Halotolerant and Resistant to High pH Hydrogenase from Haloalkaliphilic Sulfate-Reducing Bacterium Desulfonatronum thiodismutans

NASA Technical Reports Server (NTRS)

Detkova, Ekaterina N.; Pikuta, Elena V.; Hoover, Richard B.

2004-01-01

Hydrogenase is the key enzyme of energetic metabolism in cells, it catalyzing the converse reaction of hydrogen oxidation and responsible for consumption and excretion of hydrogen in bacteria. Hydrogenases are proteins containing either Nickel and Iron, or the only Iron in theirs active center. Hydrogenases have been found in many microorganisms, such as Methanogenic, acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria that could utilize the hydrogen as energy source or use it as electron sink. Hydrogenases are subject for wide physiological, biochemical, physicochemical and genetic studies due to theirs abilities produce the molecular hydrogen as alternative source of pure energy. Notwithstanding on enough large quantity of works that deal with intracellular and extrasellular enzymes of halophilic bacteria, the data about hydrogenases and theirs functions of salts practically are absent. The study of hydrogenase in cell-free extracts of extremely halophilic eubacterium Acetohalobium mabaticum showed dramatic increasing activity of the enzyme at high concentrations of NaCl and KCI (close to saturated solution). Here we present the data of free-cells extracted hydrogenase from new haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans, which grow on highly miniralized carbonate-bicarbonate medium in salinity range 1 to 7 % and at pH 7.8 - 10.5. Studied enzyme was active in Concentration range from 0 to 4.3 M NaCl with optimum at 1.0 M NaCl. At 1.0 M NaCl the enzyme activity was increased on 20 %, but with changing concentration from 2.1 M to 3.4 M the activity decreased and was kept on constant level. NaHCO3 inhibited hydrogenase activity on more then 30 %. The maximum of enzyme activity was observed at pH 9.5 with limits 7.5 and 11.5 that practically equal to pH optimum of bacterial growth. Therefore the hydrogenase of Desulfanatronum thiodismutans is tolerant to high concentrations of sodium salts and it also resistant to

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

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

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

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

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

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

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.

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.

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

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

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).

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

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.

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.

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

Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of Escherichia coli K-12

PubMed Central

2012-01-01

Background When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen (BV; Eo′ = -360 mV) as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays. Results Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo′ = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT. Conclusions The utilization of particular electron acceptors at

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.

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.

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

Protein kinase G–regulated production of H2S governs oxygen sensing

PubMed Central

Yuan, Guoxiang; Vasavda, Chirag; Peng, Ying-Jie; Makarenko, Vladislav V.; Raghuraman, Gayatri; Nanduri, Jayasri; Gadalla, Moataz M.; Semenza, Gregg L.; Kumar, Ganesh K.; Snyder, Solomon H.; Prabhakar, Nanduri R.

2015-01-01

Reflexes initiated by the carotid body, the principal O2-sensing organ, are critical for maintaining cardio-respiratory homeostasis during hypoxia. O2 sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H2S) synthesis by cystathionine-γ-lyase (CSE). We report that O2 stimulated the generation of CO, but not that of H2S, and required two cysteine residues in the heme regulatory motif (Cys265 and Cys282) of HO-2. CO stimulated protein kinase G (PKG)–dependent phosphorylation of Ser377 of CSE, inhibiting the production of H2S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H2S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O2 sensing through PKG-dependent regulation of H2S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing. PMID:25900831

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.

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.

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

H2 metabolism is widespread and diverse among human colonic microbes

PubMed Central

Wolf, Patricia G.; Biswas, Ambarish; Morales, Sergio E.; Greening, Chris; Gaskins, H. Rex

2016-01-01

ABSTRACT Microbial molecular hydrogen (H2) cycling is central to metabolic homeostasis and microbial composition in the human gastrointestinal tract. Molecular H2 is produced as an endproduct of carbohydrate fermentation and is reoxidised primarily by sulfate-reduction, acetogenesis, and methanogenesis. However, the enzymatic basis for these processes is incompletely understood and the hydrogenases responsible have not been investigated. In this work, we surveyed the genomic and metagenomic distribution of hydrogenase-encoding genes in the human colon to infer dominant mechanisms of H2 cycling. The data demonstrate that 70% of gastrointestinal microbial species listed in the Human Microbiome Project encode the genetic capacity to metabolise H2. A wide variety of anaerobically-adapted hydrogenases were present, with [FeFe]-hydrogenases predominant. We subsequently analyzed the hydrogenase gene content of stools from 20 healthy human subjects. The hydrogenase gene content of all samples was overwhelmingly dominated by fermentative and electron-bifurcating [FeFe]-hydrogenases emerging from the Bacteroidetes and Firmicutes. This study supports that H2 metabolism in the human gut is driven by fermentative H2 production and interspecies H2 transfer. However, it suggests that electron-bifurcation rather than respiration is the dominant mechanism of H2 reoxidation in the human colon, generating reduced ferredoxin to sustain carbon-fixation (e.g. acetogenesis) and respiration (via the Rnf complex). This work provides the first comprehensive bioinformatic insight into the mechanisms of H2 metabolism in the human colon. PMID:27123663

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.

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.

Mechanisms of intragastric pH sensing.

PubMed

Goo, Tyralee; Akiba, Yasutada; Kaunitz, Jonathan D

2010-12-01

Luminal amino acids and lack of luminal acidity as a result of acid neutralization by intragastric foodstuffs are powerful signals for acid secretion. Although the hormonal and neural pathways underlying this regulatory mechanism are well understood, the nature of the gastric luminal pH sensor has been enigmatic. In clinical studies, high pH, tryptic peptides, and luminal divalent metals (Ca(2+) and Mg(2+)) increase gastrin release and acid production. The calcium-sensing receptor (CaSR), first described in the parathyroid gland but expressed on gastric G cells, is a logical candidate for the gastric acid sensor. Because CaSR ligands include amino acids and divalent metals, and because extracellular pH affects ligand binding in the pH range of the gastric content, its pH, metal, and nutrient-sensing functions are consistent with physiologic observations. The CaSR is thus an attractive candidate for the gastric luminal sensor that is part of the neuroendocrine negative regulatory loop for acid secretion.

Unusual reaction of [NiFe]-hydrogenases with cyanide.

PubMed

Hexter, Suzannah V; Chung, Min-Wen; Vincent, Kylie A; Armstrong, Fraser A

2014-07-23

Cyanide reacts rapidly with [NiFe]-hydrogenases (hydrogenase-1 and hydrogenase-2 from Escherichia coli) under mild oxidizing conditions, inhibiting the electrocatalytic oxidation of hydrogen as recorded by protein film electrochemistry. Electrochemical, EPR, and FTIR measurements show that the final enzyme product, formed within a second (even under 100% H2), is the resting state known as Ni-B, which contains a hydroxido-bridged species, Ni(III)-μ(OH)-Fe(II), at the active site. "Cyanide inhibition" is easily reversed because it is simply the reductive activation of Ni-B. This paper brings back into focus an observation originally made in the 1940s that cyanide inhibits microbial H2 oxidation and addresses the interesting mechanism by which cyanide promotes the formation of Ni-B. As a much stronger nucleophile than hydroxide, cyanide binds more rapidly and promotes oxidation of Ni(II) to Ni(III); however, it is quickly replaced by hydroxide which is a far superior bridging ligand.

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.

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

Exploring the microbially-mediated soil H2 sink: A lab-based study of the physiology and related H2 consumption of isolates from the Harvard Forest

NASA Astrophysics Data System (ADS)

Rao, D.; Meredith, L. K.; Bosak, T.; Hansel, C. M.; Ono, S.; Prinn, R. G.

2012-12-01

Atmospheric hydrogen (H2) is a secondary greenhouse gas because it attenuates the removal of methane (CH4) from the atmosphere. The largest and most uncertain term in the H2 biogeochemical cycle, microbe-mediated soil uptake, is responsible for about 80% of Earth's tropospheric H2 sink. Recently, the first H2-oxidizing soil microorganisms were discovered (genus Streptomyces) whose low-threshold, high-affinity NiFe-hydrogenase functions at ambient H2 levels (approx. 530 ppb). To better understand the ecological function of this hydrogenase, we conducted a controlled laboratory study of the H2 uptake behavior in accordance with the complex life cycle development of the streptomycetes. Several strains of the genus Streptomyces containing a high-affinity NiFe- hydrogenase were isolated from soil at the Harvard Forest. The presence of this hydrogenase, detected by PCR amplification of the hydrogenase large subunit, predicted H2 uptake behavior in wild-type streptomycetes and in phylogenetically different organisms containing more distantly related versions of the gene. H2 uptake depended on the streptomyces' life cycle, reaching a maximum during spore formation. These findings reveal connections between environmental conditions, organismal life cycle, and H2 uptake. With the rise of H2-based energy sources and a potential change in the tropospheric concentration of H2, understanding the sources and sinks of this trace gas is important for the future.

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.

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

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

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.

Reinvestigation of the Steady-State Kinetics and Physiological Function of the Soluble NiFe-Hydrogenase I of Pyrococcus furiosus▿

PubMed Central

van Haaster, Daan J.; Silva, Pedro J.; Hagedoorn, Peter-Leon; Jongejan, Jaap A.; Hagen, Wilfred R.

2008-01-01

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H2 evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H2 production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low Km for H2 (∼20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the Km for NADP+ is ∼37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration. PMID:18156274

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

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

Action of inhibitors on hydrogenase in Azotobacter

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

Wilson, J.B.; Wilson, P.W.

1943-01-01

The inhibitors usually associated with the activity of the cytochrome oxidase system - cyanide and carbon monoxide - are also effective in reducing the oxidation of H/sub 2/ by intact cells of Azotobacter vinclandii. The hydrogenase system is more sensitive to CO than is the respiratory system. Oxidation of a carbon source and of hydrogen by Azotobacter cells is inhibited in a quantitatively different manner by the following compounds: sodium azide, hydroxylamine, sodium iodoacetate, and sodium fluoride. In every case, a concentration range which is definitely inhibitory for respiration has little or no effect on the hydrogenase activity. The differentialmore » inhibition by hydroxylamine explains certain observations in the literature which have been erroneously interpreted as demonstrating a specific inhibition by NH/sub 2/OH of biological nitrogen fixation. This supposed demonstration has been offered as support for the hypothesis that NH/sub 2/OH is an intermediate in the fixation reaction. The differential inhibitors can be used for detection of hydrogenase in cultures possessing a high endogenous respiration. The method is illustrated by an experiment with root nodule bacteria from pea and cowpea nodules. No hydrogenase was found in either. 8 references, 4 figures, 4 tables.« less

Activation Thermodynamics and H/D Kinetic Isotope Effect of the H ox to H red H + Transition in [FeFe] Hydrogenase

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

Ratzloff, Michael W.; Wilker, Molly B.; Mulder, David W.

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here in this paper we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The H ox→H redH + reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol -1 and a ~2.5-foldmore » kinetic isotope effect. Overall these results support electron injection from CdSe into CaI involving F-clusters, and that the H ox→H redH + step of catalytic proton reduction in CaI proceeds by a proton-dependent process.« less

Activation Thermodynamics and H/D Kinetic Isotope Effect of the H ox to H red H + Transition in [FeFe] Hydrogenase

DOE PAGES

Ratzloff, Michael W.; Wilker, Molly B.; Mulder, David W.; ...

