponectin transcription. The present study demonstrates that IGFBP-3, the hypoxia-mimetic agent cobalt chloride, and TNF-{alpha} inhibit rosiglitazone-induced adiponectin transcription in mouse embryo fibroblasts that stably express PPAR-{gamma}2. Native IGFBP-3 can bind RXR-{alpha} andmore » inhibited rosiglitazone stimulated promoter activity, whereas an IGFBP-3 mutant that does not bind RXR-{alpha} did not. These results suggest that IGFBP-3 may mediate the inhibition of adiponectin transcription by hypoxia and TNF-{alpha}, and that IGFBP-3 binding to RXR-{alpha} may be required for the observed inhibition.« less

Solar Water Splitting with a Hydrogenase Integrated in Photoelectrochemical Tandem Cells.

PubMed

Nam, Dong Heon; Zhang, Jenny; Andrei, Virgil; Kornienko, Nikolay; Heidary, Nina; Wagner, Andreas; Nakanishi, Kenichi; Sokol, Katarzyna; Slater, Barnaby; Zebger, Ingo; Hofmann, Stephan; Fontecilla-Camps, Juan; Park, Chan Beum; Reisner, Erwin

2018-06-11

Hydrogenases (H2ases) are benchmark electrocatalysts in H2 production, both in biology and (photo)catalysis in vitro. We report the tailoring of a p-type Si photocathode for optimal loading and wiring of H2ase by the introduction of a hierarchical inverse opal (IO) TiO2 interlayer. This proton reducing Si|IO-TiO2|H2ase photocathode is capable of driving overall water splitting in combination with a complementary photoanode. We demonstrate unassisted (bias-free) water-splitting by wiring Si|IO-TiO2|H2ase to a modified BiVO4 photoanode in a photoelectrochemical (PEC) cell during several hours of irradiation. Connecting the Si|IO-TiO2|H2ase to a photosystem II (PSII) photoanode provides proof-of-concept for an engineered Z-scheme that replaces the non-complementary, natural light absorber photosystem I with a complementary abiotic silicon photocathode. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Foxp3+ T cells inhibit antitumor immune memory modulated by mTOR inhibition.

PubMed

Wang, Yanping; Sparwasser, Tim; Figlin, Robert; Kim, Hyung L

2014-04-15

Inhibition of mTOR signaling enhances antitumor memory lymphocytes. However, pharmacologic mTOR inhibition also enhances regulatory T-cell (Treg) activity. To counter this effect, Treg control was added to mTOR inhibition in preclinical models. Tregs were controlled with CD4-depleting antibodies because CD4 depletion has high translational potential and already has a well-established safety profile in patients. The antitumor activity of the combination therapy was CD8 dependent and controlled growth of syngeneic tumors even when an adoptive immunotherapy was not used. Lymphocytes resulting from the combination therapy could be transferred into naïve mice to inhibit aggressive growth of lung metastases. The combination therapy enhanced CD8 memory formation as determined by memory markers and functional studies of immune recall. Removal of FoxP3-expressing T lymphocytes was the mechanism underlying immunologic memory formation following CD4 depletion. This was confirmed using transgenic DEREG (depletion of regulatory T cells) mice to specifically remove Foxp3(+) T cells. It was further confirmed with reciprocal studies where stimulation of immunologic memory because of CD4 depletion was completely neutralized by adoptively transferring tumor-specific Foxp3(+) T cells. Also contributing to tumor control, Tregs that eventually recovered following CD4 depletion were less immunosuppressive. These results provide a rationale for further study of mTOR inhibition and CD4 depletion in patients. ©2014 AACR.

Homology modeling reveals the structural background of the striking difference in thermal stability between two related [NiFe]hydrogenases.

PubMed

Szilágyi, András; Kovács, Kornél L; Rákhely, Gábor; Závodszky, Péter

2002-02-01

Hydrogenases are redox metalloenzymes in bacteria that catalyze the uptake or production of molecular hydrogen. Two homologous nickel-iron hydrogenases, HupSL and HydSL from the photosynthetic purple sulfur bacterium Thiocapsa roseopersicina, differ substantially in their thermal stabilities despite the high sequence similarity between them. The optimum temperature of HydSL activity is estimated to be at least 50 degrees C higher than that of HupSL. In this work, homology models of both proteins were constructed and analyzed for a number of structural properties. The comparison of the models reveals that the higher stability of HydSL can be attributed to increased inter-subunit electrostatic interactions: the homology models reliably predict that HydSL contains at least five more inter-subunit ion pairs than HupSL. The subunit interface of HydSL is more polar than that of HupSL, and it contains a few extra inter-subunit hydrogen bonds. A more optimized cavity system and amino acid replacements resulting in increased conformational rigidity may also contribute to the higher stability of HydSL. The results are in accord with the general observation that with increasing temperature, the role of electrostatic interactions in protein stability increases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00894-001-0071-8.

Reversible active site sulfoxygenation can explain the oxygen tolerance of a NAD+-reducing [NiFe] hydrogenase and its unusual infrared spectroscopic properties.

PubMed

Horch, Marius; Lauterbach, Lars; Mroginski, Maria Andrea; Hildebrandt, Peter; Lenz, Oliver; Zebger, Ingo

2015-02-25

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD(+)-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD(+) and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2.

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster

DOE PAGES

Suess, Daniel L. M.; Bürstel, Ingmar; De La Paz, Liliana; ...

2015-08-31

Hydrogenases catalyze the redox interconversion of protons and H 2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe–4S] H subcluster coupled to a [2Fe] H subcluster bound by CO, CN–, and azadithiolate ligands. The [2Fe] H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO) 2(CN)] synthon that is incorporated into the H cluster. Though it is thought that themore » site of synthon formation in HydG is the “dangler” Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. In order to address these questions, we show in this paper that L-cysteine (Cys) binds the auxiliary [4Fe–4S] cluster of HydG and further chelates the dangler Fe. We demonstrate that a [4Fe–4S] aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO) 2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Finally, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe 2+ binding and synthon release.« less

Control of CA3 output by feedforward inhibition despite developmental changes in the excitation-inhibition balance.

PubMed

Torborg, Christine L; Nakashiba, Toshiaki; Tonegawa, Susumu; McBain, Chris J

2010-11-17

In somatosensory cortex, the relative balance of excitation and inhibition determines how effectively feedforward inhibition enforces the temporal fidelity of action potentials. Within the CA3 region of the hippocampus, glutamatergic mossy fiber (MF) synapses onto CA3 pyramidal cells (PCs) provide strong monosynaptic excitation that exhibit prominent facilitation during repetitive activity. We demonstrate in the juvenile CA3 that MF-driven polysynaptic IPSCs facilitate to maintain a fixed EPSC-IPSC ratio during short-term plasticity. In contrast, in young adult mice this MF-driven polysynaptic inhibitory input can facilitate or depress in response to short trains of activity. Transgenic mice lacking the feedback inhibitory loop continue to exhibit both facilitating and depressing polysynaptic IPSCs, indicating that this robust inhibition is not caused by the secondary engagement of feedback inhibition. Surprisingly, eliminating MF-driven inhibition onto CA3 pyramidal cells by blockade of GABA(A) receptors did not lead to a loss of temporal precision of the first action potential observed after a stimulus but triggered in many cases a long excitatory plateau potential capable of triggering repetitive action potential firing. These observations indicate that, unlike other regions of the brain, the temporal precision of single MF-driven action potentials is dictated primarily by the kinetics of MF EPSPs, not feedforward inhibition. Instead, feedforward inhibition provides a robust regulation of CA3 PC excitability across development to prevent excessive depolarization by the monosynaptic EPSP and multiple action potential firings.

Removing polysaccharides-and saccharides-related coloring impurities in alkyl polyglycosides by bleaching with the H2O2/TAED/NaHCO3 system.

PubMed

Yanmei, Liu; Jinliang, Tao; Jiao, Sun; Wenyi, Chen

2014-11-04

The effect of H2O2/TAED/NaHCO3 system, namely NaHCO3 as alkaline agent with the (tetra acetyl ethylene diamine (TAED)) TAED-activated peroxide system, bleaching of alkyl polyglycosides solution was studied by spectrophotometry. The results showed that the optimal bleaching conditions about H2O2/TAED/NaHCO3 system bleaching of alkyl polyglycosides solution were as follows: molar ratio of TAED to H2O2 was 0.06, addition of H2O2 was 8.6%, addition of NaHCO3 was 3.2%, bleaching temperature of 50-65 °C, addition of MgO was 0.13%, and bleaching time was 8h. If too much amount of NaHCO3 was added to the system and maintained alkaline pH, the bleaching effect would be greatly reduced. Fixing molar ratio of TAED to H2O2 and increasing the amount of H2O2 were beneficial to improve the whiteness of alkyl polyglycosides, but adding too much amount of H2O2 would reduce the transparency. In the TAED-activated peroxide system, NaHCO3 as alkaline agent and buffer agent, could overcome the disadvantage of producing black precipitates when NaOH as alkaline agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease.

PubMed

Sydor, Andrew M; Liu, Jenny; Zamble, Deborah B

2011-03-01

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

ATF3 represses PPARγ expression and inhibits adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Min-Kyung; Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr

Highlights: • ATF3 decrease the expression of PPARγ and its target gene in 3T3-L1 adipocytes. • ATF3 represses the promoter activity of PPARγ2 gene. • ATF/CRE (−1537/−1530) is critical for ATF3-mediated downregulation of PPARγ. • ATF3 binds to the promoter region containing the ATF/CRE. • ER stress inhibits adipocyte differentiation through downregulation of PPARγ by ATF3. - Abstract: Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3more » in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of −2.6 Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of −2037 bp promoter, whereas it did not affect the activity of −1458 bp promoter, suggesting that ATF3 responsive element is located between the −2037 and −1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5′-TGACGTTT-3′) between −1537 and −1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3

Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

NASA Astrophysics Data System (ADS)

Tamura, Takashi; Tsunekawa, Naoki; Nemoto, Michiko; Inagaki, Kenji; Hirano, Toshiyuki; Sato, Fumitoshi

2016-01-01

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

Protonation states of intermediates in the reaction mechanism of [NiFe] hydrogenase studied by computational methods.

PubMed

Dong, Geng; Ryde, Ulf

2016-06-01

The [NiFe] hydrogenases catalyse the reversible conversion of H2 to protons and electrons. The active site consists of a Fe ion with one carbon monoxide, two cyanide, and two cysteine (Cys) ligands. The latter two bridge to a Ni ion, which has two additional terminal Cys ligands. It has been suggested that one of the Cys residues is protonated during the reaction mechanism. We have used combined quantum mechanical and molecular mechanics (QM/MM) geometry optimisations, large QM calculations with 817 atoms, and QM/MM free energy simulations, using the TPSS and B3LYP methods with basis sets extrapolated to the quadruple zeta level to determine which of the four Cys residues is more favourable to protonate for four putative states in the reaction mechanism, Ni-SIa, Ni-R, Ni-C, and Ni-L. The calculations show that for all states, the terminal Cys-546 residue is most easily protonated by 14-51 kJ/mol, owing to a more favourable hydrogen-bond pattern around this residue in the protein.

Neurogenic regulation of proximal bicarbonate and chloride reabsorption.

PubMed

Cogan, M G

1986-01-01

Although a change in renal nerve activity is known to alter proximal reabsorption, it is unclear whether reabsorption of NaHCO3 or NaCl or both are affected. Sprague-Dawley rats (n = 10) were studied using free-flow micropuncture techniques during euvolemia and following acute ipsilateral denervation. Glomerular filtration rate and single nephron glomerular filtration rate were stable. Absolute proximal bicarbonate reabsorption fell following denervation (933 +/- 40 to 817 +/- 30 pmol/min) with a parallel reduction in chloride reabsorption (1,643 +/- 116 to 1,341 +/- 129 peq/min). Urinary sodium, potassium, bicarbonate, and chloride excretion all increased significantly. To further assess the physiological significance of neurogenic modulation of proximal transport, other rats (n = 6) were subjected to acute unilateral nephrectomy (AUN). There is evidence that AUN induces a contralateral natriuresis (renorenal reflex) at least partially by causing inhibition of efferent renal nerve traffic. AUN caused significant changes in proximal NaHCO3 and NaCl reabsorption as well as in whole kidney electrolyte excretion in the same pattern as had denervation. Prior denervation of the remaining kidney prevented the proximal and whole kidney response to AUN (n = 6). In conclusion, depression of renal nerve activity inhibits both NaHCO3 and NaCl reabsorption in the rat superficial proximal convoluted tubule. The data are consistent with the hypothesis that changes in renal nerve activity modify whole kidney electrolyte excretion under physiological conditions at least partially by regulating proximal transport.

Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Gö1, both encoding a membrane-bound hydrogenase and a cytochrome b.

PubMed

Deppenmeier, U; Blaut, M; Lentes, S; Herzberg, C; Gottschalk, G

1995-01-15

DNA encompassing the structural genes of two membrane-bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505-511]. Northern-blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N-terminal amino acid sequences of vhoC and vhtC and the N-terminus of a 27-kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N-terminus of the small subunit indicate the presence of a 48-amino-acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C-terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92-97% identity. Only the C-termini of VhoC and VhtC were not similar.

Chapter Eight - Structural Characterization of Poised States in the Oxygen Sensitive Hydrogenases and Nitrogenases

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, Paul W; Mulder, David W; Artz, Jacob H.

The crystallization of FeS cluster-containing proteins has been challenging due to their oxygen sensitivity, and yet these enzymes are involved in many critical catalytic reactions. The last few years have seen a wealth of innovative experiments designed to elucidate not just structural but mechanistic insights into FeS cluster enzymes. Here, we focus on the crystallization of hydrogenases, which catalyze the reversible reduction of protons to hydrogen, and nitrogenases, which reduce dinitrogen to ammonia. A specific focus is given to the different experimental parameters and strategies that are used to trap distinct enzyme states, specifically, oxidants, reductants, and gas-treatments. Other themesmore » presented here include the recent use of Cryo-EM, and how coupling various spectroscopies to crystallization is opening up new approaches for structural and mechanistic analysis.« less

Insights into electron flux through manipulation of fermentation conditions and assessment of protein expression profiles in Clostridium thermocellum.

PubMed

Rydzak, Thomas; Grigoryan, Marina; Cunningham, Zack J; Krokhin, Oleg V; Ezzati, Peyman; Cicek, Nazim; Levin, David B; Wilkins, John A; Sparling, Richard

2014-01-01

While annotation of the genome sequence of Clostridium thermocellum has allowed predictions of pathways catabolizing cellobiose to end products, ambiguities have persisted with respect to the role of various proteins involved in electron transfer reactions. A combination of growth studies modulating carbon and electron flow and multiple reaction monitoring (MRM) mass spectrometry measurements of proteins involved in central metabolism and electron transfer was used to determine the key enzymes involved in channeling electrons toward fermentation end products. Specifically, peptides belonging to subunits of ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase (NFOR) were low or below MRM detection limits when compared to most central metabolic proteins measured. The significant increase in H2 versus ethanol synthesis in response to either co-metabolism of pyruvate and cellobiose or hypophosphite mediated pyruvate:formate lyase inhibition, in conjunction with low levels of ferredoxin-dependent hydrogenase and NFOR, suggest that highly expressed putative bifurcating hydrogenases play a substantial role in reoxidizing both reduced ferredoxin and NADH simultaneously. However, product balances also suggest that some of the additional reduced ferredoxin generated through increased flux through pyruvate:ferredoxin oxidoreductase must be ultimately converted into NAD(P)H either directly via NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB) or indirectly via NADPH-dependent hydrogenase. While inhibition of hydrogenases with carbon monoxide decreased H2 production 6-fold and redirected flux from pyruvate:ferredoxin oxidoreductase to pyruvate:formate lyase, the decrease in CO2 was only 20 % of that of the decrease in H2, further suggesting that an alternative redox system coupling ferredoxin and NAD(P)H is active in C. thermocellum in lieu of poorly expressed ferredoxin-dependent hydrogenase and NFOR.