2017-08-29

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here in this paper we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The H ox→H redH + reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol -1 and a ~2.5-foldmore » kinetic isotope effect. Overall these results support electron injection from CdSe into CaI involving F-clusters, and that the H ox→H redH + step of catalytic proton reduction in CaI proceeds by a proton-dependent process.« less

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.

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

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

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.

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.

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.

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.

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.

Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus

DOE PAGES

Wawrousek, Karen; Noble, Scott; Korlach, Jonas; ...

2014-12-05

In this article, we report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO 2 and H 2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encodingmore » both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H 2 in support of cell growth, and a regulatory hydrogenase that senses H 2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Finally and collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H 2 in support of cell growth.« less

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

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.

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.

Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803

PubMed Central

Maeda, Toshinari; Vardar, Gönül; Self, William T; Wood, Thomas K

2007-01-01

Background Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- ↔ H2 (g). Results Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a

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

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.

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

Protein film electrochemistry (PFE) is providing cutting-edge insight into the chemical principles underpinning biological hydrogen. Attached to an electrode, many enzymes exhibit "reversible" electrocatalytic behavior, meaning that a catalyzed redox reaction appears reversible or quasi-reversible when viewed by cyclic voltammetry. This efficiency is most relevant for enzymes that are inspiring advances in renewable energy, such as hydrogen-activating and CO2-reducing enzymes. Exploiting the rich repertoire of available instrumental methods, PFE experiments yield both a general snapshot and fine detail, all from tiny samples of enzyme. The dynamic electrochemical investigations blaze new trails and add exquisite detail to the information gained from structural and spectroscopic studies. This Account describes recent investigations of hydrogenases carried out in Oxford, including ideas initiated with PFE and followed through with complementary techniques, all contributing to an eventual complete picture of fast and efficient H2 activation without Pt. By immobilization of an enzyme on an electrode, catalytic electron flow and the chemistry controlling it can be addressed at the touch of a button. The buried nature of the active site means that structures that have been determined by crystallography or spectroscopy are likely to be protected, retained, and fully relevant in a PFE experiment. An electrocatalysis model formulated for the PFE of immobilized enzymes predicts interesting behavior and gives insight into why some hydrogenases are H2 producers and others are H2 oxidizers. Immobilization also allows for easy addition and removal of inhibitors along with precise potential control, one interesting outcome being that formaldehyde forms a reversible complex with reduced [FeFe]-hydrogenases, thereby providing insight into the order of electron and proton transfers. Experiments on O2-tolerant [NiFe]-hydrogenases show that O2 behaves like a reversible inhibitor: it

[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.

A model for the CO-inhibited form of [NiFe] hydrogenase: synthesis of (CO)3Fe(μ-StBu)3Ni{SC6H3-2,6-(mesityl)2} and reversible CO addition at the Ni site

PubMed Central

Ohki, Yasuhiro; Yasumura, Kazunari; Ando, Masaru; Shimokata, Satoko; Tatsumi, Kazuyuki

2010-01-01

A [NiFe] hydrogenase model compound having a distorted trigonal-pyramidal nickel center, (CO)3Fe(μ-StBu)3Ni(SDmp), 1 (Dmp = C6H3-2,6-(mesityl)2), was synthesized from the reaction of the tetranuclear Fe-Ni-Ni-Fe complex [(CO)3Fe(μ-StBu)3Ni]2(μ-Br)2, 2 with NaSDmp at -40 °C. The nickel site of complex 1 was found to add CO or CNtBu at -40 °C to give (CO)3Fe(StBu)(μ-StBu)2Ni(CO)(SDmp), 3, or (CO)3Fe(StBu)(μ-StBu)2Ni(CNtBu)(SDmp), 4, respectively. One of the CO bands of 3, appearing at 2055 cm-1 in the infrared spectrum, was assigned as the Ni-CO band, and this frequency is comparable to those observed for the CO-inhibited forms of [NiFe] hydrogenase. Like the CO-inhibited forms of [NiFe] hydrogenase, the coordination of CO at the nickel site of 1 is reversible, while the CNtBu adduct 4 is more robust. PMID:20147622

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.

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

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.

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.

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.

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

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

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.

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

Activation Thermodynamics and H/D Kinetic Isotope Effect of the Hox to HredH+ Transition in [FeFe] Hydrogenase.

PubMed

Ratzloff, Michael W; Wilker, Molly B; Mulder, David W; Lubner, Carolyn E; Hamby, Hayden; Brown, Katherine A; Dukovic, Gordana; King, Paul W

2017-09-20

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The H ox →H red H + reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol -1 and a ∼2.5-fold kinetic isotope effect. Overall, these results support electron injection from CdSe into CaI involving F-clusters, and that the H ox →H red H + step of catalytic proton reduction in CaI proceeds by a proton-dependent process.

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

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

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

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

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

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.

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.

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

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.

The roles of two O-donor ligands in the Fe2+-binding and H2O2-sensing by the Fe2+-dependent H2O2 sensor PerR.

PubMed

Ji, Chang-Jun; Yang, Yoon-Mo; Kim, Jung-Hoon; Ryu, Su-Hyun; Youn, Hwan; Lee, Jin-Won

2018-05-10

PerR is a metal-dependent peroxide sensing transcription factor which controls the expression of genes involved in peroxide resistance. The function of Bacillus subtilis PerR is mainly dictated by the regulatory metal ion (Fe 2+ or Mn 2+ ) coordinated by three N-donor ligands (His37, His91, and His93) and two O-donor ligands (Asp85 and Asp104). While H 2 O 2 sensing by PerR is mediated by Fe 2+ -dependent oxidation of N-donor ligand (either His37 or His91), one of the O-donor ligands (Asp104), but not Asp85, has been proposed as the key residue that regulates the sensitivity of PerR to H 2 O 2 . Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and H 2 O 2 sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of H 2 O 2 in the presence of excess Fe 2+ sufficient for the formation of the Fe 2+ -bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe 2+ , suggesting that Fe 2+ is not an effective corepressor for this mutant protein in vivo. Furthermore, in vitro metal titration assays indicate that D104E-PerR has a significantly reduced affinity for Fe 2+ , but not for Mn 2+ , when compared to wild type PerR. These data indicate that the type of O-donor ligand (Asp vs. Glu) at position 104 is an important determinant in providing high Fe 2+ -binding affinity required for the sensing of the physiologically relevant Fe 2+ -levels, in addition to its role in rendering PerR highly sensitive to physiological levels of H 2 O 2 . In comparison, the D85E-PerR did not show a perturbed change in Fe 2+ -binding affinity, however, it displayed a slightly decreased sensitivity to H 2 O 2 both in vivo and in vitro, suggesting that the type of O-donor ligand (Asp vs. Glu) at position 85 may be important for the fine-tuning of H 2 O 2 sensitivity. Copyright © 2018 Elsevier

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.

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.

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.

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.

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

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.

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.

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.

The H(2) sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site.

PubMed

Buhrke, Thorsten; Brecht, Marc; Lubitz, Wolfgang; Friedrich, Bärbel

2002-09-01

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

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.

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

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

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.

Metagenomic Evidence for H2 Oxidation and H2 Production by Serpentinite-Hosted Subsurface Microbial Communities

PubMed Central

Brazelton, William J.; Nelson, Bridget; Schrenk, Matthew O.

2012-01-01

Ultramafic rocks in the Earth’s mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H2). In order to assess the potential for microbial H2 utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field) and two continental serpentinite-hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland). Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H2-oxidizers. Both sites also yielded metagenomic evidence for microbial H2 production catalyzed by [FeFe]-hydrogenases in anaerobic Gram-positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood–Ljungdahl pathway. In general, our results point to H2-oxidizing Betaproteobacteria thriving in shallow, oxic–anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H2-powered primary production in serpentinite-hosted subsurface habitats. PMID:22232619

Metagenomic evidence for h(2) oxidation and h(2) production by serpentinite-hosted subsurface microbial communities.

PubMed

Brazelton, William J; Nelson, Bridget; Schrenk, Matthew O

2012-01-01

Ultramafic rocks in the Earth's mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H(2)). In order to assess the potential for microbial H(2) utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field) and two continental serpentinite-hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland). Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H(2)-oxidizers. Both sites also yielded metagenomic evidence for microbial H(2) production catalyzed by [FeFe]-hydrogenases in anaerobic Gram-positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood-Ljungdahl pathway. In general, our results point to H(2)-oxidizing Betaproteobacteria thriving in shallow, oxic-anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H(2)-powered primary production in serpentinite-hosted subsurface habitats.

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.

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

Isolation, observation, and computational modeling of proposed intermediates in catalytic proton reductions with the hydrogenase mimic Fe2(CO)6S2C6H4.

PubMed

Wright, Robert J; Zhang, Wei; Yang, Xinzheng; Fasulo, Meg; Tilley, T Don

2012-01-07

Proposed electrocatalytic proton reduction intermediates of hydrogenase mimics were synthesized, observed, and studied computationally. A new mechanism for H(2) generation appears to involve Fe(2)(CO)(6)(1,2-S(2)C(6)H(4)) (3), the dianions {[1,2-S(2)C(6)H(4)][Fe(CO)(3)(μ-CO)Fe(CO)(2)](2-) (3(2-)), the bridging hydride {[1,2-S(2)C(6)H(4)][Fe(CO)(3)(μ-CO)(μ-H)Fe(CO)(2)]}(-), 3H(-)(bridging), and the terminal hydride 3H(-)(term-stag), {[1,2-S(2)C(6)H(4)][HFe(CO)(3)Fe(CO)(3)]}(-), as intermediates. The dimeric sodium derivative of 3(2-), {[Na(2)(THF)(OEt(2))(3)][3(2-)]}(2) (4) was isolated from reaction of Fe(2)(CO)(6)(1,2-S(2)C(6)H(4)) (3) with excess sodium and was characterized by X-ray crystallography. It possesses a bridging CO and an unsymmetrically bridging dithiolate ligand. Complex 4 reacts with 4 equiv. of triflic or benzoic acid (2 equiv. per Fe center) to generate H(2) and 3 in 75% and 60% yields, respectively. Reaction of 4 with 2 equiv. of benzoic acid generated two hydrides in a 1.7 : 1 ratio (by (1)H NMR spectroscopy). Chemical shift calculations on geometry optimized structures of possible hydride isomers strongly suggest that the main product, 3H(-)(bridging), possesses a bridging hydride ligand, while the minor product is a terminal hydride, 3H(-)(term-stag). Computational studies support a catalytic proton reduction mechanism involving a two-electron reduction of 3 that severs an Fe-S bond to generate a dangling thiolate and an electron rich Fe center. The latter iron center is the initial site of protonation, and this event is followed by protonation at the dangling thiolate to give the thiol thiolate [Fe(2)H(CO)(6)(1,2-SHSC(6)H(4))]. This species then undergoes an intramolecular acid-base reaction to form a dihydrogen complex that loses H(2) and regenerates 3.

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

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.

CO-Bridged H-Cluster Intermediates in the Catalytic Mechanism of [FeFe]-Hydrogenase CaI.