3-Bromopyruvate inhibits human gastric cancer tumor growth in nude mice via the inhibition of glycolysis.

PubMed

Xian, Shu-Lin; Cao, Wei; Zhang, Xiao-Dong; Lu, Yun-Fei

2015-02-01

Tumor cells primarily depend upon glycolysis in order to gain energy. Therefore, the inhibition of glycolysis may inhibit tumor growth. Our previous study demonstrated that 3-bromopyruvate (3-BrPA) inhibited gastric cancer cell proliferation in vitro . However, the ability of 3-BrPA to suppress tumor growth in vivo, and its underlying mechanism, have yet to be elucidated. The aim of the present study was to investigate the inhibitory effect of 3-BrPA in an animal model of gastric cancer. It was identified that 3-BrPA exhibited strong inhibitory effects upon xenograft tumor growth in nude mice. In addition, the antitumor function of 3-BrPA exhibited a dose-effect association, which was similar to that of the chemotherapeutic agent, 5-fluorouracil. Furthermore, 3-BrPA exhibited low toxicity in the blood, liver and kidneys of the nude mice. The present study hypothesized that the inhibitory effect of 3-BrPA is achieved through the inhibition of hexokinase activity, which leads to the downregulation of B-cell lymphoma 2 (Bcl-2) expression, the upregulation of Bcl-2-associated X protein expression and the subsequent activation of caspase-3. These data suggest that 3-BrPA may be a novel therapy for the treatment of gastric cancer.

Light Intensity is Important for Hydrogen Production in NaHSO3-Treated Chlamydomonas reinhardtii.

PubMed

Wei, Lanzhen; Yi, Jing; Wang, Lianjun; Huang, Tingting; Gao, Fudan; Wang, Quanxi; Ma, Weimin

2017-03-01

Chlamydomonas reinhardtii is a unicellular green alga that can use light energy to produce H2 from H2O in the background of NaHSO3 treatment. However, the role of light intensity in such H2 production remains elusive. Here, light intensity significantly affected the yield of H2 production in NaHSO3-treated C. reinhardtii, which was consistent with its effects on the content of O2 and the expression and activity of hydrogenase. Further, NaHSO3 was found to be able to remove O2 via a reaction of bisulfite with superoxide anion produced at the acceptor side of PSI, and light intensity affected the reaction rate significantly. Accordingly, high light and strong light but not low light can create an anaerobic environment, which is important to activate hydrogenase and produce H2. Based on the above results, we conclude that light intensity plays an important role in removing O2 and consequently activating hydrogenase and producing H2 in NaHSO3-treated C. reinhardtii. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Molecular Dynamics Study of the Proposed Proton Transport Pathways in [FeFe]-Hydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ginovska-Pangovska, Bojana; Ho, Ming-Hsun; Linehan, John C.

2014-01-15

Possible proton channels in Clostridium pasteurianum [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to discern proposed channels, compare their properties, evaluate the functional channel, and to provide insight into the features of an active proton channel. Our simulations suggest that protons are not transported through water wires. Instead, a five-residue motif (E282, S319, E279, HOH, C299) was found to be the likely channel, consistent with experimental observations. This channel connects the surface of the enzyme and the di-thiomethylamine bridge of the catalytic H-cluster, permitting the transport of protons. The channel was found to have a persistentmore » hydrogen bonded core (residues E279 to S319), with less persistent hydrogen bonds at the ends of the channel. The hydrogen bond occupancy in this network was found to be sensitive to the protonation state of the residues in the channel, with different protonation states enhancing or stabilizing hydrogen bonding in different regions of the network. Single site mutations to non-hydrogen bonding residues break the hydrogen bonding network at that residue, consistent with experimental observations showing catalyst inactivation. In many cases, these mutations alter the hydrogen bonding in other regions of the channel which may be equally important in catalytic failure. A correlation between the protein dynamics near the proton channel and the redox partner binding regions was also found as a function of protonation state. The likely mechanism of proton movement in [FeFe]-hydrogenases is discussed based on the structural analysis presented here. This work was funded by the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of

Two Randomized Clinical Studies to Confirm Differential Plaque Removal by Sodium Bicarbonate Dentifrices in a Single Timed Brushing Model.

PubMed

Mason, Stephen; Karwal, Ritu; Bosma, Mary Lynn

2017-09-01

This study evaluated and compared plaque removal efficacy of commercially available dentifrices containing sodium bicarbonate (NaHCO3) to those without NaHCO3 in a single timed brushing clinical study model. Two randomized, examiner-blind, three-period, three-treatment, crossover studies were performed in adults with a mean Turesky modification of the Quigley-Hein Plaque Index (TPI) score of = 2.00. In Study 1, 60 subjects were randomized to commercially available dentifrices containing: (i) 67% NaHCO3 plus 1425 ppm fluoride (F) as sodium fluoride (NaF); (ii) 45% NaHCO3 plus 1425 ppm F as NaF; or (iii) 0% NaHCO3 plus silica and 1450 ppm F as NaF. In Study 2, 55 subjects were randomized to commercially available dentifrices containing: (i) 67% NaHCO3 plus 1425 ppm F as NaF; (ii) 0% NaHCO3 plus silica and 1400 ppm F as amine F/stannous F; or (iii) 0% NaHCO3 plus chlorhexidine/aluminum lactate and silica with 1360 ppm F as aluminum F. In both studies, subjects brushed their teeth for one timed minute under supervised conditions. Plaque was assessed pre- and post-brushing according to a six-site modification of the TPI. Mean TPI score was analyzed using an analysis of covariance model with treatment and study period as fixed effects, subject as a random variable, and pre-brushing score as a covariate. In both studies, mean TPI score decreased in all groups post-brushing compared with pre-brushing. In Study 1, statistically significant improvements in mean TPI score were reported with the 67% and 45% NaHCO3 dentifrices compared with the 0% NaHCO3 dentifrice (p = 0.0003 and p = 0.0005, respectively). In Study 2, improvements in mean TPI score were statistically significantly greater with the 67% NaHCO3 dentifrice compared with both 0% NaHCO3 dentifrices (p < 0.0001 for both comparisons). All dentifrices were generally well tolerated. A single timed brushing with commercially available dentifrices containing 67% or 45% NaHCO3 exerted a significantly greater effect on

Effect of H2 binding on the nonadiabatic transition probability between singlet and triplet states of the [NiFe]-hydrogenase active site.

PubMed

Kaliakin, Danil S; Zaari, Ryan R; Varganov, Sergey A

2015-02-12

We investigate the effect of H2 binding on the spin-forbidden nonadiabatic transition probability between the lowest energy singlet and triplet electronic states of [NiFe]-hydrogenase active site model, using a velocity averaged Landau-Zener theory. Density functional and multireference perturbation theories were used to provide parameters for the Landau-Zener calculations. It was found that variation of the torsion angle between the terminal thiolate ligands around the Ni center induces an intersystem crossing between the lowest energy singlet and triplet electronic states in the bare active site and in the active site with bound H2. Potential energy curves between the singlet and triplet minima along the torsion angle and H2 binding energies to the two spin states were calculated. Upon H2 binding to the active site, there is a decrease in the torsion angle at the minimum energy crossing point between the singlet and triplet states. The probability of nonadiabatic transitions at temperatures between 270 and 370 K ranges from 35% to 32% for the active site with bound H2 and from 42% to 38% for the bare active site, thus indicating the importance of spin-forbidden nonadiabatic pathways for H2 binding on the [NiFe]-hydrogenase active site.

The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes.

PubMed

Batista, Ana P; Marreiros, Bruno C; Pereira, Manuela M

2013-05-01

We have recently investigated the long-recognized relationship between complex I and group 4 [NiFe] hydrogenases and we have established the so-called Energy-converting hydrogenase related (Ehr) complex as a new member of the family. We have also observed that four subunits, homologues to NuoB, D, H and L, are common to the members of the family. We have designated this common group of subunits the universal adaptor. Taking into account the similarity of the Na(+)/H(+) antiporter-like subunits of complex I (NuoL, NuoM and NuoN) and the unique structural characteristic of the long amphipathic α helix part of NuoL, the nature of the antiporter-like subunit of the universal adaptor was questioned. Thus, in this work we further explore the properties of the universal adaptor, investigating which antiporter-like subunit is part of the universal adaptor. We observe that the universal adaptor contains an antiporter-like subunit with a long amphipathic α helix, similar to NuoL. Consequently, the long helix is a common denominator that has been conserved in all members of the family. Such conservation surely reflects the key role of such helix in the energy transduction mechanism of this family of enzymes.

XAFS of short-lived reduction products of structural and functional models of the [Fe Fe] hydrogenase H-cluster

NASA Astrophysics Data System (ADS)

Bondin, Mark I.; Borg, Stacey J.; Cheah, Mun-Hon; Best, Stephen P.

2006-11-01

Thiolate-bridged diiron compounds that are related to the active site of the [Fe-Fe] hydrogenase enzyme have been shown to act as electrocatalysts for reduction of protons. The use of XAFS for clarification of the structures of intermediates formed following reduction of related diiron carbonyl compounds is described. These measurements allow the determination of Fe-Fe and Fe-S bond lengths with good reliability and when used in conjunction with the standard bonding models this provides a means of validating the structures proposed for longer-lived ( t>20 s at -50 °C) reaction intermediates.

Ligand Displacement Reaction Paths in a Diiron Hydrogenase Active Site Model Complex.

PubMed

Blank, Jan H; Moncho, Salvador; Lunsford, Allen M; Brothers, Edward N; Darensbourg, Marcetta Y; Bengali, Ashfaq A

2016-08-26

The mechanism and energetics of CO, 1-hexene, and 1-hexyne substitution from the complexes (SBenz)2 [Fe2 (CO)6 ] (SBenz=SCH2 Ph) (1-CO), (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexene)] (1-(η(2) -1-hexene)), and (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexyne)] (1-(η(2) -1-hexyne)) were studied by using time-resolved infrared spectroscopy. Exchange of both CO and 1-hexyne by P(OEt)3 and pyridine, respectively, proceeds by a bimolecular mechanism. As similar activation enthalpies are obtained for both reactions, the rate-determining step in both cases is assumed to be the rotation of the Fe(CO)2 L (L=CO or 1-hexyne) unit to accommodate the incoming ligand. The kinetic profile for the displacement of 1-hexene is quite different than that for the alkyne and, in this case, both reaction channels, that is, dissociative (SN 1) and associative (SN 2), were found to be competitive. Because DFT calculations predict similar binding enthalpies of alkene and alkyne to the iron center, the results indicate that the bimolecular pathway in the case of the alkyne is lower in free energy than that of the alkene. In complexes of this type, subtle changes in the departing ligand characteristics and the nature of the mercapto bridge can influence the exchange mechanism, such that more than one reaction pathway is available for ligand substitution. The difference between this and the analogous study of (μ-pdt)[Fe(CO)3 ]2 (pdt=S(CH2 )3 S) underscores the unique characteristics of a three-atom S-S linker in the active site of diiron hydrogenases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Redox reactions of [FeFe]-hydrogenase models containing an internal amine and a pendant phosphine.

PubMed

Zheng, Dehua; Wang, Mei; Chen, Lin; Wang, Ning; Sun, Licheng

2014-02-03

A diiron dithiolate complex with a pendant phosphine coordinated to one of the iron centers, [(μ-SCH2)2N(CH2C6H4-o-PPh2){Fe2(CO)5}] (1), was prepared and structurally characterized. The pendant phosphine is dissociated together with a CO ligand in the presence of excess PMe3, to afford [(μ-SCH2)2N(CH2C6H4-o-PPh2){Fe(CO)2(PMe3)}2] (2). Redox reactions of 2 and related complexes were studied in detail by in situ IR spectroscopy. A series of new Fe(II)Fe(I) ([3](+) and [6](+)), Fe(II)Fe(II) ([4](2+)), and Fe(I)Fe(I) (5) complexes relevant to Hox, Hox(CO), and Hred states of the [FeFe]-hydrogenase active site were detected. Among these complexes, the molecular structures of the diferrous complex [4](2+) with the internal amine and the pendant phosphine co-coordinated to the same iron center and the triphosphine diiron complex 5 were determined by X-ray crystallography. To make a comparison, the redox reactions of an analogous complex, [(μ-SCH2)2N(CH2C6H5){Fe(CO)2(PMe3)}2] (7), were also investigated by in situ IR spectroscopy in the absence or presence of extrinsic PPh3, which has no influence on the oxidation reaction of 7. The pendant phosphine in the second coordination sphere makes the redox reaction of 2 different from that of its analogue 7.

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

PubMed Central

Lamkanfi, Mohamed; Mueller, James L.; Vitari, Alberto C.; Misaghi, Shahram; Fedorova, Anna; Deshayes, Kurt; Lee, Wyne P.; Hoffman, Hal M.

2009-01-01

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)–sensitive K+ (KATP) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking KATP subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1β secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome–associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1β secretion. PMID:19805629

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome.

PubMed

Lamkanfi, Mohamed; Mueller, James L; Vitari, Alberto C; Misaghi, Shahram; Fedorova, Anna; Deshayes, Kurt; Lee, Wyne P; Hoffman, Hal M; Dixit, Vishva M

2009-10-05

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1beta (IL-1beta) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)-sensitive K(+) (K(ATP)) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking K(ATP) subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1beta secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome-associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1beta secretion.

Inhibition of polyomavirus ori-dependent DNA replication by mSin3B.

PubMed

Xie, An-Yong; Folk, William R

2002-12-01

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.

Novel reactions of homodinuclear Ni2 complexes [Ni(RNPyS4)]2 with Fe3(CO)12 to give heterotrinuclear NiFe2 and mononuclear Fe complexes relevant to [NiFe]- and [Fe]-hydrogenases.

PubMed

Song, Li-Cheng; Cao, Meng; Wang, Yong-Xiang

2015-04-21

The homodinuclear complexes [Ni(RNPyS4)]2 (; RNPyS4 = 2,6-bis(2-mercaptophenylthiomethyl)-4-R-pyridine; R = H, MeO, Cl, Br, i-Pr) were found to be prepared by reactions of the in situ generated Li2[Ni(1,2-S2C6H4)2] with 2,6-bis[(tosyloxy)methyl]pyridine and its substituted derivatives 2,6-bis[(tosyloxy)methyl]-4-R-pyridine. Further reactions of with Fe3(CO)12 gave both heterotrinuclear complexes NiFe2(RNPyS4)(CO)5 () and mononuclear complexes Fe(RNPyS4)(CO) (), unexpectedly. Interestingly, complexes and could be regarded as models for the active sites of [NiFe]- and [Fe]-hydrogenases, respectively. All the prepared complexes were characterized by elemental analysis, spectroscopy, and particularly for some of them, by X-ray crystallography. In addition, the electrochemical properties of and as well as the electrocatalytic H2 production catalyzed by and were investigated by CV techniques.

An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

PubMed Central

Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A. A.; Baumann, Marcus E. M.; Selmer, Thorsten

2013-01-01

Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the

STAT3 inhibition as a therapeutic strategy for leukemia.

PubMed

Kanna, Rubashruti; Choudhary, Gaurav; Ramachandra, Nandini; Steidl, Ulrich; Verma, Amit; Shastri, Aditi

2017-11-22

Leukemia is characterized by selective overgrowth of malignant hematopoietic stem cells (HSC's) that interfere with HSC differentiation. Cytoreductive chemotherapy can kill rapidly dividing cancerous cells but cannot eradicate the malignant HSC pool leading to relapses. Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. STAT3 is an important transcription factor that regulates cell growth, proliferation, and inhibits apoptosis. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. Multiple strategies for interfering with STAT3 activity in leukemic cells include inhibition of STAT3 phosphorylation, interfering with STAT3 interactions, preventing nuclear transfer, inhibiting transcription and causing interference in STAT: DNA binding. A better understanding of key interactions and upstream mediators of STAT3 activity will help facilitate the development of effective cancer therapies and may result in durable remissions.