PubMed

Ratzloff, Michael W; Artz, Jacob H; Mulder, David W; Collins, Reuben T; Furtak, Thomas E; King, Paul W

2018-06-20

The [FeFe]-hydrogenases ([FeFe] H 2 ases) catalyze reversible H 2 activation at the H-cluster, which is composed of a [4Fe-4S] H subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe] H ) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H 2 bond activation have been proposed. We have examined this question in the [FeFe] H 2 ase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of H ox led to formation of H red H + ([4Fe-4S] H 2+ -Fe I -Fe I ) and H red ' ([4Fe-4S] H 1+ -Fe II -Fe I ), with both states characterized by low frequency μ-CO IR modes consistent with a fully bridged [2Fe] H . Similar μ-CO IR modes were also identified for H red H + of the [FeFe] H 2 ase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive μ-CO mode, a component of the hydride bound H-cluster IR signal, H hyd . Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed H red H + was converted to H hyd . Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of H hyd and appearance of H ox , consistent with enzymatic turnover and H 2 formation. The results show for CaI that the μ-CO of [2Fe] H remains bridging for all of the "H red " states and that H red H + is on pathway to H hyd and H 2 evolution in the catalytic mechanism. These results provide a blueprint for designing small molecule catalytic analogs.

CO-Bridged H-Cluster Intermediates in the Catalytic Mechanism of [FeFe]-Hydrogenase CaI

DOE PAGES

Ratzloff, Michael W.; Artz, Jacob H.; Mulder, David W.; ...

2018-05-23

The [FeFe]-hydrogenases ([FeFe] H 2ases) catalyze reversible H 2 activation at the H-cluster, which is composed of a [4Fe-4S] H subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe] H) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H 2 bond activation have been proposed. We have examined this question in the [FeFe] H 2ase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of H ox ledmore » to formation of H redH + ([4Fe-4S] H 2+-Fe I-Fe I) and H red' ([4Fe-4S] H 1+-Fe II-Fe I), with both states characterized by low frequency μ-CO IR modes consistent with a fully bridged [2Fe] H. Similar μ-CO IR modes were also identified for H redH + of the [FeFe] H 2ase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive μ-CO mode, a component of the hydride bound H-cluster IR signal, H hyd. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed H redH + was converted to H hyd. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of Hhyd and appearance of H ox, consistent with enzymatic turnover and H 2 formation. The results show for CaI that the μ-CO of [2Fe] H remains bridging for all of the 'H red' states and that H redH + is on pathway to H hyd and H 2 evolution in the catalytic mechanism. Here, this provides a blueprint for designing small molecule catalytic analogs« less

CO-Bridged H-Cluster Intermediates in the Catalytic Mechanism of [FeFe]-Hydrogenase CaI

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

Ratzloff, Michael W.; Artz, Jacob H.; Mulder, David W.

The [FeFe]-hydrogenases ([FeFe] H 2ases) catalyze reversible H 2 activation at the H-cluster, which is composed of a [4Fe-4S] H subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe] H) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H 2 bond activation have been proposed. We have examined this question in the [FeFe] H 2ase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of H ox ledmore » to formation of H redH + ([4Fe-4S] H 2+-Fe I-Fe I) and H red' ([4Fe-4S] H 1+-Fe II-Fe I), with both states characterized by low frequency μ-CO IR modes consistent with a fully bridged [2Fe] H. Similar μ-CO IR modes were also identified for H redH + of the [FeFe] H 2ase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive μ-CO mode, a component of the hydride bound H-cluster IR signal, H hyd. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed H redH + was converted to H hyd. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of Hhyd and appearance of H ox, consistent with enzymatic turnover and H 2 formation. The results show for CaI that the μ-CO of [2Fe] H remains bridging for all of the 'H red' states and that H redH + is on pathway to H hyd and H 2 evolution in the catalytic mechanism. Here, this provides a blueprint for designing small molecule catalytic analogs« less

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

A Tale of Two Gases: Isotope Effects Associated with the Enzymatic Production of H2 and N2O

NASA Astrophysics Data System (ADS)

Yang, H.; Gandhi, H.; Kreuzer, H. W.; Moran, J.; Hill, E. A.; McQuarters, A.; Lehnert, N.; Ostrom, N. E.; Hegg, E. L.

2014-12-01

Stable isotopes can provide considerable insight into enzymatic mechanisms and fluxes in various biological processes. In our studies, we used stable isotopes to characterize both enzyme-catalyzed H2 and N2O production. H2 is a potential alternative clean energy source and also a key metabolite in many microbial communities. Biological H2 production is generally catalyzed by hydrogenases, enzymes that combine protons and electrons to produce H2 under anaerobic conditions. In our study, H isotopes and fractionation factors (α) were used to characterize two types of hydrogenases: [FeFe]- and [NiFe]-hydrogenases. Due to differences in the active site, the α associated with H2 production for [FeFe]- and [NiFe]-hydrogenases separated into two distinct clusters (αFeFe > αNiFe). The calculated kinetic isotope effects indicate that hydrogenase-catalyzed H2 production has a preference for light isotopes, consistent with the relative bond strengths of O-H and H-H bonds. Interestingly, the isotope effects associated with H2 consumption and H2-H2O exchange reactions were also characterized, but in this case no specific difference was observed between the different enzymes. N2O is a potent greenhouse gas with a global warming potential 300 times that of CO2, and the concentration of N2O is currently increasing at a rate of ~0.25% per year. Thus far, bacterial and fungal denitrification processes have been identified as two of the major sources of biologically generated N2O. In this study, we measured the δ15N, δ18O, δ15Nα (central N atom in N2O), and δ15Nβ (terminal N atom in N2O) of N2O generated by purified fungal P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum. We observed normal isotope effects for δ18O and δ15Nα, and inverse isotope effects for bulk δ15N (the average of Nα and Nβ) and δ15Nβ. The observed isotope effects have been used in conjunction with DFT calculations to provide important insight into the mechanism of P450nor. Similar

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

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

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.

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.

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.

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.

Optimization of pH sensing using silicon nanowire field effect transistors with HfO2 as the sensing surface.

PubMed

Zafar, Sufi; D'Emic, Christopher; Afzali, Ali; Fletcher, Benjamin; Zhu, Y; Ning, Tak

2011-10-07

Silicon nanowire field effect transistor sensors with SiO(2)/HfO(2) as the gate dielectric sensing surface are fabricated using a top down approach. These sensors are optimized for pH sensing with two key characteristics. First, the pH sensitivity is shown to be independent of buffer concentration. Second, the observed pH sensitivity is enhanced and is equal to the Nernst maximum sensitivity limit of 59 mV/pH with a corresponding subthreshold drain current change of ∼ 650%/pH. These two enhanced pH sensing characteristics are attributed to the use of HfO(2) as the sensing surface and an optimized fabrication process compatible with silicon processing technology.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts.

PubMed

Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

2016-09-29

The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

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.

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

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.

Increased photosystem II stability promotes H2 production in sulfur-deprived Chlamydomonas reinhardtii

PubMed Central

Volgusheva, Alena; Styring, Stenbjörn; Mamedov, Fikret

2013-01-01

Photobiological H2 production is an attractive option for renewable solar fuels. Sulfur-deprived cells of Chlamydomonas reinhardtii have been shown to produce hydrogen with the highest efficiency among photobiological systems. We have investigated the photosynthetic reactions during sulfur deprivation and H2 production in the wild-type and state transition mutant 6 (Stm6) mutant of Chlamydomonas reinhardtii. The incubation period (130 h) was dissected into different phases, and changes in the amount and functional status of photosystem II (PSII) were investigated in vivo by electron paramagnetic resonance spectroscopy and variable fluorescence measurements. In the wild type it was found that the amount of PSII is decreased to 25% of the original level; the electron transport from PSII was completely blocked during the anaerobic phase preceding H2 formation. This block was released during the H2 production phase, indicating that the hydrogenase withdraws electrons from the plastoquinone pool. This partly removes the block in PSII electron transport, thereby permitting electron flow from water oxidation to hydrogenase. In the Stm6 mutant, which has higher respiration and H2 evolution than the wild type, PSII was analogously but much less affected. The addition of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea revealed that ∼80% of the H2 production was inhibited in both strains. We conclude that (i) at least in the earlier stages, most of the electrons delivered to the hydrogenase originate from water oxidation by PSII, (ii) a faster onset of anaerobiosis preserves PSII from irreversible photoinhibition, and (iii) mutants with enhanced respiratory activity should be considered for better photobiological H2 production. PMID:23589846

Room temperature CO and H2 sensing with carbon nanoparticles.

PubMed

Kim, Daegyu; Pikhitsa, Peter V; Yang, Hongjoo; Choi, Mansoo

2011-12-02

We report on a shell-shaped carbon nanoparticle (SCNP)-based gas sensor that reversibly detects reducing gas molecules such as CO and H(2) at room temperature both in air and inert atmosphere. Crystalline SCNPs were synthesized by laser-assisted reactions in pure acetylene gas flow, chemically treated to obtain well-dispersed SCNPs and then patterned on a substrate by the ion-induced focusing method. Our chemically functionalized SCNP-based gas sensor works for low concentrations of CO and H(2) at room temperature even without Pd or Pt catalysts commonly used for splitting H(2) molecules into reactive H atoms, while metal oxide gas sensors and bare carbon-nanotube-based gas sensors for sensing CO and H(2) molecules can operate only at elevated temperatures. A pristine SCNP-based gas sensor was also examined to prove the role of functional groups formed on the surface of functionalized SCNPs. A pristine SCNP gas sensor showed no response to reducing gases at room temperature but a significant response at elevated temperature, indicating a different sensing mechanism from a chemically functionalized SCNP sensor.

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

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

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.

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

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.

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.

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.

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.

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

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.

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

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

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

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

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.

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.

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

A [4Fe-4S]-Fe(CO)(CN)-l-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly

NASA Astrophysics Data System (ADS)

Rao, Guodong; Tao, Lizhi; Suess, Daniel L. M.; Britt, R. David

2018-05-01

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate—the first organometallic precursor to the H-cluster—validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster.

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).

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.

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

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

Phylogenetic Evidence for H2 based Electron Bifurcation In Early Life

NASA Astrophysics Data System (ADS)

Adams, M. W.; Boyd, E. S.; Schut, G.; Peters, J.

2012-12-01

Energy conservation is a fundamental underpinning of all life and is accomplished by electron transport phosphorylation and/or substrate level phosphorylation. A third mechanism, known as flavin-based electron bifurcation, has recently been established as a mechanism by which life can conserve energy. In this mechanism, a flavin-containing multienzyme complex catalyzes the thermodynamically unfavorable reduction of low potential ferredoxin using electron donors with higher potentials, such as NAD(P)H or H2. Such endergonic reactions are driven forward through the simultaneous oxidation of the electron donor with higher potential acceptors such as NAD+ or heterodisulfide. Membrane associated energy converting [NiFe]hydrogenases (Ech, Eha) link the oxidation of ferredoxin with the production of H2 and in the process conserve energy in the form of an ion (Na+/H+) gradient. Ech/Eha exhibit a modular composition represented by a Na+/H+ antiporter domain and a [NiFe] hydrogenase domain. In addition, Ech/Eha can be accompanied by a formate dehyrogenase, carbon monoxide dehydrogenase, or an FAD/NAD(P)H module that enables coupling with these substrates. Representatives of Ech/Eha have been identified among anaerobic Archaea and Bacteria, including deeply rooted methanogens, sulfur-reducing Crenarcheota/Euryarchaeota as well as Thermotogae. Ech exhibit extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family (Nuo, Fpo). Metabolically, Ech generally couple the oxidation of carbon monooxide, formate or ferredoxin to the production of H2. In contrast, the Eha complex couples the translocation of Na+ and the oxidation of H2 to the reduction of ferredoxin, which is then available for the reduction of CO2 in methanogens. In the case of Eha, the gradient of Na+/H+ produced through translocation coupled to ferredoxin oxidation can in be used to drive the phosphorylation of ADP via an ATP synthase complex, thereby representing one of the

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

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

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.

Computational Studies of pH Sensing Design Principles in Proteins

NASA Astrophysics Data System (ADS)

Garrido Ruiz, Diego

Changes in pH are important regulatory signals for biological function, under physiological and pathological conditions. Recent advances in computer simulations strategies have made the exploration of the effects of charge titrations on protein function possible. In this work, I make use of these strategies to investigate the thermodynamic coupling between conformation and protonation states that give rise to pH-dependent function. As motivation for the rest of the work, I start by presenting a collaborative investigation on a pH-sensing mutant of the EGFR tyrosine kinase common to a set of distinct cancers. From then, I reduce the complexity of the systems under study to build models where exact enumeration of states is possible to inquire about the nature of the couplings between protonation states and conformation. Finally, I discuss detailed simulations of pH-sensing proteins for which I use the expectations and insights generated with simple models to identify and interpret couplings of interest for pH-dependent behavior.

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.

Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema

PubMed Central

Peng, Ying-Jie; Makarenko, Vladislav V.; Nanduri, Jayasri; Vasavda, Chirag; Raghuraman, Gayatri; Yuan, Guoxiang; Gadalla, Moataz M.; Kumar, Ganesh K.; Snyder, Solomon H.; Prabhakar, Nanduri R.

2014-01-01

Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O2 sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H2S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H2S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H2S generation, restoring O2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats. PMID:24395806

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.

Cells with impaired mitochondrial H2O2 sensing generate less •OH radicals and live longer.

PubMed

Martins, Dorival; Titorenko, Vladimir I; English, Ann M

2014-10-01

Mitochondria are major sites of reactive oxygen species (ROS) generation, and adaptive mitochondrial ROS signaling extends longevity. We aim at linking the genetic manipulation of mitochondrial H2O2 sensing in live cells to mechanisms driving aging in the model organism, Saccharomyces cerevisiae. To this end, we compare in vivo ROS (O2(•-), H2O2 and (•)OH) accumulation, antioxidant enzyme activities, labile iron levels, GSH depletion, and protein oxidative damage during the chronological aging of three yeast strains: ccp1Δ that does not produce the mitochondrial H2O2 sensor protein, cytochrome c peroxidase (Ccp1); ccp1(W191F) that produces a hyperactive variant of this sensor protein (Ccp1(W191F)); and the isogenic wild-type strain. Since they possess elevated manganese superoxide dismutase (Sod2) activity, young ccp1Δ cells accumulate low mitochondrial superoxide (O2(•-)) levels but high H2O2 levels. These cells exhibit stable aconitase activity and contain low amounts of labile iron and hydroxyl radicals ((•)OH). Furthermore, they undergo late glutathione (GSH) depletion, less mitochondrial protein oxidative damage and live longer than wild-type cells. In contrast, young ccp1(W191F) cells accumulate little H2O2, possess depressed Sod2 activity, enabling their O2(•-) level to spike and deactivate aconitase, which, ultimately, leads to greater mitochondrial oxidative damage, early GSH depletion, and a shorter lifespan than wild-type cells. Modulation of mitochondrial H2O2 sensing offers a novel interventional approach to alter mitochondrial H2O2 levels in live cells and probe the pro- versus anti-aging effects of ROS. The strength of mitochondrial H2O2 sensing modulates adaptive mitochondrial ROS signaling and, hence, lifespan.

Computational Design of Iron Diphosphine Complexes with Pendant Amines for Hydrogenation of CO2 to Methanol: A Mimic of [NiFe] Hydrogenase.

PubMed

Chen, Xiangyang; Jing, Yuanyuan; Yang, Xinzheng

2016-06-20

Inspired by the active-site structure of the [NiFe] hydrogenase, we have computationally designed the iron complex [P(tBu) 2 N(tBu) 2 )Fe(CN)2 CO] by using an experimentally ready-made diphosphine ligand with pendant amines for the hydrogenation of CO2 to methanol. Density functional theory calculations indicate that the rate-determining step in the whole catalytic reaction is the direct hydride transfer from the Fe center to the carbon atom in the formic acid with a total free energy barrier of 28.4 kcal mol(-1) in aqueous solution. Such a barrier indicates that the designed iron complex is a promising low-cost catalyst for the formation of methanol from CO2 and H2 under mild conditions. The key role of the diphosphine ligand with pendent amine groups in the reaction is the assistance of the cleavage of H2 by forming a Fe-H(δ-) ⋅⋅⋅H(δ+) -N dihydrogen bond in a fashion of frustrated Lewis pairs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii.

PubMed

Nagy, Valéria; Vidal-Meireles, André; Tengölics, Roland; Rákhely, Gábor; Garab, Győző; Kovács, László; Tóth, Szilvia Z

2016-07-01

In nature, H2 production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over-reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress-related genes, down-regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H2 production because it supplies electrons but the evolved O2 inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50-fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn-cluster of PSII, and afterwards, it can donate electrons to tyrozin Z(+) at a slow rate. This stage is followed by donor-side-induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H2 production. We also show that ascorbate influenced H2 evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation. © 2015 John Wiley & Sons Ltd.

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).

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

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.

Energy Conservation Associated with Ethanol Formation from H2 and CO2 in Clostridium autoethanogenum Involving Electron Bifurcation

PubMed Central

Mock, Johanna; Zheng, Yanning; Mueller, Alexander P.; Ly, San; Tran, Loan; Segovia, Simon; Nagaraju, Shilpa; Köpke, Michael; Dürre, Peter

2015-01-01

ABSTRACT Most acetogens can reduce CO2 with H2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO2 and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD+ oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2 and H2. IMPORTANCE Ethanol formation from syngas (H2, CO, and CO2) and from H2 and CO2 that is catalyzed by bacteria is presently a much-discussed process for

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

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.

2D Hexagonal Boron Nitride (2D-hBN) Explored for the Electrochemical Sensing of Dopamine.

PubMed

Khan, Aamar F; Brownson, Dale A C; Randviir, Edward P; Smith, Graham C; Banks, Craig E

2016-10-04

Crystalline 2D hexagonal boron nitride (2D-hBN) nanosheets are explored as a potential electrocatalyst toward the electroanalytical sensing of dopamine (DA). The 2D-hBN nanosheets are electrically wired via a drop-casting modification process onto a range of commercially available carbon supporting electrodes, including glassy carbon (GC), boron-doped diamond (BDD), and screen-printed graphitic electrodes (SPEs). 2D-hBN has not previously been explored toward the electrochemical detection/electrochemical sensing of DA. We critically evaluate the potential electrocatalytic performance of 2D-hBN modified electrodes, the effect of supporting carbon electrode platforms, and the effect of "mass coverage" (which is commonly neglected in the 2D material literature) toward the detection of DA. The response of 2D-hBN modified electrodes is found to be largely dependent upon the interaction between 2D-hBN and the underlying supporting electrode material. For example, in the case of SPEs, modification with 2D-hBN (324 ng) improves the electrochemical response, decreasing the electrochemical oxidation potential of DA by ∼90 mV compared to an unmodified SPE. Conversely, modification of a GC electrode with 2D-hBN (324 ng) resulted in an increased oxidation potential of DA by ∼80 mV when compared to the unmodified electrode. We explore the underlying mechanisms of the aforementioned examples and infer that electrode surface interactions and roughness factors are critical considerations. 2D-hBN is utilized toward the sensing of DA in the presence of the common interferents ascorbic acid (AA) and uric acid (UA). 2D-hBN is found to be an effective electrocatalyst in the simultaneous detection of DA and UA at both pH 5.0 and 7.4. The peak separations/resolution between DA and UA increases by ∼70 and 50 mV (at pH 5.0 and 7.4, respectively, when utilizing 108 ng of 2D-hBN) compared to unmodified SPEs, with a particularly favorable response evident in pH 5.0, giving rise to a

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

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

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.

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.

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.

Detection of pH and Enzyme-Free H2O2 Sensing Mechanism by Using GdO x Membrane in Electrolyte-Insulator-Semiconductor Structure.

PubMed

Kumar, Pankaj; Maikap, Siddheswar; Qiu, Jian-Tai; Jana, Surajit; Roy, Anisha; Singh, Kanishk; Cheng, Hsin-Ming; Chang, Mu-Tung; Mahapatra, Rajat; Chiu, Hsien-Chin; Yang, Jer-Ren

2016-12-01

A 15-nm-thick GdO x membrane in an electrolyte-insulator-semiconductor (EIS) structure shows a higher pH sensitivity of 54.2 mV/pH and enzyme-free hydrogen peroxide (H2O2) detection than those of the bare SiO2 and 3-nm-thick GdO x membranes for the first time. Polycrystalline grain and higher Gd content of the thicker GdO x films are confirmed by transmission electron microscopy (TEM) and X-ray photo-electron spectroscopy (XPS), respectively. In a thicker GdO x membrane, polycrystalline grain has lower energy gap and Gd(2+) oxidation states lead to change Gd(3+) states in the presence of H2O2, which are confirmed by electron energy loss spectroscopy (EELS). The oxidation/reduction (redox) properties of thicker GdO x membrane with higher Gd content are responsible for detecting H2O2 whereas both bare SiO2 and thinner GdO x membranes do not show sensing. A low detection limit of 1 μM is obtained due to strong catalytic activity of Gd. The reference voltage shift increases with increase of the H2O2 concentration from 1 to 200 μM owing to more generation of Gd(3+) ions, and the H2O2 sensing mechanism has been explained as well.

[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.

Importance of the Active Site "Canopy" Residues in an O2-Tolerant [NiFe]-Hydrogenase.

PubMed

Brooke, Emily J; Evans, Rhiannon M; Islam, Shams T A; Roberts, Gerri M; Wehlin, Sara A M; Carr, Stephen B; Phillips, Simon E V; Armstrong, Fraser A

2017-01-10

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a "canopy" above the bimetallic center, closest to the site at which exogenous agents CO and O 2 interact, substrate H 2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H 2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N-metal distance of 4.4 Å): its replacement with lysine lowers the H 2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H 2 /D 2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H 2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H 2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O 2 tolerance by stabilizing the oxidized resting Ni III -OH state ("Ni-B"). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.

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.

Highly Enhanced H2 Sensing Performance of Few-Layer MoS2/SiO2/Si Heterojunctions by Surface Decoration of Pd Nanoparticles.

PubMed

Hao, Lanzhong; Liu, Yunjie; Du, Yongjun; Chen, Zhaoyang; Han, Zhide; Xu, Zhijie; Zhu, Jun

2017-10-17

A novel few-layer MoS 2 /SiO 2 /Si heterojunction is fabricated via DC magnetron sputtering technique, and Pd nanoparticles are further synthesized on the device surface. The results demonstrate that the fabricated sensor exhibits highly enhanced responses to H 2 at room temperature due to the decoration of Pd nanoparticles. For example, the Pd-decorated MoS 2 /SiO 2 /Si heterojunction shows an excellent response of 9.2 × 10 3 % to H 2 , which is much higher than the values for the Pd/SiO 2 /Si and MoS 2 /SiO 2 /Si heterojunctions. In addition, the H 2 sensing properties of the fabricated heterojunction are dependent largely on the thickness of the Pd-nanoparticle layer and there is an optimized Pd thickness for the device to achieve the best sensing characteristics. Based on the microstructure characterization and electrical measurements, the sensing mechanisms of the Pd-decorated MoS 2 /SiO 2 /Si heterojunction are proposed. These results indicate that the Pd decoration of few-layer MoS 2 /SiO 2 /Si heterojunctions presents an effective strategy for the scalable fabrication of high-performance H 2 sensors.