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

PubMed

Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A; Lenz, Oliver

2011-01-01

The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H+/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K(I) values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

PubMed

McTernan, Patrick M; Chandrayan, Sanjeev K; Wu, Chang-Hao; Vaccaro, Brian J; Lancaster, W Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L; Tainer, John A; Adams, Michael W W

2014-07-11

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Influence of calcium ions on the crystallization of sodium bicarbonate

NASA Astrophysics Data System (ADS)

Zhu, Yi; Demilie, Paul; Davoine, Perrine; Cartage, Thierry; Delplancke-Ogletree, Marie-Paule

2005-02-01

In industrial crystallization of sodium bicarbonate (sodium hydrogenocarbonate), the presence of calcium ions in solutions is unavoidable due to the production process. The understanding of the Ca 2+ role in NaHCO 3 crystallization would be helpful for improving the quality of the final products. The influence of calcium ions on NaHCO 3 crystallization was investigated in a 5-l mixed suspension mixed product removal crystallizer under controlled conditions. A density meter was used for continuous supersaturation monitoring. After a steady state had been reached, different CaCl 2 amounts were added at a constant flow rate. It was found that limited calcium ion levels in the system reduce drastically the nucleation frequency of NaHCO 3 and has a limited influence on crystal growth rate. The supersaturation measurements and other methods confirmed this phenomenon. The relationship between the Ca 2+ influence on NaHCO 3 crystallization, the calcium carbonate solubility and its metastable zone in concentrated NaHCO 3 solution was established. In fact, Ca 2+ has a maximum effect on NaHCO 3 crystallization kinetics when the saturation of calcium carbonate in NaHCO 3 solution has been reached, and the effect is constant in the metastable zone. The excess of Ca 2+ precipitates in NaHCO 3 solution as CaCO 3, as observed by energy dispersive X-ray and X-ray diffraction. This explained why an increasing Ca 2+ concentration in the solution has a limited influence on NaHCO 3 crystal size distribution and habit, but decreases the crystal purity. It is also confirmed that an impurity as Ca 2+ has no influence on the equilibrium NaHCO 3-Na 2CO 3.

Direct Comparison of the Performance of a Bio-inspired Synthetic Nickel Catalyst and a [NiFe]-Hydrogenase, Both Covalently Attached to Electrodes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Macia, Patricia; Dutta, Arnab; Lubitz, Wolfgang

2015-10-12

The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we’ve come in mimicking the impressive enzymatic performance. Here we directly compare the catalytic properties of the [Ni(PCy2NGly2)2]2+ complex with the [NiFe]-hydrogenase from Desulfobivrio vulgaris Miyazaki F (DvMF) immobilized to a functionalized electrode under identical conditions. At pH=7, the enzyme has higher performance in both activity and overpotential, and is more stable, whilemore » at low pH, the molecular catalyst outperforms the enzyme in all respects. The Ni complex also has increased tolerance to CO. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bioinspired complexes in fuel cells. AD and WJS acknowledge the Office of Science Early Career Research Program through the US Department of Energy (US DOE), Office of Science, Office of Basic Energy Sciences (BES), and Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the US DOE.« less

Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.

PubMed

Huggenberger, Reto; Ullmann, Stefan; Proulx, Steven T; Pytowski, Bronislaw; Alitalo, Kari; Detmar, Michael

2010-09-27

The role of lymphangiogenesis in inflammation has remained unclear. To investigate the role of lymphatic versus blood vasculature in chronic skin inflammation, we inhibited vascular endothelial growth factor (VEGF) receptor (VEGFR) signaling by function-blocking antibodies in the established keratin 14 (K14)-VEGF-A transgenic (Tg) mouse model of chronic cutaneous inflammation. Although treatment with an anti-VEGFR-2 antibody inhibited skin inflammation, epidermal hyperplasia, inflammatory infiltration, and angiogenesis, systemic inhibition of VEGFR-3, surprisingly, increased inflammatory edema formation and inflammatory cell accumulation despite inhibition of lymphangiogenesis. Importantly, chronic Tg delivery of the lymphangiogenic factor VEGF-C to the skin of K14-VEGF-A mice completely inhibited development of chronic skin inflammation, epidermal hyperplasia and abnormal differentiation, and accumulation of CD8 T cells. Similar results were found after Tg delivery of mouse VEGF-D that only activates VEGFR-3 but not VEGFR-2. Moreover, intracutaneous injection of recombinant VEGF-C156S, which only activates VEGFR-3, significantly reduced inflammation. Although lymphatic drainage was inhibited in chronic skin inflammation, it was enhanced by Tg VEGF-C delivery. Together, these results reveal an unanticipated active role of lymphatic vessels in controlling chronic inflammation. Stimulation of functional lymphangiogenesis via VEGFR-3, in addition to antiangiogenic therapy, might therefore serve as a novel strategy to treat chronic inflammatory disorders of the skin and possibly also other organs.

Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

PubMed

Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

2004-06-01

There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

Response to Long-Term NaHCO3-Derived Alkalinity in Model Lotus japonicus Ecotypes Gifu B-129 and Miyakojima MG-20: Transcriptomic Profiling and Physiological Characterization

PubMed Central

Rocco, Rubén; Bordenave, Cesar D.; Escaray, Francisco J.; Antonelli, Cristian; Calzadilla, Pablo; Gárriz, Andrés; Serna, Eva; Carrasco, Pedro; Menendez, Ana B.

2014-01-01

The current knowledge regarding transcriptomic changes induced by alkalinity on plants is scarce and limited to studies where plants were subjected to the alkaline salt for periods not longer than 48 h, so there is no information available regarding the regulation of genes involved in the generation of a new homeostatic cellular condition after long-term alkaline stress. Lotus japonicus is a model legume broadly used to study many important physiological processes including biotic interactions and biotic and abiotic stresses. In the present study, we characterized phenotipically the response to alkaline stress of the most widely used L. japonicus ecotypes, Gifu B-129 and MG-20, and analyzed global transcriptome of plants subjected to 10 mM NaHCO3 during 21 days, by using the Affymetrix Lotus japonicus GeneChip®. Plant growth assessment, gas exchange parameters, chlorophyll a fluorescence transient (OJIP) analysis and metal accumulation supported the notion that MG-20 plants displayed a higher tolerance level to alkaline stress than Gifu B-129. Overall, 407 and 459 probe sets were regulated in MG-20 and Gifu B-129, respectively. The number of probe sets differentially expressed in roots was higher than that of shoots, regardless the ecotype. Gifu B-129 and MG-20 also differed in their regulation of genes that could play important roles in the generation of a new Fe/Zn homeostatic cellular condition, synthesis of plant compounds involved in stress response, protein-degradation, damage repair and root senescence, as well as in glycolysis, gluconeogenesis and TCA. In addition, there were differences between both ecotypes in the expression patterns of putative transcription factors that could determine distinct arrangements of flavonoid and isoflavonoid compounds. Our results provided a set of selected, differentially expressed genes deserving further investigation and suggested that the L. japonicus ecotypes could constitute a useful model to search for common and

Tamoxifen Inhibition of Kv7.2/Kv7.3 Channels

PubMed Central

Ferrer, Tania; Aréchiga-Figueroa, Ivan Arael; Shapiro, Mark S.; Tristani-Firouzi, Martin; Sanchez-Chapula, José A.

2013-01-01

KCNQ genes encode five Kv7 K+ channel subunits (Kv7.1–Kv7.5). Four of these (Kv7.2–Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels. PMID:24086693

Tamoxifen inhibition of kv7.2/kv7.3 channels.

PubMed

Ferrer, Tania; Aréchiga-Figueroa, Ivan Arael; Shapiro, Mark S; Tristani-Firouzi, Martin; Sanchez-Chapula, José A

2013-01-01

KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels.

Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmad, Israr; Muneer, Kashiff M.; Tamimi, Iman A.

2013-07-01

The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1β and IL-18 secretion. The NLRP3 (NACHT, LRR, and pyrin domain-containing protein 3) inflammasome is constitutively assembled and activated in human melanoma cells. We have examined the inhibitory effect of thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone), a major ingredient of black seed obtained from the plant Nigella sativa on metastatic human (A375) and mouse (B16F10) melanoma cell lines. We have assessed whether thymoquinone inhibits metastasis of melanoma cells by targeting NLRP3 subunit of inflammasomes. Using an in vitro cell migration assay, we found that thymoquinone inhibited the migration of both humanmore » and mouse melanoma cells. The inhibitory effect of thymoquinone on metastasis was also observed in vivo in B16F10 mouse melanoma model. The inhibition of migration of melanoma cells by thymoquinone was accompanied by a decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by thymoquinone resulted in inhibition of IL-1β and IL-18. Treatment of mouse melanoma cells with thymoquinone also inhibited NF-κB activity. Furthermore, inhibition of reactive oxygen species (ROS) by thymoquinone resulted in partial inactivation of NLRP3 inflammasome. Thus, thymoquinone exerts its inhibitory effect on migration of human and mouse melanoma cells by inhibition of NLRP3 inflammasome. Thus, our results indicate that thymoquinone can be a potential immunotherapeutic agent not only as an adjuvant therapy for melanoma, but also, in the control and prevention of metastatic melanoma. - Highlights: • Thymoquinone causes inhibition of migration of melanoma cells. • Thymoquinone causes inhibition of metastasis in vivo. • Thymoquinone causes inhibition of migration by activation of NLRP3 inflammasome.« less

Glycogen Synthase Kinase-3 (GSK3) Inhibition Induces Prosurvival Autophagic Signals in Human Pancreatic Cancer Cells*

PubMed Central

Marchand, Benoît; Arsenault, Dominique; Raymond-Fleury, Alexandre; Boisvert, François-Michel; Boucher, Marie-Josée

2015-01-01

Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation. PMID:25561726

Sodium bicarbonate use and the risk of hypernatremia in thoracic aortic surgical patients with metabolic acidosis following deep hypothermic circulatory arrest

PubMed Central

Ghadimi, Kamrouz; Gutsche, Jacob T.; Ramakrishna, Harish; Setegne, Samuel L.; Jackson, Kirk R.; Augoustides, John G.; Ochroch, E. Andrew; Weiss, Stuart J.; Bavaria, Joseph E.; Cheung, Albert T.

2016-01-01

Objective: Metabolic acidosis after deep hypothermic circulatory arrest (DHCA) for thoracic aortic operations is commonly managed with sodium bicarbonate (NaHCO3). The purpose of this study was to determine the relationships between total NaHCO3 dose and the severity of metabolic acidosis, duration of mechanical ventilation, duration of vasoactive infusions, and Intensive Care Unit (ICU) or hospital length of stay (LOS). Methods: In a single center, retrospective study, 87 consecutive elective thoracic aortic operations utilizing DHCA, were studied. Linear regression analysis was used to test for the relationships between the total NaHCO3 dose administered through postoperative day 2, clinical variables, arterial blood gas values, and short-term clinical outcomes. Results: Seventy-five patients (86%) received NaHCO3. Total NaHCO3 dose averaged 136 ± 112 mEq (range: 0.0–535 mEq) per patient. Total NaHCO3 dose correlated with minimum pH (r = 0.41, P < 0.0001), minimum serum bicarbonate (r = −0.40, P < 0.001), maximum serum lactate (r = 0.46, P = 0.007), duration of metabolic acidosis (r = 0.33, P = 0.002), and maximum serum sodium concentrations (r = 0.29, P = 0.007). Postoperative hypernatremia was present in 67% of patients and peaked at 12 h following DHCA. Eight percent of patients had a serum sodium ≥ 150 mEq/L. Total NaHCO3 dose did not correlate with anion gap, serum chloride, not the duration of mechanical ventilator support, vasoactive infusions, ICU or hospital LOS. Conclusion: Routine administration of NaHCO3 was common for the management of metabolic acidosis after DHCA. Total dose of NaHCO3 was a function of the severity and duration of metabolic acidosis. NaHCO3 administration contributed to postoperative hypernatremia that was often severe. The total NaHCO3 dose administered was unrelated to short-term clinical outcomes. PMID:27397449

Inhibition of GSK3 dependent tau phosphorylation by metals.

PubMed

Gómez-Ramos, Alberto; Domínguez, Jorge; Zafra, Delia; Corominola, Helena; Gomis, Ramon; Guinovart, Joan J; Avila, Jesús

2006-04-01

One of the main pathological characteristics of Alzheimer's disease is the presence in the brain of the patients of an aberrant structure, the paired helical filaments, composed of hyperphosphorylated tau. The level of tau phosphorylation has been correlated with the capacity for tau aggregation. Thus, the mechanism for tau phosphorylation could be important to clarify those pathological features in Alzheimer's disease. Tau protein could be modified by different kinases, being GSK3 the one that could modify more sites of that protein. GSK3 activity could be modulate by the presence of metals like magnesium that can be required for the proper function of the kinase, whereas, metals like manganesum or lithium inhibit the activity of the kinase. Many works have been done to study the inhibition of GSK3 by lithium, a specific inhibitor of that kinase. More recently, it has been indicated that sodium tungstate could also inhibit GSK3 through a different mechanism. In this review, we discuss the effect of these two metals, lithium and tungstate, on GSK3 (or tau I kinase) activity.

Mineral intake independent from gastric irritation or pica by cell-dehydrated rats.

PubMed

Constancio, Juliana; Pereira-Derderian, Daniela T B; Menani, José V; De Luca, Laurival A

2011-10-24

Gavage of 2 M NaCl (IG 2 M NaCl), a procedure to induce cell-dehydration-and water and 0.15 M NaCl intake in a two-bottle choice test-is also a potential gastric irritant. In this study, we assessed whether mineral intake induced by IG 2 M NaCl is associated with gastric irritation or production of pica in the rat. We first determined the amount of mineral solution (0.15 M NaCl, 0.15 M NaHCO3, 0.01 M KCl and 0.05 mM CaCl2) and water ingested in response to IG 2 M NaCl in a five-bottle test. Then, we used mineral solutions (0.01 M KCl and 0.15 M NaHCO3), whose intakes were significantly increased compared to controls, and water in three-bottle tests to test the gastric irritation hypothesis. The IG 2 M NaCl induced KCl and NaHCO3 intake that was not inhibited by gavage with gastric protectors Al(OH)3 or NaHCO3. IG 2 M NaCl or gavage of 0.6 N acetic acid induced mild irritation, hyperemia, of the glandular part of the stomach. A gavage of 50% ethanol induced strong irritation seen as pinpoint ulcerations. Neither ethanol nor acetic acid induced any fluid intake. Neither IG 2 M NaCl nor acetic acid induced kaolin intake, a marker of pica in laboratory rats. Ethanol did induce kaolin intake. These results suggest that IG 2 M NaCl induced a mineral fluid intake not selective for sodium and independent from gastric irritation or pica. Copyright © 2011 Elsevier Inc. All rights reserved.