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

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.

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.

pH sensing and regulation in cancer.

PubMed

Damaghi, Mehdi; Wojtkowiak, Jonathan W; Gillies, Robert J

2013-12-17

Cells maintain intracellular pH (pHi) within a narrow range (7.1-7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the pHi, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs). In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including cofilin and talin regulated actin (de-)polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate, and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H(+)-sensors, suggesting a mechanistic disconnect between intra- and extracellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.

Palladium and platinum-based nanoparticle functional sensor layers for selective H2 sensing

DOEpatents

Ohodnicki, Jr., Paul R.; Baltrus, John P.; Brown, Thomas D.

2017-07-04

The disclosure relates to a plasmon resonance-based method for H.sub.2 sensing in a gas stream utilizing a hydrogen sensing material. The hydrogen sensing material is comprises Pd-based or Pt-based nanoparticles having an average nanoparticle diameter of less than about 100 nanometers dispersed in an inert matrix having a bandgap greater than or equal to 5 eV, and an oxygen ion conductivity less than approximately 10.sup.-7 S/cm at a temperature of 700.degree. C. Exemplary inert matrix materials include SiO.sub.2, Al.sub.2O.sub.3, and Si.sub.3N.sub.4 as well as modifications to modify the effective refractive indices through combinations and/or doping of such materials. The hydrogen sensing material utilized in the method of this disclosure may be prepared using means known in the art for the production of nanoparticles dispersed within a supporting matrix including sol-gel based wet chemistry techniques, impregnation techniques, implantation techniques, sputtering techniques, and others.

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.

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

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.

Regioselectivity of H Cluster Oxidation

PubMed Central

2011-01-01

The H2-evolving potential of [FeFe] hydrogenases is severely limited by the oxygen sensitivity of this class of enzymes. Recent experimental studies on hydrogenase from C. reinhardtii point to O2-induced structural changes in the [Fe4S4] subsite of the H cluster. Here, we investigate the mechanistic basis of this observation by means of density functional theory. Unexpectedly, we find that the isolated H cluster shows a pathological catalytic activity for the formation of reactive oxygen species such as O2– and HO2–. After protonation of O2–, an OOH radical may coordinate to the Fe atoms of the cubane, whereas H2O2 specifically reacts with the S atoms of the cubane-coordinating cysteine residues. Both pathways are accompanied by significant structural distortions that compromise cluster integrity and thus catalytic activity. These results explain the experimental observation that O2-induced inhibition is accompanied by distortions of the [Fe4S4] moiety and account for the irreversibility of this process. PMID:22106822

Potential for microbial H2 and metal transformations associated with novel bacteria and archaea in deep terrestrial subsurface sediments

PubMed Central

Hernsdorf, Alex W; Amano, Yuki; Miyakawa, Kazuya; Ise, Kotaro; Suzuki, Yohey; Anantharaman, Karthik; Probst, Alexander; Burstein, David; Thomas, Brian C; Banfield, Jillian F

2017-01-01

Geological sequestration in deep underground repositories is the prevailing proposed route for radioactive waste disposal. After the disposal of radioactive waste in the subsurface, H2 may be produced by corrosion of steel and, ultimately, radionuclides will be exposed to the surrounding environment. To evaluate the potential for microbial activities to impact disposal systems, we explored the microbial community structure and metabolic functions of a sediment-hosted ecosystem at the Horonobe Underground Research Laboratory, Hokkaido, Japan. Overall, we found that the ecosystem hosted organisms from diverse lineages, including many from the phyla that lack isolated representatives. The majority of organisms can metabolize H2, often via oxidative [NiFe] hydrogenases or electron-bifurcating [FeFe] hydrogenases that enable ferredoxin-based pathways, including the ion motive Rnf complex. Many organisms implicated in H2 metabolism are also predicted to catalyze carbon, nitrogen, iron and sulfur transformations. Notably, iron-based metabolism is predicted in a novel lineage of Actinobacteria and in a putative methane-oxidizing ANME-2d archaeon. We infer an ecological model that links microorganisms to sediment-derived resources and predict potential impacts of microbial activity on H2 consumption and retardation of radionuclide migration. PMID:28350393

Potential for microbial H2 and metal transformations associated with novel bacteria and archaea in deep terrestrial subsurface sediments.

PubMed

Hernsdorf, Alex W; Amano, Yuki; Miyakawa, Kazuya; Ise, Kotaro; Suzuki, Yohey; Anantharaman, Karthik; Probst, Alexander; Burstein, David; Thomas, Brian C; Banfield, Jillian F

2017-08-01

Geological sequestration in deep underground repositories is the prevailing proposed route for radioactive waste disposal. After the disposal of radioactive waste in the subsurface, H 2 may be produced by corrosion of steel and, ultimately, radionuclides will be exposed to the surrounding environment. To evaluate the potential for microbial activities to impact disposal systems, we explored the microbial community structure and metabolic functions of a sediment-hosted ecosystem at the Horonobe Underground Research Laboratory, Hokkaido, Japan. Overall, we found that the ecosystem hosted organisms from diverse lineages, including many from the phyla that lack isolated representatives. The majority of organisms can metabolize H 2 , often via oxidative [NiFe] hydrogenases or electron-bifurcating [FeFe] hydrogenases that enable ferredoxin-based pathways, including the ion motive Rnf complex. Many organisms implicated in H 2 metabolism are also predicted to catalyze carbon, nitrogen, iron and sulfur transformations. Notably, iron-based metabolism is predicted in a novel lineage of Actinobacteria and in a putative methane-oxidizing ANME-2d archaeon. We infer an ecological model that links microorganisms to sediment-derived resources and predict potential impacts of microbial activity on H 2 consumption and retardation of radionuclide migration.

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.

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

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.

[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.

Investigation of Gas-Sensing Property of Acid-Deposited Polyaniline Thin-Film Sensors for Detecting H2S and SO2

PubMed Central

Dong, Xingchen; Zhang, Xiaoxing; Wu, Xiaoqing; Cui, Hao; Chen, Dachang

2016-01-01

Latent insulation defects introduced in manufacturing process of gas-insulated switchgears can lead to partial discharge during long-time operation, even to insulation fault if partial discharge develops further. Monitoring of decomposed components of SF6, insulating medium of gas-insulated switchgear, is a feasible method of early-warning to avoid the occurrence of sudden fault. Polyaniline thin-film with protonic acid deposited possesses wide application prospects in the gas-sensing field. Polyaniline thin-film sensors with only sulfosalicylic acid deposited and with both hydrochloric acid and sulfosalicylic acid deposited were prepared by chemical oxidative polymerization method. Gas-sensing experiment was carried out to test properties of new sensors when exposed to H2S and SO2, two decomposed products of SF6 under discharge. The gas-sensing properties of these two sensors were compared with that of a hydrochloric acid deposited sensor. Results show that the hydrochloric acid and sulfosalicylic acid deposited polyaniline thin-film sensor shows the most outstanding sensitivity and selectivity to H2S and SO2 when concentration of gases range from 10 to 100 μL/L, with sensitivity changing linearly with concentration of gases. The sensor also possesses excellent long-time and thermal stability. This research lays the foundation for preparing practical gas-sensing devices to detect H2S and SO2 in gas-insulated switchgears at room temperature. PMID:27834895

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.

VhuD Facilitates Electron Flow from H2 or Formate to Heterodisulfide Reductase in Methanococcus maripaludis

PubMed Central

Costa, Kyle C.; Lie, Thomas J.; Xia, Qin

2013-01-01

Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H2 via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F420-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth. PMID:24039260

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.

Disposable, Paper-Based, Inkjet-Printed Humidity and H2S Gas Sensor for Passive Sensing Applications

PubMed Central

Quddious, Abdul; Yang, Shuai; Khan, Munawar M.; Tahir, Farooq A.; Shamim, Atif; Salama, Khaled N.; Cheema, Hammad M.

2016-01-01

An inkjet-printed, fully passive sensor capable of either humidity or gas sensing is presented herein. The sensor is composed of an interdigitated electrode, a customized printable gas sensitive ink and a specialized dipole antenna for wireless sensing. The interdigitated electrode printed on a paper substrate provides the base conductivity that varies during the sensing process. Aided by the porous nature of the substrate, a change in relative humidity from 18% to 88% decreases the electrode resistance from a few Mega-ohms to the kilo-ohm range. For gas sensing, an additional copper acetate-based customized ink is printed on top of the electrode, which, upon reaction with hydrogen sulphide gas (H2S) changes, both the optical and the electrical properties of the electrode. A fast response time of 3 min is achieved at room temperature for a H2S concentration of 10 ppm at a relative humidity (RH) of 45%. The passive wireless sensing is enabled through an antenna in which the inner loop takes care of conductivity changes in the 4–5 GHz band, whereas the outer-dipole arm is used for chipless identification in the 2–3 GHz band. PMID:27929450

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.

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.

H2O2-Sensitive Isoforms of Drosophila melanogaster TRPA1 Act in Bitter-Sensing Gustatory Neurons to Promote Avoidance of UV During Egg-Laying

PubMed Central

Guntur, Ananya R.; Gou, Bin; Gu, Pengyu; He, Ruo; Stern, Ulrich; Xiang, Yang; Yang, Chung-Hui

2017-01-01

The evolutionarily conserved TRPA1 channel can sense various stimuli including temperatures and chemical irritants. Recent results have suggested that specific isoforms of Drosophila TRPA1 (dTRPA1) are UV-sensitive and that their UV sensitivity is due to H2O2 sensitivity. However, whether such UV sensitivity served any physiological purposes in animal behavior was unclear. Here, we demonstrate that H2O2-sensitive dTRPA1 isoforms promote avoidance of UV when adult Drosophila females are selecting sites for egg-laying. First, we show that blind/visionless females are still capable of sensing and avoiding UV during egg-laying when intensity of UV is high yet within the range of natural sunlight. Second, we show that such vision-independent UV avoidance is mediated by a group of bitter-sensing neurons on the proboscis that express H2O2-sensitive dTRPA1 isoforms. We show that these bitter-sensing neurons exhibit dTRPA1-dependent UV sensitivity. Importantly, inhibiting activities of these bitter-sensing neurons, reducing their dTRPA1 expression, or reducing their H2O2-sensitivity all significantly reduced blind females’ UV avoidance, whereas selectively restoring a H2O2-sensitive isoform of dTRPA1 in these neurons restored UV avoidance. Lastly, we show that specifically expressing the red-shifted channelrhodopsin CsChrimson in these bitter-sensing neurons promotes egg-laying avoidance of red light, an otherwise neutral cue for egg-laying females. Together, these results demonstrate a physiological role of the UV-sensitive dTRPA1 isoforms, reveal that adult Drosophila possess at least two sensory systems for detecting UV, and uncover an unexpected role of bitter-sensing taste neurons in UV sensing. PMID:27932542

H2O2-Sensitive Isoforms of Drosophila melanogaster TRPA1 Act in Bitter-Sensing Gustatory Neurons to Promote Avoidance of UV During Egg-Laying.