Inhibition of Interferon Regulatory Factor 3 Activation by Paramyxovirus V Protein

PubMed Central

Irie, Takashi; Kiyotani, Katsuhiro; Igarashi, Tomoki; Yoshida, Asuka

2012-01-01

The V protein of Sendai virus (SeV) suppresses innate immunity, resulting in enhancement of viral growth in mouse lungs and viral pathogenicity. The innate immunity restricted by the V protein is induced through activation of interferon regulatory factor 3 (IRF3). The V protein has been shown to interact with melanoma differentiation-associated gene 5 (MDA5) and to inhibit beta interferon production. In the present study, we infected MDA5-knockout mice with V-deficient SeV and found that MDA5 was largely unrelated to the innate immunity that the V protein suppresses in vivo. We therefore investigated the target of the SeV V protein. We previously reported interaction of the V protein with IRF3. Here we extended the observation and showed that the V protein appeared to inhibit translocation of IRF3 into the nucleus. We also found that the V protein inhibited IRF3 activation when induced by a constitutive active form of IRF3. The V proteins of measles virus and Newcastle disease virus inhibited IRF3 transcriptional activation, as did the V protein of SeV, while the V proteins of mumps virus and Nipah virus did not, and inhibition by these proteins correlated with interaction of each V protein with IRF3. These results indicate that IRF3 is important as an alternative target of paramyxovirus V proteins. PMID:22532687

X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli

PubMed Central

Volbeda, Anne; Amara, Patricia; Darnault, Claudine; Mouesca, Jean-Marie; Parkin, Alison; Roessler, Maxie M.; Armstrong, Fraser A.; Fontecilla-Camps, Juan C.

2012-01-01

The crystal structure of the membrane-bound O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H2-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O2-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oϵ of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H2-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O2 to H2O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O2 reduction, located near the active site. PMID:22431599

Monomethylarsonous acid (MMA+3) Inhibits IL-7 Signaling in Mouse Pre-B Cells

PubMed Central

Ezeh, Peace C.; Xu, Huan; Lauer, Fredine T.; Liu, Ke Jian; Hudson, Laurie G.; Burchiel, Scott W.

2016-01-01

Our previously published data show that As+3 in vivo and in vitro, at very low concentrations, inhibits lymphoid, but not myeloid stem cell development in mouse bone marrow. We also showed that the As+3 metabolite, monomethylarsonous acid (MMA+3), was responsible for the observed pre-B cell toxicity caused by As+3. Interleukin-7 (IL-7) is the primary growth factor responsible for pre-lymphoid development in mouse and human bone marrow, and Signal Transducer and Activator of Transcription 5 (STAT5) is a transcriptional factor in the IL-7 signaling pathway. We found that MMA+3 inhibited STAT5 phosphorylation at a concentration as low as 50 nM in mouse bone marrow pre-B cells. Inhibition of STAT5 phosphorylation by As+3 occurred only at a concentration of 500 nM. In the IL-7 dependent mouse pre-B 2E8 cell line, we also found selective inhibition of STAT5 phosphorylation by MMA+3, and this inhibition was dependent on effects on JAK3 phosphorylation. IL-7 receptor expression on 2E8 cell surface was also suppressed by 50 nM MMA+3 at 18 h. As further evidence for the inhibition of STAT5, we found that the induction of several genes required in B cell development, cyclin D1, E2A, EBF1, and PAX5, were selectively inhibited by MMA+3. Since 2E8 cells lack the enzymes responsible for the conversion of As+3 to MMA+3 in vitro, the results of these studies suggest that As+3 induced inhibition of pre-B cell formation in vivo is likely dependent on the formation of MMA+3 which in turn inhibits IL-7 signaling at several steps in mouse pre-B cells. PMID:26518055

Advances in the Function and Regulation of Hydrogenase in the Cyanobacterium Synechocystis PCC6803

PubMed Central

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2014-01-01

In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H2 production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production. PMID:25365180

Inhibition of APOBEC3G Activity Impedes Double-Strand DNA Repair

PubMed Central

Prabhu, Ponnandy; Shandilya, Shivender; Britan-Rosich, Elena; Nagler, Adi; Schiffer, Celia A.; Kotler, Moshe

2015-01-01

The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in dsDNA damage, such as ionizing irradiation (IR) and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases sensitivity of lymphoma cells to IR. In the current study, we show that additional peptides derived from Vif, A3G and A3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, while, replacing a single amino acid in the LYYF motif completely abrogate inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break (DSB) repair after radiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit DSB repair halts their propagation. These results suggest that A3G may be a potential therapeutic target amenable to peptide and peptidomimetic inhibition. PMID:26460502

Inhibition of APOBEC3G activity impedes double-stranded DNA repair.

PubMed

Prabhu, Ponnandy; Shandilya, Shivender M D; Britan-Rosich, Elena; Nagler, Adi; Schiffer, Celia A; Kotler, Moshe

2016-01-01

The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to ionizing radiation. In the current study, we show that additional peptides derived from Vif, A3G, and APOBEC3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, whereas replacing a single residue in the LYYF motif completely abrogates inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break repair after irradiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit double-strand break repair halts their propagation. These results suggest that A3G may be a potential therapeutic target that is amenable to peptide and peptidomimetic inhibition. © 2015 FEBS.

Momilactone B Inhibits Ketosis In Vitro by Regulating the ANGPTL3-LPL Pathway and Inhibiting HMGCS2.

PubMed

Kang, Dong Young; S P, Nipin; Darvin, Pramod; Joung, Youn Hee; Byun, Hyo Joo; Do, Chang Hee; Park, Kyung Do; Park, Mi Na; Cho, Kwang Hyun; Yang, Young Mok

2017-07-03

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.

Inhibition of glycogen synthase kinase 3beta during heart failure is protective.

PubMed

Hirotani, Shinichi; Zhai, Peiyong; Tomita, Hideharu; Galeotti, Jonathan; Marquez, Juan Pablo; Gao, Shumin; Hong, Chull; Yatani, Atsuko; Avila, Jesús; Sadoshima, Junichi

2007-11-26

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus

Biomimetics of [NiFe]-Hydrogenase: Nickel- or Iron-Centered Proton Reduction Catalysis?

PubMed

Tang, Hao; Hall, Michael B

2017-12-13

The [NiFe] hydrogenase (H2ase) has been characterized in the Ni-R state with a hydride bridging between Fe and Ni but displaced toward the Ni. In nearly all of the synthetic Ni-R models reported so far, the hydride ligand is either displaced toward Fe, or terminally bound to Fe. Recently, a structural and functional [NiFe]-H2ase mimic ( Nat. Chem. 2016 , 8 , 1054 - 1060 ) was reported to produce H 2 catalytically via EECC mechanism through a Ni-centered hydride intermediate like the enzyme. Here, a comprehensive DFT study shows a much lower energy route via an E[ECEC] mechanism through an Fe-centered hydride intermediate. Although catalytic H 2 production occurs at the potential corresponding to the complex's second reduction, a third electron is needed to induce the second proton addition from the weak acid. The first two-electron reductions and a proton addition produce a semibridging hydride with a short Fe-H bond like other structured [NiFe]-biomimetics, but this species is not basic enough to add another proton from the weak acid without the third electron. The calculated mechanism provides insight into the origin of this structure in the enzyme.

Sodium bicarbonate secretion indicated by ultrastructural cytochemical localization of HCO3(-), Cl-, and Na+ ions on rat bile duct brush cells.

PubMed

Ogata, Takuro

2005-12-01

Brush cells are widely distributed in the digestive and respiratory apparatus, but their function is still unknown. Because brush cells (BC) are found in organs secreting NaHCO3, it was hypothesized that these cells may secrete NaHCO3. To test this possibility, rat common bile duct epithelia were examined by ultrastructural cytochemical methods for localizing HCO3(-), Cl-, and Na+ ions. All three ion precipitates were few in or on BCs of rats without stimulation. Lead carbonate precipitates, which localized HCO3(-) ions by the lead nitrate-osmium method, increased markedly on the surface of the microvilli (MV) of BCs after secretin or meal stimulation, but similar precipitates were few on the luminal surface of principal cells (PCs). Silver chloride precipitates, which indicate the presence of Cl- ions by the silver-osmium method, increased in the apical cytoplasm and in MV of BCs after secretin or meal stimulation, but they were few in PCs. Sodium pyroantimonate precipitates, which localize Na+ ions by the potassium pyroantimonate-osmium method, increased on the surface of the MV, along the basolateral membrane, and in the apical cytoplasm of BCs after secretin or meal stimulation, but they were few in PCs. These results strongly suggest that BCs may be a significant source of NaHCO3 secretion.

Monomethylarsonous acid (MMA+3) Inhibits IL-7 Signaling in Mouse Pre-B Cells.

PubMed

Ezeh, Peace C; Xu, Huan; Lauer, Fredine T; Liu, Ke Jian; Hudson, Laurie G; Burchiel, Scott W

2016-02-01

Our previously published data show that As(+3) in vivo and in vitro, at very low concentrations, inhibits lymphoid, but not myeloid stem cell development in mouse bone marrow. We also showed that the As(+3) metabolite, monomethylarsonous acid (MMA(+3)), was responsible for the observed pre-B cell toxicity caused by As(+3). Interleukin-7 (IL-7) is the primary growth factor responsible for pre-lymphoid development in mouse and human bone marrow, and Signal Transducer and Activator of Transcription 5 (STAT5) is a transcriptional factor in the IL-7 signaling pathway. We found that MMA(+3) inhibited STAT5 phosphorylation at a concentration as low as 50 nM in mouse bone marrow pre-B cells. Inhibition of STAT5 phosphorylation by As(+3) occurred only at a concentration of 500 nM. In the IL-7 dependent mouse pre-B 2E8 cell line, we also found selective inhibition of STAT5 phosphorylation by MMA(+3), and this inhibition was dependent on effects on JAK3 phosphorylation. IL-7 receptor expression on 2E8 cell surface was also suppressed by 50 nM MMA(+3) at 18 h. As further evidence for the inhibition of STAT5, we found that the induction of several genes required in B cell development, cyclin D1, E2A, EBF1, and PAX5, were selectively inhibited by MMA(+3). Since 2E8 cells lack the enzymes responsible for the conversion of As(+3) to MMA(+3) in vitro, the results of these studies suggest that As(+3) induced inhibition of pre-B cell formation in vivo is likely dependent on the formation of MMA(+3) which in turn inhibits IL-7 signaling at several steps in mouse pre-B cells. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Class IA PI3K inhibition inhibits cell growth and proliferation in mantle cell lymphoma.

PubMed

Tabe, Yoko; Jin, Linhua; Konopleva, Marina; Shikami, Masato; Kimura, Shinya; Andreeff, Michael; Raffeld, Mark; Miida, Takashi

2014-01-01

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway preferentially occurs in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of this disease. In this study, we investigated the role of PI3K isoforms on proliferation of aggressive MCL cells. The changes in cell viability, cell cycle distribution and apoptosis induction by the PI3K isoform-selective inhibitors were evaluated. The molecular basis underlying the effects of the specific inhibition of PI3K isoforms was investigated by Western blot analysis. Our results demonstrated that a class IA PI3K isoform is most commonly involved in the constitutive activation of Akt in aggressive MCL. Treatment with a p110α isoform-specific inhibitor induced prominent cell cycle arrest followed by apoptosis through complete abolishment of phosphorylated (p)-Akt and its downstream targets. An inhibitor of isoform p110δ induced moderate cell cycle arrest with downregulation of p-Akt and p-S6K. A dual inhibitor of p110α and p110δ GDC-0941 caused more prominent cell growth inhibition compared to selective p110α or p110δ inhibitors. Inhibition of the class IB PI3K isoform p110γ did not cause cell cycle arrest or induce apoptosis in MCL cells. These findings suggest that the therapeutic ablation of class IA PI3K may be a promising strategy for the treatment of refractory, aggressive MCL. Copyright © 2013 S. Karger AG, Basel.

An iodocyclization approach to substituted 3-iodothiophenes.

PubMed

Gabriele, Bartolo; Mancuso, Raffaella; Salerno, Giuseppe; Larock, Richard C

2012-09-07

A novel approach to 3-iodothiophenes by direct iodocyclization of alkynylthiol derivatives is presented. A variety of 1-mercapto-3-yn-2-ols 5 (readily available from alkynylation of the corresponding alpha-mercapto ketones or alpha-mercapto esters) were smoothly converted into the corresponding 3-iodothiophene derivatives 6 in good yields by reaction with molecular iodine in the presence of NaHCO(3) at room temperature in MeCN as the solvent.

N2 and P3 modulation during partial inhibition in a modified go/nogo task.

PubMed

Nguyen, An T; Moyle, Jonson J; Fox, Allison M

2016-09-01

The neural response following the partial inhibition of responses can provide insight into the processes underlying response inhibition. We examined the N2 and P3 on trials where participants correctly responded to go stimuli, successfully inhibited their response to nogo stimuli, and nogo trials where they initiated but did not complete their response (partial inhibitions) in an adult sample (N=24, M(age)=21.17, SD(age)=3.52). An enhanced and delayed N2 was observed on partially inhibited compared to successfully inhibited nogo trials. Further analysis showed that this modulation was error-related. An enhanced central P3 was observed following successful inhibitions compared to correct go trials, but not following partial inhibitions. The results suggest that the central P3 enhancement is specific to the complete and successful inhibition of responses. Therefore, the absence of a central P3 on partial inhibitions could reflect insufficient inhibition or a monitored failure in inhibiting the response. Although, our findings provide support for the role of P3 in response inhibition, it raises questions about the processes involved in the subsequent inhibition or correction of the erroneous response. Further research examining the neural response following both partial and unsuccessful inhibitions could provide insight regarding these processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Dimethyl sulfoxide inhibits NLRP3 inflammasome activation.

PubMed

Ahn, Huijeong; Kim, Jeeyoung; Jeung, Eui-Bae; Lee, Geun-Shik

2014-04-01

Dimethyl sulfoxide (DMSO) is an amphipathic molecule that is commonly/widely used as a solvent for biological compounds. In addition, DMSO has been studied as a medication for the treatment of inflammation, cystitis, and arthritis. Based on the anti-inflammatory characteristics of DMSO, we elucidated the effects of DMSO on activation of inflammasomes, which are cytoplasmic multi-protein complexes that mediate the maturation of interleukin (IL)-1β by activating caspase-1 (Casp1). In the present study, we prove that DMSO attenuated IL-1β maturation, Casp1 activity, and ASC pyroptosome formation via NLRP3 inflammasome activators. Further, NLRC4 and AIM2 inflammasome activity were not affected, suggesting that DMSO is a selective inhibitor of the NLRP3 inflammasomes. The anti-inflammatory effect of DMSO was further confirmed in animal, LPS-endotoxin sepsis and inflammatory bowel disease models. In addition, DMSO inhibited LPS-mediating IL-1s transcription. Taken together, DMSO shows anti-inflammatory characteristics, attenuates NLRP3 inflammasome activation, and mediates inhibition of IL-1s transcription. Crown Copyright © 2013. Published by Elsevier GmbH. All rights reserved.

Naloxone inhibits nod-like receptor protein 3 inflammasome.

PubMed

Lin, Han-Yu; Chang, Ya-Ying; Kao, Ming-Chang; Huang, Chun-Jen

2017-11-01

Naloxone, an opioid receptor antagonist, possesses potent anti-inflammation effects. We previously confirmed the effects of naloxone on inhibiting upregulation of inflammatory cytokine interleukin-1β (IL-1β). Production of mature form IL-1β is mediated by the nod-like receptor protein 3 (NLRP3) inflammasome, a multiprotein complex composed of NLRP3, and the adaptor protein apoptosis-associated speck-like protein contains a caspase recruitment domain (ASC). We elucidated whether naloxone could inhibit the activation of NLRP3 inflammasome. To induce IL-1β production and NLRP3 inflammasome activation, the human monocytic leukemia cell line THP-1 cells were first primed with lipopolysaccharide (LPS, 1 μg/mL) and then activated with adenosine triphosphate (ATP, 1 mM). For NLRP3 transcription, THP-1 cells were only treated with LPS priming. Enzyme-link immunosorbent assay data revealed that the concentration of IL-1β in THP-1 cells treated with LPS plus ATP was significantly higher than that in THP-1 cells treated with LPS plus ATP plus naloxone (0.1 μM) (P < 0.001). Real-time quantitative reverse transcription and polymerase chain reaction data also revealed that NLRP3 mRNA concentration in THP-1 cells treated with LPS was significantly higher than that in THP-1 cells treated with LPS plus naloxone (P = 0.001). ASC speck formation, that is, ASC assembles into a large protein complex, is an indicator for NLRP3 inflammasome activation. Our data revealed that the percentage of cells containing ASC specks in THP-1 cells treated with LPS plus ATP was also significantly higher than that in THP-1 cells treated with LPS plus ATP plus naloxone (P < 0.001). Naloxone inhibits NLRP3 inflammasome activation. Copyright © 2017 Elsevier Inc. All rights reserved.

Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer

PubMed Central

Roper, Jatin; Sinnamon, Mark J.; Coffee, Erin M.; Belmont, Peter; Keung, Lily; Georgeon-Richard, Larissa; Wang, Wei Vivian; Faber, Anthony C.; Yun, Jihye; Yilmaz, Omer H.; Bronson, Roderick T.; Martin, Eric S.; Tsichlis, Philip N.; Hung, Kenneth E.

2014-01-01

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer. PMID:24576621

Stat3 inhibition activates tumor macrophages and abrogates glioma growth in mice.

PubMed

Zhang, Leying; Alizadeh, Darya; Van Handel, Michelle; Kortylewski, Marcin; Yu, Hua; Badie, Behnam

2009-10-01

As the main effector-cell population of the central nervous system, microglia (MG) are considered to play an important immunoregulatory function in a number of pathological conditions such as inflammation, trauma, degenerative disease, and brain tumors. Recent studies, however, have suggested that the anti-neoplastic function of MG may be suppressed in malignant brain tumors. Considering the proposed suppressive role of signal transducers and activators of transcription 3 (Stat3) in antitumor immunity, we evaluated the role of Stat3 inhibition on MG and macrophage (MP) activation and tumor growth in a murine glioma model. N9 MG cells were exposed to GL261 glioma conditioned medium (GL261-CM) and evaluated for Stat3 activity and cytokine expression. Furthermore, the role of Stat3 inhibition on MG and MP activation was studied both in vitro and in vivo. Finally, the effect of Stat3 inhibition on tumor growth was assessed in intracranial GL261 gliomas. GL261-CM increased Stat3 activity in N9 cells in vitro and resulted in overexpression of IL-10 and IL-6, and downregulation of IL1-beta, a pro-inflammatory cytokine. Inhibition of Stat3 by CPA-7 or siRNA reversed glioma-induced cytokine expression profile in N9 cells. Furthermore, inactivation of Stat3 in intracranial GL261 tumors by siRNA resulted in MG/MP activation and tumor growth inhibition. Glioma-induced MG and MP suppression may be mediated thorough Stat3. Inhibition of Stat3 function in tumor MG/MP may result in their activation and can potentially be used as an adjunct immunotherapy approach for gliomas.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

PubMed

Marklein, Diana; Graab, Ulrike; Naumann, Ivonne; Yan, Tiandong; Ridzewski, Rosalie; Nitzki, Frauke; Rosenberger, Albert; Dittmann, Kai; Wienands, Jürgen; Wojnowski, Leszek; Fulda, Simone; Hahn, Heidi

2012-01-01

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

PubMed Central

Marklein, Diana; Graab, Ulrike; Naumann, Ivonne; Yan, Tiandong; Ridzewski, Rosalie; Nitzki, Frauke; Rosenberger, Albert; Dittmann, Kai; Wienands, Jürgen; Wojnowski, Leszek; Fulda, Simone; Hahn, Heidi

2012-01-01

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines. PMID:23300809

The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes.

PubMed

Paik, S R; Yokoyama, K; Yoshida, M; Ohta, T; Kagawa, Y; Allison, W S

1993-12-01

The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C. Half maximal stimulation is observed at about 3 microM dye. At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye. The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E. coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

Combined effects of high pressure and sodium hydrogen carbonate treatment on beef: improvement of texture and color

NASA Astrophysics Data System (ADS)

Ohnuma, Shun; Kim, Yun-Jung; Suzuki, Atsushi; Nishiumi, Tadayuki

2013-06-01

We investigated the effect of combined high pressure and sodium hydrogen carbonate (NaHCO3) treatment on the physical properties and color of silverside Australian beef. Meat samples were pressurized at 100-500 MPa and the water content, weight reduction, rupture stress, and meat color were determined. The water content of meat treated with NaHCO3 and high pressure (300 MPa) reached a maximum of 70.1%. Weight reduction tended to decrease with high pressure treatment at 300 MPa. Meats treated with NaHCO3 and high pressure at 400 MPa showed a>50% decrease in hardness. Whitening of the meat was reduced by the combined high pressure and NaHCO3 treatment. Therefore, the combined high pressure and NaHCO3 treatment is effective for improvement of beef quality.

Computational study of the electronic structure and magnetic properties of the Ni-C state in [NiFe] hydrogenases including the second coordination sphere.

PubMed

Kampa, Mario; Lubitz, Wolfgang; van Gastel, Maurice; Neese, Frank

2012-12-01

[NiFe] hydrogenases catalyze the reversible formation of H(2). The [NiFe] heterobimetallic active site is rich in redox states. Here, we investigate the key catalytic state Ni-C of Desulfovibrio vulgaris Miyazaki F hydrogenase using a cluster model that includes the truncated amino acids of the entire second coordination sphere of the enzyme. The optimized geometries, computed g tensors, hyperfine coupling constants, and IR stretching frequencies all agree well with experimental values. For the hydride in the bridging position, only a single minimum on the potential energy surface is found, indicating that the hydride bridges and binds to both nickel and iron. The influence of the second coordination sphere on the electronic structure is investigated by comparing results from the large cluster models with truncated models. The largest interactions of the second coordination sphere with the active site concern the hydrogen bonds with the cyanide ligands, which modulate the bond between iron and these ligands. Secondly, the electronic structure of the active site is found to be sensitive to the protonation state of His88. This residue forms a hydrogen bond with the spin-carrying sulfur atom of Cys549, which in turn tunes the spin density at the nickel and coordinating sulfur atoms. In addition, the unequal distribution of spin density over the equatorial cysteine residues results from different orientations of the cysteine side chains, which are kept in their particular orientation by the secondary structure of the protein.

Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

PubMed Central

Hollmann, Markus W; Ritter, Carsten H; Henle, Philipp; de Klaver, Manuela; Kamatchi, Ganesan L; Durieux, Marcel E

2001-01-01

Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca2+-activated Cl−-currents (ICl(Ca)), elicited by acetyl-β-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [3H]-quinuclydinyl benzylate ([3H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein α subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC50 of 370 nM, approximately 21 fold higher than the IC50 (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the Gq- protein α subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA. PMID:11325812

Lithium Suppresses Astrogliogenesis by Neural Stem and Progenitor Cells by Inhibiting STAT3 Pathway Independently of Glycogen Synthase Kinase 3 Beta

PubMed Central

Zhu, Zhenzhong; Kremer, Penny; Tadmori, Iman; Ren, Yi; Sun, Dongming; He, Xijing; Young, Wise

2011-01-01

Transplanted neural stem and progenitor cells (NSCs) produce mostly astrocytes in injured spinal cords. Lithium stimulates neurogenesis by inhibiting GSK3b (glycogen synthetase kinase 3-beta) and increasing WNT/beta catenin. Lithium suppresses astrogliogenesis but the mechanisms were unclear. We cultured NSCs from subventricular zone of neonatal rats and showed that lithium reduced NSC production of astrocytes as well as proliferation of glia restricted progenitor (GRP) cells. Lithium strongly inhibited STAT3 (signal transducer and activator of transcription 3) activation, a messenger system known to promote astrogliogenesis and cancer. Lithium abolished STAT3 activation and astrogliogenesis induced by a STAT3 agonist AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), suggesting that lithium suppresses astrogliogenesis by inhibiting STAT3. GSK3β inhibition either by a specific GSK3β inhibitor SB216763 or overexpression of GID5-6 (GSK3β Interaction Domain aa380 to 404) did not suppress astrogliogenesis and GRP proliferation. GSK3β inhibition also did not suppress STAT3 activation. Together, these results indicate that lithium inhibits astrogliogenesis through non-GSK3β-mediated inhibition of STAT. Lithium may increase efficacy of NSC transplants by increasing neurogenesis and reducing astrogliogenesis. Our results also may explain the strong safety record of lithium treatment of manic depression. Millions of people take high-dose (>1 gram/day) lithium carbonate for a lifetime. GSK3b inhibition increases WNT/beta catenin, associated with colon and other cancers. STAT3 inhibition may reduce risk for cancer. PMID:21931595

Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Xiaopeng; Du, Jie; Hua, Song

Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly,more » combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation.« less

The activation of the [NiFe]-hydrogenase from Allochromatium vinosum. An infrared spectro-electrochemical study.

PubMed

Bleijlevens, Boris; van Broekhuizen, Fleur A; De Lacey, Antonio L; Roseboom, Winfried; Fernandez, Victor M; Albracht, Simon P J

2004-09-01

The membrane-bound [NiFe]-hydrogenase from Allochromatium vinosum can occur in several inactive or active states. This study presents the first systematic infrared characterisation of the A. vinosum enzyme, with emphasis on the spectro-electrochemical properties of the inactive/active transition. This transition involves an energy barrier, which can be overcome at elevated temperatures. The reduced Ready enzyme can exist in two different inactive states, which are in an apparent acid-base equilibrium. It is proposed that a hydroxyl ligand in a bridging position in the Ni-Fe site is protonated and that the formed water molecule is subsequently removed. This enables the active site to bind hydrogen in a bridging position, allowing the formation of the fully active state of the enzyme. It is further shown that the active site in enzyme reduced by 1 bar H(2) can occur in three different electron paramagnetic resonance (EPR)-silent states with a different degree of protonation.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jan, Yi-Hua; Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu; Baker, Angela A.

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling,more » a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.« less

An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties.

PubMed

van der Linden, Eddy; Burgdorf, Tanja; de Lacey, Antonio L; Buhrke, Thorsten; Scholte, Marcel; Fernandez, Victor M; Friedrich, Bärbel; Albracht, Simon P J

2006-03-01

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni-Fe site of the soluble NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI(2)) with aerobic specific H(2)-NAD(+) activities of 150-185 mumol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.

TANKYRASE Inhibition Enhances the Antiproliferative Effect of PI3K and EGFR Inhibition, Mutually Affecting β-CATENIN and AKT Signaling in Colorectal Cancer.

PubMed

Solberg, Nina T; Waaler, Jo; Lund, Kaja; Mygland, Line; Olsen, Petter A; Krauss, Stefan

2018-03-01

Overactivation of the WNT/β-CATENIN signaling axis is a common denominator in colorectal cancer. Currently, there is no available WNT inhibitor in clinical practice. Although TANKYRASE (TNKS) inhibitors have been proposed as promising candidates, there are many colorectal cancer models that do not respond positively to TNKS inhibition in vitro and in vivo Therefore, a combinatorial therapeutic approach combining a TNKS inhibitor (G007-LK) with PI3K (BKM120) and EGFR (erlotinib) inhibitors in colorectal cancer was investigated. The data demonstrate that TNKS inhibition enhances the effect of PI3K and EGFR inhibition in the TNKS inhibitor-sensitive COLO320DM, and in the nonsensitive HCT-15 cell line. In both cell lines, combined TNKS/PI3K/EGFR inhibition is more effective at reducing growth than a dual TNKS/MEK inhibition. TNKS/PI3K/EGFR inhibition affected in a context-dependent manner components of the WNT/β-CATENIN, AKT/mTOR, EGFR, and RAS signaling pathways. TNKS/PI3K/EGFR inhibition also efficiently reduced growth of both COLO320DM and HCT-15 tumor xenografts in vivo At the highest doses, tumor xenograft growth was halted without affecting the body weight of the tested animals. Implications: Combining TNKS inhibitors with PI3K and EGFR inhibition may expand the therapeutic arsenal against colorectal cancers. Mol Cancer Res; 16(3); 543-53. ©2017 AACR . ©2017 American Association for Cancer Research.

Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

PubMed Central

Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-an; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

2016-01-01

Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation. PMID:26947250

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

NASA Technical Reports Server (NTRS)

Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

2003-01-01

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

PubMed

Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

2010-03-01

The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells.

The flavonol quercetin-3-glucoside inhibits cyanidin-3-glucoside absorption in vitro.

PubMed

Walton, Michaela C; McGhie, Tony K; Reynolds, Gordon W; Hendriks, Wouter H

2006-06-28

At present, little is known about the mechanisms responsible for intestinal absorption of anthocyanins (ACNs). For example, it has not yet been established if ACNs are absorbed through an active transport mechanism, such as the sodium-dependent glucose transporter (SGLT1), or by passive diffusion. Previously, we found that the absorption of ACNs differs between regions of the digestive tract and is maximal in the jejunum, suggesting that an active transport mechanism is involved. In the present study, we examined the effect of d-glucose (main substrate of SGLT1), phloridzin (inhibitor of SGLT1), and quercetin-3-glucose (Q3G, a flavonol) on the absorption of cyanidin-3-glucoside (C3G; approximately 5 micromol/L) by mouse jejunum mounted in Ussing chambers. We found that the presence of either D-glucose (10, 20, and 40 mmol/L) or phloridzin (50, 100, and 200 micromol/L) resulted in a small but insignificant inhibition of C3G disappearance from the mucosal solution (decrease of disappearance with glucose, 33%; with phloridzin, 18%; NS). However, when the flavonol Q3G (50 micromol/L) was added to the mucosal solution together with the C3G, the disappearance of C3G was significantly decreased (74%; p < 0.001), and Q3G disappeared instead. In addition, we found phloretin and quercetin, the aglycones of phloridzin and Q3G, respectively, present in the mucosal solution and tissue extracts, indicating hydrolysis of these compounds by the enterocytes of the jejunum. In contrast, the aglycone cyanidin was not detected at all. Our results show that in the mouse small intestine, ACN absorption is not solely dependent on the activity of the SGLT1 transporter, as d-glucose and phloridzin had only a slight effect on uptake. Q3G, however, clearly inhibited C3G disappearance. These results suggest that there might be a competitive inhibition between C3G and Q3G absorption. It is possible that an absorption mechanism other than the SGLT1 is involved, which has a structural preference

Selenoprotein W enhances skeletal muscle differentiation by inhibiting TAZ binding to 14-3-3 protein.

PubMed

Jeon, Yeong Ha; Park, Yong Hwan; Lee, Jea Hwang; Hong, Jeong-Ho; Kim, Ick Young

2014-07-01

Selenoprotein W (SelW) is expressed in various tissues, particularly in skeletal muscle. We have previously reported that SelW is up-regulated during C2C12 skeletal muscle differentiation and inhibits binding of 14-3-3 to its target proteins. 14-3-3 reduces myogenic differentiation by inhibiting nuclear translocation of transcriptional co-activator with PDZ-binding motif (TAZ). Phosphorylation of TAZ at Ser89 is required for binding to 14-3-3, leading to cytoplasmic retention of TAZ and a delay in myogenic differentiation. Here, we show that myogenic differentiation was delayed in SelW-knockdown C2C12 cells. Down-regulation of SelW also increased TAZ binding to 14-3-3, which eventually resulted in decreasing translocation of TAZ to the nucleus. However, phosphorylation of TAZ at Ser89 was not affected. Although phosphorylation of TAZ at Ser89 was sustained by the phosphatase inhibitor okadaic acid, nuclear translocation of TAZ was increased by ectopic expression of SelW. This result was due to decreased binding of TAZ to 14-3-3. We also found that the interaction between TAZ and MyoD was increased by ectopic expression of SelW. Taken together, these findings strongly demonstrate that SelW enhances C2C12 cell differentiation by inhibiting TAZ binding to 14-3-3. Copyright © 2014 Elsevier B.V. All rights reserved.