PubMed

Guntur, Ananya R; Gou, Bin; Gu, Pengyu; He, Ruo; Stern, Ulrich; Xiang, Yang; Yang, Chung-Hui

2017-02-01

The evolutionarily conserved TRPA1 channel can sense various stimuli including temperatures and chemical irritants. Recent results have suggested that specific isoforms of Drosophila TRPA1 (dTRPA1) are UV-sensitive and that their UV sensitivity is due to H 2 O 2 sensitivity. However, whether such UV sensitivity served any physiological purposes in animal behavior was unclear. Here, we demonstrate that H 2 O 2 -sensitive dTRPA1 isoforms promote avoidance of UV when adult Drosophila females are selecting sites for egg-laying. First, we show that blind/visionless females are still capable of sensing and avoiding UV during egg-laying when intensity of UV is high yet within the range of natural sunlight. Second, we show that such vision-independent UV avoidance is mediated by a group of bitter-sensing neurons on the proboscis that express H 2 O 2 -sensitive dTRPA1 isoforms. We show that these bitter-sensing neurons exhibit dTRPA1-dependent UV sensitivity. Importantly, inhibiting activities of these bitter-sensing neurons, reducing their dTRPA1 expression, or reducing their H 2 O 2 -sensitivity all significantly reduced blind females' UV avoidance, whereas selectively restoring a H 2 O 2 -sensitive isoform of dTRPA1 in these neurons restored UV avoidance. Lastly, we show that specifically expressing the red-shifted channelrhodopsin CsChrimson in these bitter-sensing neurons promotes egg-laying avoidance of red light, an otherwise neutral cue for egg-laying females. Together, these results demonstrate a physiological role of the UV-sensitive dTRPA1 isoforms, reveal that adult Drosophila possess at least two sensory systems for detecting UV, and uncover an unexpected role of bitter-sensing taste neurons in UV sensing. Copyright © 2017 by the Genetics Society of America.

The Interplay of Proton, Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii*

PubMed Central

Doebbe, Anja; Keck, Matthias; La Russa, Marco; Mussgnug, Jan H.; Hankamer, Ben; Tekçe, Ercan; Niehaus, Karsten; Kruse, Olaf

2010-01-01

To obtain a detailed picture of sulfur deprivation-induced H2 production in microalgae, metabolome analyses were performed during key time points of the anaerobic H2 production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H2 production process. This work reports on the differential analysis of metabolic profiles of the high H2-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H2 production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H2 production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast. PMID:20581114

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

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.

The indium oxide micro and nanopyramids: Morphology materializing and H2S sensing properties

NASA Astrophysics Data System (ADS)

Shariati, Mohsen

2015-07-01

Indium oxide (In2O3) pyramidal nano and microstructures were prepared by a thermal evaporation and condensation method. The preannealing step affected the nanostructures morphologies and their sensing capability. The nanosize structures have been fabricated in nucleated preorganized situation. By changing from prepared sites to undesired sites, the morphology was deteriorated. The synthesized In2O3 structures were characterized by field emission scanning electron microscopy (FESEM) and the X-ray diffraction (XRD) measurements. The FESEM images showed that nanostructures with 100-250 nm in size were fabricated. The XRD patterns indicated that most of the samples are crystalline. Then, the fabricated structures were investigated for H2S gas sensing. The nanocrystal pyramids were found to be sensitive to as low as 100 ppb of H2S gas at room temperature and microcrystal ones to 300 ppb. The nanopyramids demonstrated that they were very sensitive to gas presence and their response and recovery time were in a few seconds.

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.

Doping and defect-induced germanene: A superior media for sensing H2S, SO2, and CO2 gas molecules

NASA Astrophysics Data System (ADS)

Monshi, M. M.; Aghaei, S. M.; Calizo, I.

2017-11-01

First-principles calculations based on density functional theory (DFT) have been employed to investigate the structural, electronic, and gas-sensing properties of pure, defected, and doped germanene nanosheets. Our calculations have revealed that while a pristine germanene nanosheet adsorbs CO2 weakly, H2S moderately, and SO2 strongly, the introduction of vacancy defects increases the sensitivity significantly which is promising for future gas-sensing applications. Mulliken population analysis imparts that an appreciable amount of charge transfer occurs between gas molecules and a germanene nanosheet which supports our results for adsorption energies of the systems. The enhancement of the interactions between gas molecules and the germanene nanosheet has been further investigated by density of states. Projected density of states provides detailed insight of the gas molecule's contribution in the gas-sensing system. Additionally, the influences of substituted dopant atoms such as B, N, and Al in the germanene nanosheet have also been considered to study the impact on its gas sensing ability. There was no significant improvement found in the doped gas sensing capability of germanene over the vacancy defects, except for CO2 upon adsorption on N-doped germanene.

Controllable synthesis of green and blue fluorescent carbon nanodots for pH and Cu(2+) sensing in living cells.

PubMed

Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Zhao, Bo; Wen, Xiangping; Zhang, Guomei; Dong, Chuan; Shuang, Shaomin

2016-03-15

We report a controllable strategy for fabrication of green and blue fluorescent carbon nanodots (CDs), and demonstrate their applications for pH and Cu(2+) sensing in living cells. Green and blue fluorescent CDs have been synthesized by hydrothermal method and pyrolysis of leeks, respectively, providing an easy way for the production of CDs without the request of tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. Green fluorescent CDs (G-CDs) exhibit high tolerance to pH values and external cations. Blue fluorescent CDs (B-CDs) can be applied to pH and Cu(2+) sensing. The linear range of Cu(2+) detection is 0.01-10.00 μM and the detection limit is 0.05 μM. For pH detection, there is a good linearity in the pH range of 3.5-10.0. The linear and rapid response of B-CDs to Cu(2+) and pH is valuable for Cu(2+) and pH sensing in living cells. Confocal fluorescent imaging of human cervical carcinoma cells indicates that B-CDs could visualize Cu(2+) and pH fluctuations in living cells with negligible autofluorescence. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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.

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

Acidic pH sensing in the bacterial cytoplasm is required for Salmonella virulence.

PubMed

Choi, Jeongjoon; Groisman, Eduardo A

2016-09-01

pH regulates gene expression, biochemical activities and cellular behaviors. A mildly acidic pH activates the master virulence regulatory system PhoP/PhoQ in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The sensor PhoQ harbors an extracytoplasmic domain implicated in signal sensing, and a cytoplasmic domain controlling activation of the regulator PhoP. We now report that, surprisingly, a decrease in Salmonella's own cytoplasmic pH induces transcription of PhoP-activated genes even when the extracytoplasmic pH remains neutral. Amino acid substitutions in PhoQ's cytoplasmic domain hindered activation by acidic pH and attenuated virulence in mice, but did not abolish activation by low Mg(2+) or the antimicrobial peptide C18G. Conversely, removal of PhoQ's extracytoplasmic domains prevented the response to the latter PhoQ-activating signals but not to acidic pH. PhoP-dependent genes were minimally induced by acidic pH in the non-pathogenic species Salmonella bongori but were activated by low Mg(2+) and C18G as in pathogenic S. enterica. Our findings indicate that the sensor PhoQ enables S. enterica to respond to both host- and bacterial-derived signals that alter its cytoplasmic pH. © 2016 John Wiley & Sons Ltd.

Effects of annealing temperature on the H2-sensing properties of Pd-decorated WO3 nanorods

NASA Astrophysics Data System (ADS)

Lee, Sangmin; Lee, Woo Seok; Lee, Jae Kyung; Hyun, Soong Keun; Lee, Chongmu; Choi, Seungbok

2018-03-01

The temperature of the post-annealing treatment carried out after noble metal deposition onto semiconducting metal oxides (SMOs) must be carefully optimized to maximize the sensing performance of the metal-decorated SMO sensors. WO3 nanorods were synthesized by thermal evaporation of WO3 powders and decorated with Pd nanoparticles using a sol-gel method, followed by an annealing process. The effects of the annealing temperature on the hydrogen gas-sensing properties of the Pd-decorated WO3 nanorods were then examined; the optimal annealing temperature, leading to the highest response of the WO3 nanorod sensor to H2, was determined to be 600 °C. Post-annealing at 600 °C resulted in nanorods with the highest surface area-to-volume ratio, as well as in the optimal size and the largest number of deposited Pd nanoparticles, leading to the highest response and the shortest response/recovery times toward H2. The improved H2-sensing performance of the Pd-decorated WO3 nanorod sensor, compared to a sensor based on pristine WO3 nanorods, is attributed to the enhanced catalytic activity, increased surface area-to-volume ratio, and higher amounts of surface defects.

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

PubMed Central

Gutiérrez‐Sanz, Óscar; Natale, Paolo; Márquez, Ileana; Marques, Marta C.; Zacarias, Sonia; Pita, Marcos; Pereira, Inês A. C.

2016-01-01

Abstract ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0‐ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode‐assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products. PMID:26991333

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

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

Dually Fluorescent Sensing of pH and Dissolved Oxygen Using a Membrane Made from Polymerizable Sensing Monomers.

PubMed

Tian, Yanqing; Shumway, Bradley R; Youngbull, A Cody; Li, Yongzhong; Jen, Alex K-Y; Johnson, Roger H; Meldrum, Deirdre R

2010-06-03

Using a thermal polymerization approach and polymerizable pH and oxygen sensing monomers with green and red emission spectra, respectively, new pH, oxygen, and their dual sensing membranes were prepared using poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) as a matrix. The sensors were grafted on acrylate-modified quartz glass and characterized under different pH values, oxygen concentrations, ion strengths, temperatures and cell culture media. The pH and oxygen sensors were excited using the same excitation wavelength and exhibited well-separated emission spectra. The pH-sensing films showed good response over the pH range 5.5 to 8.5, corresponding to pK(a) values in the biologically-relevant range between 6.9 and 7.1. The oxygen-sensing films exhibited linear Stern-Volmer quenching responses to dissolved oxygen. As the sensing membranes were prepared using thermally initiated polymerization of sensing moiety-containing monomers, no leaching of the sensors from the membranes to buffers or medium was observed. This advantageous characteristic accounts in part for the sensors' biocompatibility without apparent toxicity to HeLa cells after 40 hours incubation. The dual-sensing membrane was used to measure pH and dissolved oxygen simultaneously. The measured results correlated with the set-point values.

Ratiometric photoluminescence sensing based on Ti3C2 MXene quantum dots as an intracellular pH sensor.

PubMed

Chen, Xu; Sun, Xueke; Xu, Wen; Pan, Gencai; Zhou, Donglei; Zhu, Jinyang; Wang, He; Bai, Xue; Dong, Biao; Song, Hongwei

2018-01-18

Intracellular pH sensing is of importance and can be used as an indicator for monitoring the evolution of various diseases and the health of cells. Here, we developed a new class of surface-functionalized MXene quantum dots (QDs), Ti 3 C 2 , by the sonication cutting and hydrothermal approach and further explored their intracellular pH sensing. The functionalized Ti 3 C 2 QDs exhibit bright excitation-dependent blue photoluminescence (PL) originating from the size effect and surface defects. Meanwhile, Ti 3 C 2 QDs demonstrate a high PL response induced by the deprotonation of the surface defects. Furthermore, combining the highly pH sensitive Ti 3 C 2 QDs with the pH insensitive [Ru(dpp) 3 ]Cl 2 , we developed a ratiometric pH sensor to quantitatively monitor the intracellular pH values. These novel MXene quantum dots can serve as a promising platform for developing practical fluorescent nanosensors.