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ching-Chu

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0–15 μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis ofmore » 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. - Highlights: • Andrographolide is a diterpenoid

INHIBITION OF INDOLEAMINE 2,3-DIOXYGENASE DOES NOT IMPEDE ORAL TOLERANCE

EPA Science Inventory

Rationale: Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, regulates immune tolerance through inhibition of T-cell proliferation. Pharmacologic inhibition of IDO, which causes fetal rejection and increased tumor resistance in mice, may prove useful in cancer...

Differences in antiproliferative effect of STAT3 inhibition in HCC cells with versus without HBV expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Yun; Zhou, Lin; Xie, Haiyang

2015-06-05

Chronic infection with hepatitis B virus (HBV) plays an important role in the etiology of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3) inactivation could inhibit the tumor growth of HCC. In this study, differential antiproliferative effect of STAT3 inhibition was observed with HBV-related HCC cells being more resistant than non-HBV-related HCC cells. Resistance of HBV-related HCC cells to STAT3 inhibition was positively correlated to the expression of HBV. Enhanced ERK activation after STAT3 blockade was detected in HBV-related HCC cells but not in non-HBV-related HCC cells. Combined ERK and STAT3 inhibition eliminates the discrepancy between themore » two types of HCC cells. Moderate reduced HBV expression was found after STAT3 inhibition. These findings disclose a discrepancy in cellular response to STAT3 inhibition between non-HBV-related and HBV-related HCC cells and underscore the complexity of antiproliferative effect of STAT3 inactivation in HBV-related HCC cells. - Highlights: • HBV endows HCC cells with resistance to STAT3 inactivation on proliferation. • Abnormal ERK activation after STAT3 inhibition in HBV-related HCC cells. • Combined ERK and STAT3 inhibition eliminates the discrepancy. • STAT3 inhibition moderately reduces HBV expression.« less

Influence of Sulfur Metalation on the Accessibility of the Ni(II/I) Couple in [N,N'-Bis(2-mercaptoethyl)-1,5-diazacyclooctanato]nickel(II): Insight into the Redox Properties of [NiFe]-Hydrogenase.

PubMed

Musie, Ghezai; Farmer, Patrick J.; Tuntulani, Thawatchai; Reibenspies, Joseph H.; Darensbourg, Marcetta Y.

1996-04-10

A redox model study of [NiFe] hydrogenase has examined a series of five polymetallics based on the metalation of the dithiolate complex [1,5-bis(mercaptoethyl)-1,5-diazacyclooctane]Ni(II), Ni-1. Crystal structures of three polymetallics of the series have been reported earlier: [(Ni-1)(2)()Ni]Cl(2)(), [(Ni-1)(2)()FeCl(2)()](2)(), and [(Ni-1)(3)()(ZnCl)(2)()]Cl(2)(). Two are described here: [(Ni-1)(2)()Pd]Cl(2)().2H(2)()Ocrystallizes in the monoclinic system, space group P2(1)/c with cell constants a = 12.212(4) Å, b = 7.642(2) Å, c = 16.625(3) Å, beta = 107.69(2) degrees, V = 1443.230(0) Å(3), Z = 2, R = 0.051, and R(w) = 0.056. [(Ni-1)(2)()CoCl]PF(6)() crystallizes in the triclinic system, space group P&onemacr;, with cell constants a = 8.14(2) Å, b = 13.85(2) Å, c = 15.67(2) Å, alpha = 113.59(10) degrees, beta = 101.84(14) degrees, gamma = 94.0(2) degrees, V = 1561.620(0)Å(3), Z = 2, R = 0.072, and R(w) = 0.077. In all Ni-1 serves as a bidentate metallothiolate ligand with a "hinge" angle in the range 105-118 degrees and Ni-M distances of 2.7- 3.7 Å. The most accessible redox event is shown by EPR and electrochemistry to reside in the N(2)S(2)Ni unit and is the Ni(II/I) couple. Charge neutralization of the thiolate sulfurs by metalation can (dependent on the interacting metal) stabilize the Ni(I) state as efficiently as methylation forming a thioether. The implication of these results for the heterometallic active site of [NiFe]-hydrogenase as structured from Desulfovibrio gigas (Volbeda, A., et al. Nature, 1995, 373, 580), the generality of the Ni(&mgr;-SR)(2)M hinge structure, and a possible explanation for the unusual redox potentials are discussed.

GSK-3 Inhibition Sensitizes Acute Myeloid Leukemia Cells to 1,25D-Mediated Differentiation

PubMed Central

Gupta, Kalpana; Stefan, Tammy; Ignatz-Hoover, James; Moreton, Stephen; Parizher, Gary; Saunthararajah, Yogen; Wald, David N.

2017-01-01

1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment. PMID:26964622

[Effects of different salt and alkali stresses on ion distribution in Red globe/Beta grapevines].

PubMed

Du, Yuan-peng; Jin, Xue-juan; Guo, Shu-hua; Fu, Qing-qing; Zhai, Heng

2015-06-01

The potted Red globe/Beta grapevines were selected to irrigated with NaCl, Na2SO4, NaHCO3, NH4Cl, (NH4)2SO4. Hence, the ions which induced leaf etiolation were screened and the impacts of different salt and alkali on ion distribution in different organs of grapevines were investigated. It was found that NaHCO3 exerted the greatest effects on grapevines, leaf etiolation at 14 days after treatment. By contrast, NaCl and NH4Cl treatments induced leaf etiolation at 28 days after treatment. The Na+ content in all the detected organs were significantly increased under NaHCO3 and NaCl treatment, and Na+ content in root under NaHCO3 treatment was 6.4 times as that in control root. NaHCO3 and NaCl treatments significantly decreased K+ content in the organs with the exception of leaf. NaHCO3 treatment significantly decreased K/Na in different organs, which declined to 0.1 in root. By contrast, NaCl treatment significantly decreased K/Na in the detected organs with exception of stem. Besides, the transport of Ca2+, Mg2+, Fe2+ to aboveground organs was significantly decreased by NaHCO3 and NaCl treatments. K/Na ratio in the detected organs were decreased under NH4Cl, (NH4) 2SO4 and Na2SO4 treatments, especially under NH4 Cl treatment. Taken together, NaHCO3 was the primary factor resulting in leaf etiolation, followed by NaCl and NH4Cl, while (NH4) 2SO4 and Na2SO4 produced impacts.

pH-Dependent isotope exchange and hydrogenation catalysed by water-soluble NiRu complexes as functional models for [NiFe]hydrogenases.

PubMed

Kure, Bunsho; Matsumoto, Takahiro; Ichikawa, Koji; Fukuzumi, Shunichi; Higuchi, Yoshiki; Yagi, Tatsuhiko; Ogo, Seiji

2008-09-21

The pH-dependent hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes and hydrogenation of the carbonyl compounds have been investigated with water-soluble bis(mu-thiolate)(mu-hydride)NiRu complexes, Ni(II)(mu-SR)(2)(mu-H)Ru(II) {(mu-SR)(2) = N,N'-dimethyl-N,N'-bis(2-mercaptoethyl)-1,3-propanediamine}, as functional models for [NiFe]hydrogenases. In acidic media (at pH 4-6), the mu-H ligand of the Ni(II)(mu-SR)(2)(mu-H)Ru(II) complexes has H(+) properties, and the complexes catalyse the hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes. A mechanism of the hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes through a low-valent Ni(I)(mu-SR)(2)Ru(I) complex is proposed. In contrast, in neutral-basic media (at pH 7-10), the mu-H ligand of the Ni(II)(mu-SR)(2)(mu-H)Ru(II) complexes acts as H(-), and the complexes catalyse the hydrogenation of carbonyl compounds.

Cloning, characterization, and chromosomal mapping of a human electroneutral Na(+)-driven Cl-HCO3 exchanger.

PubMed

Grichtchenko, I I; Choi, I; Zhong, X; Bray-Ward, P; Russell, J M; Boron, W F

2001-03-16

The electroneutral Na(+)-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pH(i)) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBank accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); approximately 50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; approximately 73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na(+)-driven anion exchanger (NDAE1) from Drosophila. Northern blot analysis of NDCBE1 shows a robust approximately 12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed in Xenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pH(i) and [Na(+)](i) (monitored with microelectrodes) that require HCO3(-) and are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The pH(i) increase also requires extracellular Na(+). The Na(+):HCO3(-) stoichiometry is 1:2. Forward-running NDCBE1 mediates a 36Cl efflux that requires extracellular Na(+) and HCO3(-) and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl(-). Thus, NDCBE1 encodes a human, electroneutral Na(+)-driven Cl-HCO3 exchanger.

Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris

USGS Publications Warehouse

Lovley, D.R.; Widman, P.K.; Woodward, J.C.; Phillips, E.J.P.

1993-01-01

The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium- contaminated waters and waste streams.

Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.

PubMed

Yang, Wei; Hosford, Sarah R; Traphagen, Nicole A; Shee, Kevin; Demidenko, Eugene; Liu, Stephanie; Miller, Todd W

2018-03-01

Hyperactivation of the PI3K pathway has been implicated in resistance to antiestrogen therapies in estrogen receptor α (ER)-positive breast cancer, prompting the development of therapeutic strategies to inhibit this pathway. Autophagy has tumor-promoting and -suppressing roles and has been broadly implicated in resistance to anticancer therapies, including antiestrogens. Chloroquine (CQ) is an antimalarial and amebicidal drug that inhibits autophagy in mammalian cells and human tumors. Herein, we observed that CQ inhibited proliferation and autophagy in ER + breast cancer cells. PI3K inhibition with GDC-0941 (pictilisib) induced autophagy. Inhibition of autophagy using CQ or RNA interference potentiated PI3K inhibitor-induced apoptosis. Combined inhibition of PI3K and autophagy effectively induced mitochondrial membrane depolarization, which required the BH3-only proapoptotic proteins Bim and PUMA. Treatment with GDC-0941, CQ, or the combination, significantly suppressed the growth of ER + breast cancer xenografts in mice. In an antiestrogen-resistant xenograft model, GDC-0941 synergized with CQ to provide partial, but durable, tumor regression. These findings warrant clinical evaluation of therapeutic strategies to target ER, PI3K, and autophagy for the treatment of ER + breast cancer.-Yang, W., Hosford, S. R., Traphagen, N. A., Shee, K., Demidenko, E., Liu, S., Miller, T. W. Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.

Synthesis, Purification, and Characterization of a [mu]-(1,3-Propanedithiolato)-Hexacarbonyldiiron

ERIC Educational Resources Information Center

Works, Carmen F.

2007-01-01

A project which exposes students to biologically important transition-metal chemistry is illustrated by taking an example of the iron-carbonyl compound, [mu]-(1,3-Propanedithiolaro)-hexa-carbonyldiiron as a structural model for an iron-only hydro-genase. The project provides the students with experience of Schlenk line techniques, purification,…

Antioxidants Inhibit Formation of 3-Monochloropropane-1,2-diol Esters in Model Reactions.

PubMed

Li, Chang; Jia, Hanbing; Shen, Mingyue; Wang, Yuting; Nie, Shaoping; Chen, Yi; Zhou, Yongqiang; Wang, Yuanxing; Xie, Mingyong

2015-11-11

The capacities of six antioxidants to inhibit the formation of 3-monochloropropane-1,2 diol (3-MCPD) esters were examined in this study. Inhibitory capacities of the antioxidants were investigated both in chemical models containing the precursors (tripalmitoyl glycerol, 1,2-dipalmitoyl-sn-glycerol, monopalmitoyl glycerol, and sodium chloride) of 3-MCPD esters and in oil models (rapeseed oil and sodium chloride). Six antioxidants, butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), tert-butyl hydroquinone (TBHQ), propyl gallate (PG), L-ascorbyl palmitate (AP), and α-tocopherol (VE), were found to exhibit inhibiting capacities on 3-MCPD ester formation both in chemical models and in oil models. TBHQ provided the highest inhibitory capacity both in chemical models and in oil models; 44% of 3-MCPD ester formation was inhibited in the presence of TBHQ (66 mg/kg of oil) after heating of rapeseed oil at 230 °C for 30 min, followed by PG and AP. BHT, BHA, and VE appeared to have weaker inhibitory abilities in both models. VE exhibited the lowest inhibition rate; 22% of 3-MCPD esters were inhibited in the presence of VE (172 mg/kg of oil) after heating of rapeseed oil at 230 °C for 30 min. In addition, the inhibition rates of PG and VE decreased dramatically with an increase in temperature or heating time. The results suggested that some antioxidants, such as TBHQ, PG, and AP, could be the potential inhibitors of 3-MCPD esters in practice.

Improvement of texture and palatability of chicken breast: effect of high hydrostatic pressure and sodium hydrogen carbonate

NASA Astrophysics Data System (ADS)

Tabe, Kanae; Kim, Yun-Jung; Ohnuma, Shun; Ogoshi, Hiro; Suzuki, Atsushi; Nishiumi, Tadayuki

2013-06-01

Chicken breast is not preferred in Japan because it is not juicy. In this study, the effect of combined high pressure and sodium hydrogen carbonate (NaHCO3) treatment on the texture and palatability of chicken breast was investigated. The sample used was broiler chicken breast. Meat samples were soaked in.0-.4 M NaHCO3 solution and then pressurized at 100-400 MPa. After pressurization, the samples were heated for 30 min at 80°C and cooled down in ice-cold water. High pressure and NaHCO3 treatment of broiler chicken breast resulted in increased water content, and decreased weight reduction and rupture stress. Moreover, meat exposed to 200 MPa pressurization and.3 M NaHCO3 treatment was judged tender, juicy and of good taste by sensory evaluation. The combination of high pressure and NaHCO3 treatment can be effectively used for broiler chicken breast production.

Inhibition of TRPC3 downregulates airway hyperresponsiveness, remodeling of OVA-sensitized mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lingwei; Li, Jie; Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou

Airway hyperresponsiveness (AHR), airway remodeling and inflammation are the fundamental pathological alterations that occur in asthma. Transient receptor potential canonical 3 (TRPC3) has been implicated in diverse functions of airway smooth muscle cells (ASMCs) in asthma. However, the underlying mechanisms remain incompletely understood. We investigated the mRNA and protein expression of TRPC3 in ASMCs from normal and OVA-sensitized mouse. And the effects of inhibition or knockdown of TRPC3 with Ethyl-1- (4- (2,3,3-trichloroacrylamide) phenyl) −5 - (trifluoromethyl) -1H -pyrazole -4-carboxylate (Pyr3) and lentiviral shRNA on OVA-sensitized mouse AHR, airway remodeling, circulating inflammatory cytokines, cell proliferation and migration. We found that TRPC3 mRNAmore » and protein expression levels were significantly increased in ASMCs from OVA-sensitized mouse. Inhibiting TRPC3 with continuous subcutaneous administration of Pyr3 decreased enhanced pause (Penh) of OVA-sensitized mouse. Meanwhile, both Pyr3 and lentiviral shRNA treatment of ASMCs in OVA-sensitized mouse significantly decreased their proliferation and migration. These results suggest that TRPC3 plays a critical role in asthma and represents a promising new target for asthma treatment. - Highlights: • Penh, airway remodeling and the gene expression and protein of TRPC3 are increased in OVA-sensitized mice. • Inhibition of TRPC3 suppresses the OVA-sensitized mice Penh and airway remodeling. • Inhibition of TRPC3 decreases OVA-sensitized mice ASMC proliferation and migration.« less

RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle

PubMed Central

Safdie, Gracia; Liewald, Jana F.; Kagan, Sarah; Battat, Emil; Gottschalk, Alexander; Treinin, Millet

2016-01-01

Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation–inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition. PMID:27489343

Cortisol inhibits CSF2 and CSF3 via DNA methylation and inhibits invasion in first-trimester trophoblast cells

PubMed Central

Smith, Arianna; Witte, Elizabeth; McGee, Devin; Knott, Jason; Narang, Kavita; Racicot, Karen

2018-01-01

Problem Heightened maternal stress affects trophoblast function and increases risk for adverse pregnancy outcomes. Methods of Study Studies were performed using the first-trimester trophoblast cell line, Sw.71. Cytokines were quantified using qPCR and ELISA. Epigenetic regulation of cytokines was characterized by inhibiting histone deacetylation (1 μmol/L suberoylanilide hydroxamic acid [SAHA]) or methylation (5 μmol/L 5-azacytidine), or with chromatin immunoprecipitation (ChIP) with a pan-acetyl histone-3 antibody. Invasion assays used Matrigel chambers. Results Cortisol inhibited expression of CSF2 (GM-CSF) and CSF3 (G-CSF) in trophoblast cells. Cortisol-associated inhibition was dependent on DNA methylation and was not affected by acetylation. There was also a modest decrease in trophoblast invasion, not dependent on loss of CSFs. Conclusion In first-trimester trophoblast cells, the physiological glucocorticoid, cortisol, inhibited two cytokines with roles in placental development and decreased trophoblast invasion. Cortisol-associated changes in trophoblast function could increase the risk for immune-mediated abortion or other adverse pregnancy outcomes. PMID:28846166

Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

2014-09-30

Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There aremore » correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.« less

Modulation of active site electronic structure by the protein matrix to control [NiFe] hydrogenase reactivity.

PubMed

Smith, Dayle M A; Raugei, Simone; Squier, Thomas C

2014-11-21

Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni-Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

Differential inhibition of Ca2+ channels in mature rat cerebellar Purkinje cells by sFTX-3.3 and FTX-3.3.

PubMed

Dupere, J R; Moya, E; Blagbrough, I S; Usowicz, M M

1996-01-01

Synthetic funnel web spider toxin (sFTX-3.3) is a polyamine amide analogue of FTX, a toxin fraction isolated from the venom of the funnel web spider, Agelenopsis aperta, that blocks P-type Ca2+ channels. The structures of these polyamine containing compounds are not identical: sFTX-3.3 contains an amide carbonyl oxygen that is absent from the predicted structure of native FTX. Recently, a compound called FTX-3.3 was synthesized with the structure predicted for native FTX. We have compared the effects of polyamine amide sFTX-3.3 and polyamine FTX-3.3, on Ca2+ channel currents in the soma of mature rat cerebellar Purkinje neurons, in which the predominant Ca2+ channels are defined as P-type. Differential inhibition by sFTX-3.3 and FTX-3.3 revealed three populations of Ca2+ channels. One group, mediating approximately 66% of the current, was blocked by sFTX-3.3 with an IC50 (concentration producing half maximal inhibition) of 33 nM or by FTX-3.3 with an IC50 of 55 pM. A second population (5-25% of the total current) was inhibited by sFTX-3.3 with an IC50 of 33 nM, but was insensitive to FTX-3.3, while a third (10-30%) was blocked by FTX-3.3 with an IC50 of 125 nM and was resistant to sFTX-3.3. These channels also showed distinctive current-voltage relationships. Our results suggest that P-type Ca2+ channels in mature rat cerebellar Purkinje cells may be subdivided according to pharmacological and biophysical properties.

Review of prehospital sodium bicarbonate use for cyclic antidepressant overdose

PubMed Central

Calkins, T; Chan, T; Clark, R; Stepanski, B; Vilke, G

2003-01-01

Methods: A three year retrospective observational review of records was performed using the San Diego County Quality Assurance Network database for prehospital providers. All adult patients who were treated with NaHCO3 by paramedics for a CA overdose were included. Demographic data, presenting cardiovascular and neurological symptoms, paramedic treatments, and any changes in status were reviewed. Results: Twenty one patients were treated by paramedics with NaHCO3 for CA overdose. Seventeen patients (80%) presented with mental status changes, including 11 presenting with a GCS<8. Seven of the 21 (33%) presented with a cardiac arrhythmia expected to possibly respond to NaHCO3 treatment. Seven of the 21 (33%) were hypotensive, and five (24%) patients had reported seizure activity. Only 2 of the 21 patients (10%) treated with NaHCO3 had recorded improvements after administration of the drug, while the other 19 remained stable without any deterioration. Sixteen of 21 patients (76%) were given NaHCO3 for indications on standing order, while five patients were treated outside the standing order indications by base physician order with none of the five patients having any change in status ater treatment. Conclusions: After prehospital NaHCO3 use in patients with CA overdose, there were no complications reported, two patients improved in status and the others remained unchanged. Base hospital physician orders of NaHCO3 for indications beyond the standing orders were not associated with changes in patient status. PMID:12954700

3'-hydroxy-ε,ε-caroten-3-one inhibits the differentiation of 3T3-L1 cells to adipocytes.

PubMed

Kotake-Nara, Eiichi; Hase, Megumi; Kobayashi, Miyuki; Nagao, Akihiko

2016-01-01

An oxidative metabolite of lutein, 3'-hydroxy-ε,ε-caroten-3-one, inhibited the differentiation of 3T3-L1 cells to adipocytes and the subsequent triacylglycerol production, but lutein did not. The α,β-unsaturated carbonyl structure of 3'-hydroxy-ε,ε-caroten-3-one was considered to participate in the inhibitory effect, suggesting that this lutein metabolite has the potential to prevent metabolic syndrome.

Paired box 7 inhibits differentiation in 3T3-L1 preadipocytes.

PubMed

Izumi, Wakana; Takuma, Yuko; Ebihara, Ryo; Mizunoya, Wataru; Tatsumi, Ryuichi; Nakamura, Mako

2018-06-13

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3-L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets. © 2018 Japanese Society of Animal Science.

Cyanidin-3-glucoside reverses ethanol-induced inhibition of neurite outgrowth: role of glycogen synthase kinase 3 Beta.

PubMed

Chen, Gang; Bower, Kimberly A; Xu, Mei; Ding, Min; Shi, Xianglin; Ke, Zun-Ji; Luo, Jia

2009-05-01

Ethanol is a potent teratogen for the developing central nervous system (CNS), and fetal alcohol syndrome (FAS) is the most common nonhereditary cause of mental retardation. Ethanol disrupts neuronal differentiation and maturation. It is important to identify agents that provide neuroprotection against ethanol neurotoxicity. Using an in vitro neuronal model, mouse Neuro2a (N2a) neuroblastoma cells, we demonstrated that ethanol inhibited neurite outgrowth and the expression of neurofilament (NF) proteins. Glycogen synthase kinase 3beta (GSK3beta), a multifunctional serine/threonine kinase negatively regulated neurite outgrowth of N2a cells; inhibiting GSK3beta activity by retinoic acid (RA) and lithium induced neurite outgrowth, while over-expression of a constitutively active S9A GSK3beta mutant prevented neurite outgrowth. Ethanol inhibited neurite outgrowth by activating GSK3beta through the dephosphorylation of GSK3beta at serine 9. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family rich in many edible berries and other pigmented fruits, enhanced neurite outgrowth by promoting p-GSK3beta(Ser9). More importantly, C3G reversed ethanol-mediated activation of GSK3beta and inhibition of neurite outgrowth as well as the expression of NF proteins. C3G also blocked ethanol-induced intracellular accumulation of reactive oxygen species (ROS). However, the antioxidant effect of C3G appeared minimally involved in its protection. Our study provides a potential avenue for preventing or ameliorating ethanol-induced damage to the developing CNS.

Six-Month Evaluation of a Sodium Bicarbonate-Containing Toothpaste for Reduction of Established Gingivitis: A Randomized USA-Based Clinical Trial.

PubMed

Jose, Anto; Pratten, Jonathan; Bosma, Mary-Lynn; Milleman, Kimberly R; Milleman, Jeffery L; Wang, Nan

2018-03-01

Short-term use of sodium bicarbonate (NaHCO3)-containing toothpaste reduces plaque and improves clinical measures of gingivitis. To examine this over a longer period, we compared efficacy and tolerability of twice-daily brushing for 24 weeks with 67% or 0% NaHCO3-containing toothpastes in USA-based participants with moderate gingivitis (Clinicaltrials.gov:NCT02207400). This was a six-month, randomized, examiner-blind, parallel-group, clinical trial. Investigators randomized adults with blood in expectorate after brushing and ≥ 20 gingival bleeding sites to 67% NaHCO3 (n = 123; n = 107 completed study) or 0% NaHCO3 (n = 123; n = 109 completed study) toothpastes. Primary efficacy variables included between-treatment differences in number of bleeding sites and Modified Gingival Index (MGI) score at 24 weeks. Secondary efficacy variables included Bleeding Index and Turesky modification of the Quigley-Hein Plaque Index (overall and interproximal sites) at six, 12, and 24 weeks. A subset of 50 participants underwent sampling to assess plaque microbiology over the course of treatment. Compared with the 0% NaHCO3 toothpaste, the 67% NaHCO3 toothpaste produced statistically significant improvements at Week 24 in number of bleeding sites (46.7% difference) and MGI (33.9% difference), and for all other endpoints (all p < 0.0001). There was no significant between-treatment difference in the proportion of participants harboring opportunistic pathogens. Products were generally well tolerated, with two and five treatment-related adverse events reported in the 67% and 0% NaHCO3 toothpaste groups, respectively. Gingival bleeding, gingivitis, and plaque indices were significantly improved at six, 12, and 24 weeks with twice-daily brushing with 67% NaHCO3-containing toothpaste in participants with moderate gingivitis. Copyright© by the YES Group, Inc.

Tribbles 3 inhibits brown adipocyte differentiation and function by suppressing insulin signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Ha-Won; Choi, Ran Hee; McClellan, Jamie L.

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function. Tribbles 3 (TRB3) is a pseudo kinase that has been shown to regulate metabolism and insulin signaling in multiple tissues but the role of TRB3 in BAT has not been studied. In this study, we found thatmore » TRB3 expression was present in BAT and overexpression of TRB3 in brown preadipocytes impaired differentiation and decreased expression of BAT markers. Furthermore, TRB3 overexpression resulted in significantly lower oxygen consumption rates for basal and proton leakage, indicating decreased BAT activity. Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin-stimulated IRS1 and Akt phosphorylation, whereas TRB3KO mice displayed improved IRS1 and Akt phosphorylation. Finally, deletion of IRS1 abolished the function of TRB3 to regulate BAT differentiation and metabolism. These data demonstrate that TRB3 inhibits insulin signaling in BAT, resulting in impaired differentiation and function. - Highlights: • TRB3 is expressed in brown adipose tissue and its expression is increased during differentiation. • Overexpression of TRB3 inhibits differentiation and its activity. • Overexpression of TRB3 in brown preadipocytes inhibits insulin signaling. • TRB3KO mice displays improved insulin signaling in brown adipose tissue. • Insulin signaling is required for the effects of TRB3 to regulate brown adipose tissue

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3-cell-induced osteoclast formation.

PubMed

Araujo, John C; Poblenz, Ann; Corn, Paul; Parikh, Nila U; Starbuck, Michael W; Thompson, Jerry T; Lee, Francis; Logothetis, Christopher J; Darnay, Bryant G

2009-11-01

Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop*

PubMed Central

Monteil, Arnaud; Chausson, Patrick; Boutourlinsky, Katia; Mezghrani, Alexandre; Huc-Brandt, Sylvaine; Blesneac, Iulia; Bidaud, Isabelle; Lemmers, Céline; Leresche, Nathalie; Lambert, Régis C.; Lory, Philippe

2015-01-01

Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (Δ1-Cav3.2; Ser423–Pro542) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, Δ1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of Δ1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels. PMID:25931121

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taketazu, F.; Chiba, S.; Shibuya, K.

1991-02-01

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

Decursin chemosensitizes human multiple myeloma cells through inhibition of STAT3 signaling pathway.

PubMed

Kim, Hyun Jung; Kim, Sung-Moo; Park, Kyung-Ran; Jang, Hyeung-Jin; Na, Young-Soon; Ahn, Kyoo Seok; Kim, Sung-Hoon; Ahn, Kwang Seok

2011-02-01

Recent reports have indicated that decursin can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this experiment, we investigated how decursin could potentiate the cytotoxic effects of bortezomib in human multiple myeloma cells. We found that decursin inhibited cell viability in U266, MM.1S and ARH77 cells, but not in peripheral blood mononuclear cells (PBMC). Decursin-induced apoptosis through the activation of caspase-8, -9, and -3 in U266 cells. This correlated with the down-regulating of cyclin D1, bcl-2, bcl-xL, survivin, and the vascular endothelial growth factor (VEGF), which are all regulated by the activation of signal transducers and the activator of transcription 3 (STAT3). Indeed, decursin inhibited constitutive STAT3 activation through inhibition of the activation of Janus-activated kinase 2 (JAK2) in U266 cells. In addition, decursin inhibited interleukin-6-inducible STAT3 activation in a time-dependent manner in MM.1S cells. Interestingly, decursin significantly potentiated the apoptotic effects of bortezomib in U266 cells. These effects of decursin were correlated with the suppression of constitutive STAT3 activation in U266 cells. Overall, these results suggest that decursin is a novel blocker of STAT3 activation and it may be a potential candidate for overcoming chemo-resistance through suppression of this signaling. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

STAT3 Oligonucleotide Inhibits Tumor Angiogenesis in Preclinical Models of Squamous Cell Carcinoma

PubMed Central

Klein, Jonah D.; Sano, Daisuke; Sen, Malabika; Myers, Jeffrey N.; Grandis, Jennifer R.; Kim, Seungwon

2014-01-01

Purpose Signal transducer and activator of transcription 3 (STAT3) has shown to play a critical role in head and neck squamous cell carcinoma (HNSCC) and we have recently completed clinical trials of STAT3 decoy oligonucleotide in patients with recurrent or metastatic HNSCC. However, there is limited understanding of the role of STAT3 in modulating other aspects of tumorigenesis such as angiogenesis. In this study, we aimed to examine the effects of STAT3 decoy oligonucleotide on tumor angiogenesis. Experimental Design A STAT3 decoy oligonucleotide and small interfering RNA (siRNA) were used to inhibit STAT3 in endothelial cells in vitro and in vivo. The biochemical effects of STAT3 inhibition were examined in conjunction with the consequences on proliferation, migration, apoptotic staining, and tubule formation. Additionally, we assessed the effects of STAT3 inhibition on tumor angiogenesis using murine xenograft models. Results STAT3 decoy oligonucleotide decreased proliferation, induces apoptosis, decreased migration, and decreased tubule formation of endothelial cells in vitro. The STAT3 decoy oligonucleotide also inhibited tumor angiogenesis in murine tumor xenografts. Lastly, our data suggest that the antiangiogenic effects of STAT3 decoy oligonucleotide were mediatedthrough the inhibition of both STAT3 and STAT1. Conclusions The STAT3 decoy oligonucleotidewas found to be an effective antiangiogenic agent, which is likely to contribute to the overall antitumor effects of this agent in solid tumors.Taken together with the previously demonstrated antitumor activity of this agent, STAT3 decoy oligonucleotide represents a promising single agent approach to targeting both the tumor and vascular compartments in various malignancies. PMID:24404126

Sodium bicarbonate causes dose-dependent increases in cerebral blood flow in infants and children with single ventricle physiology

PubMed Central

Buckley, Erin M.; Naim, Maryam Y.; Lynch, Jennifer M.; Goff, Donna A.; Schwab, Peter J.; Diaz, Laura K.; Nicolson, Susan C.; Montenegro, Lisa M.; Lavin, Natasha A.; Durduran, Turgut; Spray, Thomas L.; Gaynor, J. William; Putt, Mary E.; Yodh, A.G.; Fogel, Mark A.; Licht, Daniel J.