Pt-decorated GaN nanowires with significant improvement in H2 gas-sensing performance at room temperature.

PubMed

Abdullah, Q N; Yam, F K; Hassan, Z; Bououdina, M

2015-12-15

Superior sensitivity towards H2 gas was successfully achieved with Pt-decorated GaN nanowires (NWs) gas sensor. GaN NWs were fabricated via chemical vapor deposition (CVD) route. Morphology (field emission scanning electron microscopy and transmission electron microscopy) and crystal structure (high resolution X-ray diffraction) characterizations of the as-synthesized nanostructures demonstrated the formation of GaN NWs having a wurtzite structure, zigzaged shape and an average diameter of 30-166nm. The Pt-decorated GaN NWs sensor shows a high response of 250-2650% upon exposure to H2 gas concentration from 7 to 1000ppm respectively at room temperature (RT), and then increases to about 650-4100% when increasing the operating temperature up to 75°C. The gas-sensing measurements indicated that the Pt-decorated GaN NWs based sensor exhibited efficient detection of H2 at low concentration with excellent sensitivity, repeatability, and free hysteresis phenomena over a period of time of 100min. The large surface-to-volume ratio of GaN NWs and the catalytic activity of Pt metal are the most influential factors leading to the enhancement of H2 gas-sensing performances through the improvement of the interaction between the target molecules (H2) and the sensing NWs surface. The attractive low-cost, low power consumption and high-performance of the resultant decorated GaN NWs gas sensor assure their uppermost potential for H2 gas sensor working at low operating temperature. Copyright © 2015 Elsevier Inc. All rights reserved.

Fabrication of a LRET-based upconverting hybrid nanocomposite for turn-on sensing of H2O2 and glucose

NASA Astrophysics Data System (ADS)

Wu, Shuang; Kong, Xiang-Juan; Cen, Yao; Yuan, Jing; Yu, Ru-Qin; Chu, Xia

2016-04-01

Blood glucose detecting has aroused considerable attention because diabetes mellitus has become a worldwide publish health problem. Herein, we construct an exceptionally simple upconverting hybrid nanocomposite, composed of DNA-templated Ag nanoparticles (DNA-AgNPs) and NaYF4:Yb/Tm@NaYF4 core-shell upconversion nanoparticles (UCNPs), for the sensing of H2O2 and glucose. In this design, UCNPs with bared surface act as the donor, and DNA-AgNPs serve as efficient quenchers. DNA-AgNPs can be directly assembled on the bared surface of UCNPs, which further decreases the distance of donor-to-acceptor. The formation of DNA-AgNPs/UCNP nanocomposite results in luminescence quenching of UCNP by DNA-AgNPs through luminescence resonance energy transfer (LRET). Upon H2O2 addition, AgNPs can be etched and transformed into Ag+, leading to inhibition of the LRET process and causing the recovery of upconversion luminescence. Based on the conversion of glucose into H2O2 by glucose oxidase, the DNA-AgNPs/UCNP nanocomposite can also be exploited for glucose sensing. Moreover, due to the non-autofluorescence offered by UCNPs, the approach developed can be applied to monitor glucose levels in human serum samples with satisfactory results.Blood glucose detecting has aroused considerable attention because diabetes mellitus has become a worldwide publish health problem. Herein, we construct an exceptionally simple upconverting hybrid nanocomposite, composed of DNA-templated Ag nanoparticles (DNA-AgNPs) and NaYF4:Yb/Tm@NaYF4 core-shell upconversion nanoparticles (UCNPs), for the sensing of H2O2 and glucose. In this design, UCNPs with bared surface act as the donor, and DNA-AgNPs serve as efficient quenchers. DNA-AgNPs can be directly assembled on the bared surface of UCNPs, which further decreases the distance of donor-to-acceptor. The formation of DNA-AgNPs/UCNP nanocomposite results in luminescence quenching of UCNP by DNA-AgNPs through luminescence resonance energy transfer (LRET). Upon H

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

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.

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase*

PubMed Central

Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L.; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

2015-01-01

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. PMID:25666617

A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.

PubMed

Abou-Hamdan, Abbas; Ceccaldi, Pierre; Lebrette, Hugo; Gutiérrez-Sanz, Oscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; De Lacey, Antonio L; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

2015-03-27

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Arginine-Containing Ligands Enhance H-2 Oxidation Catalyst Performance

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

Dutta, Arnab; Roberts, John A.; Shaw, Wendy J.

2014-06-16

In H2 fuel cells, performance depends on factors controlling turnover frequency and energy efficiency in the electrocatalytic oxidation of H2. Nature uses the hydrogenase enzymes to oxidize H2 at high turnover frequencies (up to 20,000 s-1) and low overpotentials (<100 mV), while the fastest synthetic catalyst reported to date only oxidizes H2 at 50 s-1 under 1 atm H2. Here we report a water-soluble complex incorporating the amino acid arginine, [NiII(PCy2NArg2)2]6+, that operates at 210 s-1 (180 mV overpotential) under 1 atm H2 and 144,000 s-1 (460 mV overpotential) under 133 atm H2. The complex functions from pH 0-14 withmore » rates increasing at lower pH values. The arginine groups impart water solubility and play a critical role in enhancing turnover frequency, most consistent with an intramolecular Arg-Arg interaction that controls the structure of the catalyst active site. This work was funded by the Office of Science Early Career Research Program through the US DOE, BES (AD, WJS), and the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US DOE, BES (JASR). PNNL is operated by Battelle for the US DOE.« less

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.

Effect of ordered mesoporous carbon contact layer on the sensing performance of sputtered RuO2 thin film pH sensor.

PubMed

Lonsdale, W; Maurya, D K; Wajrak, M; Alameh, K

2017-03-01

The effect of contact layer on the pH sensing performance of a sputtered RuO 2 thin film pH sensor is investigated. The response of pH sensors employing RuO 2 thin film electrodes on screen-printed Pt, carbon and ordered mesoporous carbon (OMC) contact layers are measured over a pH range from 4 to 10. Working electrodes with OMC contact layer are found to have Nernstian pH sensitivity (-58.4mV/pH), low short-term drift rate (5.0mV/h), low hysteresis values (1.13mV) and fast reaction times (30s), after only 1h of conditioning. A pH sensor constructed with OMC carbon contact layer displays improved sensing performance compared to Pt and carbon-based counterparts, making this electrode more attractive for applications requiring highly-accurate pH sensing with reduced conditioning time. Copyright © 2016 Elsevier B.V. All rights reserved.

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

A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.

PubMed

Feng, Lihui; Rutherford, Steven T; Papenfort, Kai; Bagert, John D; van Kessel, Julia C; Tirrell, David A; Wingreen, Ned S; Bassler, Bonnie L

2015-01-15

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response. Copyright © 2015 Elsevier Inc. All rights reserved.

Electron Bifurcation Involved in the Energy Metabolism of the Acetogenic Bacterium Moorella thermoacetica Growing on Glucose or H2 plus CO2

PubMed Central

Huang, Haiyan; Wang, Shuning; Moll, Johanna

2012-01-01

Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO2 molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fdred2−) molecules. In the reductive part, 2 CO2 molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO2 are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO2 reduction to acetic acid are generated by the reduction of 2 NADP+ molecules with 1 NADH and 1 Fdred2− catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD+ and Fd with 2 H2 molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H2-CO2-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H2-CO2-grown cells additionally contain high specific activities of an NADP+-dependent hydrogenase that catalyzes the reduction of NADP+ with H2. This activity is hardly detectable in glucose-grown cells. PMID:22582275

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).

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.

Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes.

PubMed

Pi, Jian; Jawed, Muhammad; Wang, Jun; Xu, Li; Yan, Yunjun

2016-01-01

In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3. Copyright © 2015 Elsevier Inc. All rights reserved.

Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

PubMed

Kondo, Ayano; Yamamoto, Shogo; Nakaki, Ryo; Shimamura, Teppei; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Yoshida, Tetsuo; Aburatani, Hiroyuki; Osawa, Tsuyoshi

2017-02-28

Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Purification and Characterization of [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

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

Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.

2011-08-02

The γ-proteobacterium Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that was implicated in both H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned into a protein expression vector. The resulting plasmid was transformed into a MR-1 mutant deficient in H2 formation. Expression of MR-1 [NiFe]-H2ase in trans restored the mutant’s ability to produce H2 at 37% of that for wild type. Following expression, MR-1 [NiFe]-H2ase was purified to near homogeneity. The purified MR-1 [NiFe]-H2ase could couplemore » H2 oxidation to reduction of Tc(VII) and methyl viologen directly. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated Tc(VII) but not methyl viologen reductions. Under the conditions tested, Tc(VII) reduction was complete in Tris buffer but not in HEPES buffer. The reduced Tc(IV) was soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc(IV) precipitates formed in HEPES buffer were packed with crystallites. Although X-ray absorption near-edge spectroscopy measurements confirmed that the reduction products found in both buffers were Tc(IV), extended X-ray adsorption fine-structure measurements revealed that these products were very different. While the product in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2•nH2O. These results shows for the first time that MR-1 [NiFe]-H2ase is a bidirectional enzyme that catalyzes both H2 formation and oxidation as well as Tc(VII) reduction directly by coupling H2 oxidation.« less

Sensing Characteristics of Flame-Spray-Made Pt/ZnO Thick Films as H2 Gas Sensor

PubMed Central

Tamaekong, Nittaya; Liewhiran, Chaikarn; Wisitsoraat, Anurat; Phanichphant, Sukon

2009-01-01

Hydrogen sensing of thick films of nanoparticles of pristine, 0.2, 1.0 and 2.0 atomic percentage of Pt concentration doped ZnO were investigated. ZnO nanoparticles doped with 0.2–2.0 at.% Pt were successfully produced in a single step by flame spray pyrolysis (FSP) technique using zinc naphthenate and platinum(II) acetylacetonate as precursors dissolved in xylene. The particle properties were analyzed by XRD, BET, SEM and TEM. Under the 5/5 (precursor/oxygen) flame condition, ZnO nanoparticles and nanorods were observed. The crystallite sizes of ZnO spheroidal and hexagonal particles were found to be ranging from 5 to 20 nm while ZnO nanorods were seen to be 5–20 nm wide and 20–40 nm long. ZnO nanoparticles paste composed of ethyl cellulose and terpineol as binder and solvent respectively was coated on Al2O3 substrate interdigitated with gold electrodes to form thin films by spin coating technique. The thin film morphology was analyzed by SEM technique. The gas sensing properties toward hydrogen (H2) was found that the 0.2 at.% Pt/ZnO sensing film showed an optimum H2 sensitivity of ∼164 at hydrogen concentration in air of 1 volume% at 300 °C and a low hydrogen detection limit of 50 ppm at 300 °C operating temperature. PMID:22399971

Nanoparticle/Polymer assembled microcapsules with pH sensing property.