2013-01-01

Background Sodium bicarbonate (NaHCO3) is a common treatment for metabolic acidemia, however little definitive information exists regarding its treatment efficacy and cerebral hemodynamic effects. This pilot observational study quantifies relative changes in cerebral blood flow (rCBF) and oxy and deoxy-hemoglobin concentrations (ΔHbO2 and ΔHb) due to bolus administration of NaHCO3 in patients with mild base deficits. Methods Infants and children with hypoplastic left heart syndrome (HLHS) were recruited prior to cardiac surgery. NaHCO3 was given as needed for treatment of base deficit. Diffuse optical spectroscopies were employed for 15 minutes post-injection to non-invasively monitor ΔHb, ΔHbO2 and rCBF relative to baseline prior to NaHCO3 administration. Results Twenty-two anesthetized and mechanically ventilated HLHS patients (1 day to 4 years old) received a median (interquartile range) dose of 1.1 (0.8, 1.8) mEq/kg NaHCO3 administered intravenously over 10–20 seconds to treat a base deficit of −4 (−6, −3) mEq/l. NaHCO3 caused significant dose-dependent increases in rCBF, however population averaged ΔHb or Δ4HbO2 compared to controls were not significant. Conclusions Dose-dependent increases in cerebral blood flow (CBF) caused by bolus NaHCO3 are an important consideration in vulnerable populations wherein risk of rapid CBF fluctuations does not outweigh the benefit of treating a base deficit. PMID:23403802

Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease.

PubMed

Tomlinson, Suzanne M; Watowich, Stanley J

2008-11-11

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.

Novel quinazoline-based sulfonamide derivative (3D) induces apoptosis in colorectal cancer by inhibiting JAK2-STAT3 pathway.

PubMed

Al-Obeed, Omar; Vaali-Mohammed, Mansoor-Ali; Eldehna, Wagdy M; Al-Khayal, Khayal; Mahmood, Amer; Abdel-Aziz, Hatem A; Zubaidi, Ahmed; Alafeefy, Ahmed; Abdulla, Maha; Ahmad, Rehan

2018-01-01

Colorectal cancer (CRC) is a major worldwide health problem owing to its high prevalence and mortality rate. Developments in screening, prevention, biomarker, personalized therapies and chemotherapy have improved detection and treatment. However, despite these advances, many patients with advanced metastatic tumors still succumb to the disease. New anticancer agents are needed for treating advanced stage CRC as most of the deaths occur due to cancer metastasis. A recently developed novel sulfonamide derivative 4-((2-(4-(dimethylamino) phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3D) has shown potent antitumor effect; however, the mechanism underlying the antitumor effect remains unknown. 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis. Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability. Taken together, these findings

Hydrogen production by the naked active site of the di-iron hydrogenases in water.

PubMed

Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

2009-10-01

We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster.

Photolysis of water for H2 production with the use of biological and artificial catalysts

NASA Astrophysics Data System (ADS)

Hall, D. O.; Adams, M. W. W.; Morris, P.; Rao, K. K.

1980-02-01

An aqueous mixture of chloroplasts, hydrogenase and electron transfer catalyst on illumination liberates H2, the source of the H atoms being water. The rate and duration of H2 production from such a system depends on the stability of chloroplast and hydrogenase activities in light and oxygen. Both chloroplasts and hydrogenases can be stabilized to a certain degree by immobilization in gels or by incubation in bovine serum albumin. Natural electron carriers of hydrogenases are ferredoxin, cytochrome c3 and NAD. Viologen dyes and synthetic iron-sulphur particles (Jeevanu) can substitute for the biological carriers. Methyl viologen, photoreduced in the presence of chloroplasts, can liberate H2 in combination with Pt (Adam's catalyst). An aqueous solution of proflavine can be photoreduced in the presence of organic electron donors such as EDTA, cysteine, dithiothreitol, etc.; the reduced proflavine can subsequently liberate H2 with MV-Pt, MV-hydrogenase, ferredoxin-hydrogenase or cytochrome-hydrogenase systems.

Differential sensitivity of porcine endogenous retrovirus to APOBEC3-mediated inhibition.

PubMed

Park, Sung-Han; Kim, Jin Ha; Jung, Yong-Tae

2015-08-01

Pigs are considered to be suitable xenotransplantation organ donors. However, the risk of pathogen transmission from pigs to humans is a major concern in the transplantation of porcine tissues. The porcine endogenous retroviruses (PERVs) PERV-A, PERV-A/C, and PERV-B can infect human cells, but PERV-C is an ecotropic virus infecting only pig cells. Thus, several strategies have been proposed to reduce PERV transmission in xenograft recipients. Human APOBEC3G (huA3G) is a single-strand DNA cytosine deaminase, which inactivates the coding capacity of the virus by deamination of cDNA cytosines to uracils. This reaction occurs within the (-) DNA strand during reverse transcription, resulting in a G-to-A mutation in the (+) strand. While recent data have shown that PERV-B is severely inhibited by huA3G and porcine A3Z2-Z3 (poA3F) in a pseudotype assay, little is known about PERV-C. Here, we compare the antiretroviral activities of huA3G, huA3F and poA3Z2-Z3 against PERV-C. Our data show that APOBEC3 was packaged into PERV-C particles and inhibited PERV-C replication in a dose-dependent manner. PERV-C infectivity was strongly inhibited by poA3Z2-Z3, but it did not markedly reduce PERV-B infectivity. This suggests that PERV-C Gag interacts efficiently with poA3Z2-Z3. In addition, we constructed stably huA3G- and poA3Z2-Z3-expressing 293-PERV-PK-CIRCE cells (human 293 cells infected with PK15-derived PERVs) to examine whether PERV is resistant to poA3Z2-Z3 in a virus-spreading assay. The stably expressed huA3G and poA3Z2-Z3 were more packaging-competent than transiently expressed APOBEC3 proteins. These results suggest that poA3Z2-Z3 can inhibit PERV replication in a pseudotype assay as well as in a virus-spreading assay.

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation

PubMed Central

Araujo, John C.; Poblenz, Ann; Corn, Paul G.; Parikh, Nila U.; Starbuck, Michael W.; Thompson, Jerry T.; Lee, Francis; Logothetis, Christopher J.; Darnay, Bryant G.

2013-01-01

Purpose Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Results Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. Experimental design We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Conclusion Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases. PMID:19855158

RNCR3 knockdown inhibits diabetes mellitus-induced retinal reactive gliosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chang; Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai; The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing

Retinal reactive gliosis is an important pathological feature of diabetic retinopathy. Identifying the underlying mechanisms causing reactive gliosis will be important for developing new therapeutic strategies for treating diabetic retinopathy. Herein, we show that long noncoding RNA-RNCR3 knockdown significantly inhibits retinal reactive gliosis. RNCR3 knockdown leads to a marked reduction in the release of several cytokines. RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration, as shown by less apoptotic retinal cells and ameliorative visual function. RNCR3 knockdown could also decrease Müller glial cell viability and proliferation, and reduce the expression of glial reactivity-related genes including GFAP and vimentin in vitro. Collectively, thismore » study shows that RNCR3 knockdown may be a promising strategy for the prevention of diabetes mellitus-induced retinal neurodegeneration. - Highlights: • RNCR3 knockdown inhibits retinal reactive gliosis. • RNCR3 knockdown causes a significant change in cytokine profile. • RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration. • RNCR3 knockdown affects Müller glial cell function in vitro.« less

Strategically timing inhibition of phosphatidylinositol 3-kinase to maximize therapeutic index in estrogen receptor alpha-positive, PIK3CA-mutant breast cancer

PubMed Central

Yang, Wei; Hosford, Sarah R.; Dillon, Lloye M.; Shee, Kevin; Liu, Stephanie C.; Bean, Jennifer R.; Salphati, Laurent; Pang, Jodie; Zhang, Xiaolin; Nannini, Michelle A.; Demidenko, Eugene; Bates, Darcy; Lewis, Lionel D.; Marotti, Jonathan D.; Eastman, Alan R.; Miller, Todd W.

2016-01-01

Purpose Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)-positive breast cancer in combination with anti-estrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER+ breast cancer will provide rationale for treatment scheduling to maximize therapeutic index. Experimental Design Anti-estrogen-sensitive and -resistant ER+ human breast cancer cell lines, and mice bearing PIK3CA-mutant xenografts were treated with the anti-estrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. Results Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6–9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared to fulvestrant alone. Conclusions Continuous and metronomic PI3K inhibition elicit robust anti-cancer effects in ER+, PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors

Stat3 orchestrates interaction between endothelial and tumor cells and inhibition of Stat3 suppresses brain metastasis of breast cancer cells.

PubMed

Lee, Hsueh-Te; Xue, Jianfei; Chou, Ping-Chieh; Zhou, Aidong; Yang, Phillip; Conrad, Charles A; Aldape, Kenneth D; Priebe, Waldemar; Patterson, Cam; Sawaya, Raymond; Xie, Keping; Huang, Suyun

2015-04-30

Brain metastasis is a major cause of morbidity and mortality in patients with breast cancer. Our previous studies indicated that Stat3 plays an important role in brain metastasis. Here, we present evidence that Stat3 functions at the level of the microenvironment of brain metastases. Stat3 controlled constitutive and inducible VEGFR2 expression in tumor-associated brain endothelial cells. Furthermore, inhibition of Stat3 by WP1066 decreased the incidence of brain metastases and increased survival in a preclinical model of breast cancer brain metastasis. WP1066 inhibited Stat3 activation in tumor-associated endothelial cells, reducing their infiltration and angiogenesis. WP1066 also inhibited breast cancer cell invasion. Our results indicate that WP1066 can inhibit tumor angiogenesis and brain metastasis mediated by Stat3 in endothelial and tumor cells.

A glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase.

PubMed

Dementin, Sébastien; Burlat, Bénédicte; De Lacey, Antonio L; Pardo, Alejandro; Adryanczyk-Perrier, Géraldine; Guigliarelli, Bruno; Fernandez, Victor M; Rousset, Marc

2004-03-12

Kinetic, EPR, and Fourier transform infrared spectroscopic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H(2)/ortho-H(2) conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover numbers for the oxidation of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H(2) oxidation and hydrogen isotope exchange activities, but did not change the para-H(2)/ortho-H(2) conversion activity. The spectroscopic properties of this mutant were totally different, especially in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.

ATF3 activates Stat3 phosphorylation through inhibition of p53 expression in skin cancer cells.

PubMed

Hao, Zhen-Feng; Ao, Jun-Hong; Zhang, Jie; Su, You-Ming; Yang, Rong-Ya

2013-01-01

ATF3, a member of the ATF/CREB family of transcription factors, has been found to be selectively induced by calcineurin/NFAT inhibition and to enhance keratinocyte tumor formation, although the precise role of ATF3 in human skin cancer and possible mechanisms remain unknown. In this study, clinical analysis of 30 skin cancer patients and 30 normal donors revealed that ATF3 was accumulated in skin cancer tissues. Functional assays demonstrated that ATF3 significantly promoted skin cancer cell proliferation. Mechanically, ATF3 activated Stat3 phosphorylation in skin cancer cell through regulation of p53 expression. Moreover, the promotion effect of ATF3 on skin cancer cell proliferation was dependent on the p53-Stat3 signaling cascade. Together, the results indicate that ATF3 might promote skin cancer cell proliferation and enhance skin keratinocyte tumor development through inhibiting p53 expression and then activating Stat3 phosphorylation.

Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum.

PubMed

Matare, Tapiwa; Nziramasanga, Pasipanodya; Gwanzura, Lovemore; Robertson, Valerie

2017-01-01

The potential of NaHCO 3 versus human serum to induce germ tube formation in Candida albicans was investigated. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO 3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20  μ L inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO 3 concentration. Parallel germ tube formation using human serum was done in test tubes. The optimum concentration of NaHCO 3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO 3 . Tris-maleate buffered NaHCO 3 induced germ tube formation in C. albicans isolates at rates similar to human serum.

New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies.

PubMed

Patel, Dhilon S; Bharatam, Prasad V

2006-01-01

Glycogen Synthase Kinase-3 is a regulatory serine/threonine kinase, which is being targeted for the treatment of a number of human diseases including type-2 diabetes mellitus, neurodegenerative diseases, cancer and chronic inflammation. Selective GSK-3 inhibition is an important requirement owing to the possibility of side effects arising from other kinases. A pharmacophore mapping strategy is employed in this work to identify new leads for selective GSK-3 inhibition. Ligands known to show selective GSK-3 inhibition were employed in generating a pharmacophore map using distance comparison method (DISCO). The derived pharmacophore map was validated using (i) important interactions involved in selective GSK-3 inhibitions, and (ii) an in-house database containing different classes of GSK-3 selective, non-selective and inactive molecules. New Lead identification was carried out by performing virtual screening using validated pharmacophoric query and three chemical databases namely NCI, Maybridge and Leadquest. Further data reduction was carried out by employing virtual filters based on (i) Lipinski's rule of 5 (ii) van der Waals bumps and (iii) restricting the number of rotatable bonds to seven. Final screening was carried out using FlexX based molecular docking study.

New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies

NASA Astrophysics Data System (ADS)

Patel, Dhilon S.; Bharatam, Prasad V.

2006-01-01

Glycogen Synthase Kinase-3 is a regulatory serine/threonine kinase, which is being targeted for the treatment of a number of human diseases including type-2 diabetes mellitus, neurodegenerative diseases, cancer and chronic inflammation. Selective GSK-3 inhibition is an important requirement owing to the possibility of side effects arising from other kinases. A pharmacophore mapping strategy is employed in this work to identify new leads for selective GSK-3 inhibition. Ligands known to show selective GSK-3 inhibition were employed in generating a pharmacophore map using distance comparison method (DISCO). The derived pharmacophore map was validated using (i) important interactions involved in selective GSK-3 inhibitions, and (ii) an in-house database containing different classes of GSK-3 selective, non-selective and inactive molecules. New Lead identification was carried out by performing virtual screening using validated pharmacophoric query and three chemical databases namely NCI, Maybridge and Leadquest. Further data reduction was carried out by employing virtual filters based on (i) Lipinski's rule of 5 (ii) van der Waals bumps and (iii) restricting the number of rotatable bonds to seven. Final screening was carried out using FlexX based molecular docking study.

Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages.

PubMed

Choi, Seoung-Ryoung; Britigan, Bradley E; Moran, David M; Narayanasamy, Prabagaran

2017-01-01

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages

PubMed Central

Choi, Seoung-ryoung; Britigan, Bradley E.; Moran, David M.

2017-01-01

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs. PMID:28542623

Structural basis of kynurenine 3-monooxygenase inhibition

PubMed Central

Amaral, Marta; Levy, Colin; Heyes, Derren J.; Lafite, Pierre; Outeiro, Tiago F.; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S.

2013-01-01

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (i.e. kynurenine pathway), leads to amelioration of Huntington’s disease-relevant phenotypes in yeast, fruit fly, and mouse models1–5, as well as a mouse model of Alzheimer’s disease3. KMO is a FAD-dependent monooxygenase, and is located in the outer mitochondrial membrane where it converts L-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders6, as well as cancer7,8, and several peripheral inflammatory conditions9. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained hitherto unknown. Here we report the first crystal structure of KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active site structure, preventing productive binding of the substrate kynurenine. Functional assays and targeted mutagenesis revealed that the active site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO:UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington’s, Alzheimer’s, and Parkinson’s diseases. PMID:23575632

Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3

PubMed Central

Rabbani, M. A. G.; Ribaudo, Michael; Guo, Ju-Tao

2016-01-01

ABSTRACT A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-β. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. IMPORTANCE The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3. PMID:27707917

Simultaneous Inhibition of EGFR and PI3K Enhances Radiosensitivity in Human Breast Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Ping; Zhang Qing; Torossian, Artour

2012-07-01

Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used tomore » investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF{sub SF2}) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF{sub SF2} at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may