PubMed

Zhang, Pan; Song, Xiaoxue; Tong, Weijun; Gao, Changyou

2014-10-01

The dual-labeled microcapsules via nanoparticle/polymer assembly based on polyamine-salt aggregates can be fabricated for the ratiometric intracellular pH sensing. After deposition of SiO2 nanoparticles on the poly(allylamine hydrochloride)/multivalent anionic salt aggregates followed by silicic acid treatment, the generated microcapsules are stable in a wide pH range (3.0 ∼ 8.0). pH sensitive dye and pH insensitive dye are simultaneously labeled on the capsules, which enable the ratiometric pH sensing. Due to the rough and positively charged surface, the microcapsules can be internalized by several kinds of cells naturally. Real-time measurement of intracellular pH in several living cells shows that the capsules are all located in acidic organelles after being taken up. Furthermore, the negatively charged DNA and dyes can be easily encapsulated into the capsules via charge interaction. The microcapsules with combination of localized pH sensing and drug loading abilities have many advantages, such as following the real-time transportation and processing of the carriers in cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of H2 photoproduction by marine green alga Tetraselmis subcordiformis integrated with an alkaline fuel cell.

PubMed

Guo, Zhen; Li, Ying; Guo, Haiyan

2016-03-01

To investigate the feasibility of coupling carbonyl cyanide m-chlorophenylhydrazone-regulated photohydrogen production by Tetraselmis subcordiformis in a photobioreactor to an alkaline fuel cell (AFC). H2 evolution kinetics in the AFC integrated process was characterized. The duration of H2 evolution was prolonged and its yield was improved about 1.5-fold (to 78 ± 5 ml l(-1)) compared with that of the process without AFC. Improved H2 yield was possibly caused by removal of H2 feedback inhibition by H2 consumption in situ. Decreases in the H2 production rate correlated with the gradual deactivation of PSII and hydrogenase activities. The H2 yield was closely associated with catabolism of starch and protein. A marine green algal CO2-supplemented culture integrated with in situ H2-consumption by an AFC system was developed as a viable protocol for the H2 production.

Evolution of resistive switching mechanism through H2O2 sensing by using TaOx-based material in W/Al2O3/TaOx/TiN structure

NASA Astrophysics Data System (ADS)

Chakrabarti, Somsubhra; Panja, Rajeswar; Roy, Sourav; Roy, Anisha; Samanta, Subhranu; Dutta, Mrinmoy; Ginnaram, Sreekanth; Maikap, Siddheswar; Cheng, Hsin-Ming; Tsai, Ling-Na; Chang, Ya-Ling; Mahapatra, Rajat; Jana, Debanjan; Qiu, Jian-Tai; Yang, Jer-Ren

2018-03-01

Understanding of resistive switching mechanism through H2O2 sensing and improvement of switching characteristics by using TaOx-based material in W/Al2O3/TaOx/TiN structure have been reported for the first time. Existence of amorphous Al2O3/TaOx layer in the RRAM devices has been confirmed by transmission electron microscopy. By analyzing the oxidation states of Ta2+/Ta5+ for TaOx switching material and W0/W6+ for WOx layer at the W/TaOx interface through X-ray photoelectron spectroscopy and H2O2 sensing, the reduction-oxidation mechanism under Set/Reset occurs only in the TaOx layer for the W/Al2O3/TaOx/TiN structures. This leads to higher Schottky barrier height at the W/Al2O3 interface (0.54 eV vs. 0.46 eV), higher resistance ratio, and long program/erase endurance of >108 cycles with 100 ns pulse width at a low operation current of 30 μA. Stable retention of more than 104 s at 85 °C is also obtained. Using conduction mechanism and reduction-oxidation reaction, current-voltage characteristic has been simulated. Both TaOx and WOx membranes have high pH sensitivity values of 47.65 mV/pH and 49.25 mV/pH, respectively. Those membranes can also sense H2O2 with a low concentration of 1 nM in an electrolyte-insulator-semiconductor structure because of catalytic activity, while the Al2O3 membrane does not show sensing. The TaOx material in W/Al2O3/TaOx/TiN structure does not show only a path towards high dense, small size memory application with understanding of switching mechanism but also can be used for H2O2 sensors.

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.

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

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle.

PubMed

Fusi, L; Brunello, E; Yan, Z; Irving, M

2016-10-31

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway.

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

PubMed Central

Fusi, L.; Brunello, E.; Yan, Z.; Irving, M.

2016-01-01

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway. PMID:27796302

Endoscopic sensing of alveolar pH.

PubMed

Choudhury, D; Tanner, M G; McAughtrie, S; Yu, F; Mills, B; Choudhary, T R; Seth, S; Craven, T H; Stone, J M; Mati, I K; Campbell, C J; Bradley, M; Williams, C K I; Dhaliwal, K; Birks, T A; Thomson, R R

2017-01-01

Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation.

Endoscopic sensing of alveolar pH

PubMed Central

Choudhury, D.; Tanner, M. G.; McAughtrie, S.; Yu, F.; Mills, B.; Choudhary, T. R.; Seth, S.; Craven, T. H.; Stone, J. M.; Mati, I. K.; Campbell, C. J.; Bradley, M.; Williams, C. K. I.; Dhaliwal, K.; Birks, T. A.; Thomson, R. R.

2016-01-01

Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation. PMID:28101415

Induction of a proton gradient across a gold-supported biomimetic membrane by electroenzymatic H2 oxidation.

PubMed

Gutiérrez-Sanz, Óscar; Tapia, Cristina; Marques, Marta C; Zacarias, Sonia; Vélez, Marisela; Pereira, Inês A C; De Lacey, Antonio L

2015-02-23

Energy-transduction mechanisms in living organisms, such as photosynthesis and respiration, store light and chemical energy in the form of an electrochemical gradient created across a lipid bilayer. Herein we show that the proton concentration at an electrode/phospholipid-bilayer interface can be controlled and monitored electrochemically by immobilizing a membrane-bound hydrogenase. Thus, the energy derived from the electroenzymatic oxidation of H2 can be used to generate a proton gradient across the supported biomimetic membrane. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

15N Hyperpolarization of Imidazole-15N2 for Magnetic Resonance pH Sensing via SABRE-SHEATH

PubMed Central

2016-01-01

15N nuclear spins of imidazole-15N2 were hyperpolarized using NMR signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH). A 15N NMR signal enhancement of ∼2000-fold at 9.4 T is reported using parahydrogen gas (∼50% para-) and ∼0.1 M imidazole-15N2 in methanol:aqueous buffer (∼1:1). Proton binding to a 15N site of imidazole occurs at physiological pH (pKa ∼ 7.0), and the binding event changes the 15N isotropic chemical shift by ∼30 ppm. These properties are ideal for in vivo pH sensing. Additionally, imidazoles have low toxicity and are readily incorporated into a wide range of biomolecules. 15N-Imidazole SABRE-SHEATH hyperpolarization potentially enables pH sensing on scales ranging from peptide and protein molecules to living organisms. PMID:27379344

15N Hyperpolarization of Imidazole-15N2 for Magnetic Resonance pH Sensing via SABRE-SHEATH.

PubMed

Shchepin, Roman V; Barskiy, Danila A; Coffey, Aaron M; Theis, Thomas; Shi, Fan; Warren, Warren S; Goodson, Boyd M; Chekmenev, Eduard Y

2016-06-24

15 N nuclear spins of imidazole- 15 N 2 were hyperpolarized using NMR signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH). A 15 N NMR signal enhancement of ∼2000-fold at 9.4 T is reported using parahydrogen gas (∼50% para-) and ∼0.1 M imidazole- 15 N 2 in methanol:aqueous buffer (∼1:1). Proton binding to a 15 N site of imidazole occurs at physiological pH (p K a ∼ 7.0), and the binding event changes the 15 N isotropic chemical shift by ∼30 ppm. These properties are ideal for in vivo pH sensing. Additionally, imidazoles have low toxicity and are readily incorporated into a wide range of biomolecules. 15 N-Imidazole SABRE-SHEATH hyperpolarization potentially enables pH sensing on scales ranging from peptide and protein molecules to living organisms.

Colorimetric and Fluorescent Bimodal Ratiometric Probes for pH Sensing of Living Cells.

PubMed

Liu, Yuan-Yuan; Wu, Ming; Zhu, Li-Na; Feng, Xi-Zeng; Kong, De-Ming

2015-06-01

pH measurement is widely used in many fields. Ratiometric pH sensing is an important way to improve the detection accuracy. Herein, five water-soluble cationic porphyrin derivatives were synthesized and their optical property changes with pH value were investigated. Their pH-dependent assembly/disassembly behaviors caused significant changes in both absorption and fluorescence spectra, thus making them promising bimodal ratiometric probes for both colorimetric and fluorescent pH sensing. Different substituent identity and position confer these probes with different sensitive pH-sensing ranges, and the substituent position gives a larger effect. By selecting different porphyrins, different signal intensity ratios and different fluorescence excitation wavelengths, sensitive pH sensing can be achieved in the range of 2.1-8.0. Having demonstrated the excellent reversibility, good accuracy and low cytotoxicity of the probes, they were successfully applied in pH sensing inside living cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hierarchical hollow microsphere and flower-like indium oxide: Controllable synthesis and application as H{sub 2}S cataluminescence sensing materials

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

Cai, Pingyang, E-mail: cpyxx@163.com; Bai, Wei, E-mail: weibaiscu@gmail.com; Zhang, Lichun, E-mail: lichun0203@yahoo.cn

Graphical abstract: Hierarchical hollow microsphere and flower-like In{sub 2}O{sub 3} were controllable fabricated through a novel and simple hydrothermal process, and the former showed superior cataluminescence sensing performance to H{sub 2}S. Highlights: ► In{sub 2}O{sub 3} hierarchical hollow sphere were prepared via a hydrothermal route. ► The growth process of In{sub 2}O{sub 3} hierarchical hollow sphere has been investigated. ► The sensor based on prepared In{sub 2}O{sub 3} shows good sensing performance to H{sub 2}S. -- Abstract: In the present work, In{sub 2}O{sub 3} hierarchical hollow microsphere and flower-like microstructure were achieved controllably by a hydrothermal process in the sodiummore » dodecyl sulfate (SDS)-N,N-dimethyl-formamide (DMF) system. XRD, SEM, HRTEM and N{sub 2} adsorption measurements were used to characterize the as-prepared indium oxide materials and the possible mechanism for the microstructures formation was briefly discussed. The cataluminescence gas sensor based on the as-prepared In{sub 2}O{sub 3} was utilized to detect H{sub 2}S concentrations in flowing air. Comparative gas sensing results revealed that the sensor based on hierarchical hollow microsphere exhibited much higher sensing sensitivity in detecting H{sub 2}S gas than the sensor based on flower-like microstructure. The present gas sensor had a fast response time of 5 s and a recovery time of less than 25 s, furthermore, the cataluminescence intensity vs. H{sub 2}S concentration was linear in range of 2–20 μg mL{sup −1} with a detection limit of 0.5 μg mL{sup −1}. The present highly sensitive, fast-responding, and low-cost In{sub 2}O{sub 3}-based gas sensor for H{sub 2}S would have many practical applications.« less

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.

Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.

PubMed

Justus, Calvin R; Dong, Lixue; Yang, Li V

2013-12-05

The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

Low-Frequency Noise in Layered ReS2 Field Effect Transistors on HfO2 and Its Application for pH Sensing.

PubMed

Liao, Wugang; Wei, Wei; Tong, Yu; Chim, Wai Kin; Zhu, Chunxiang

2018-02-28

Layered rhenium disulfide (ReS 2 ) field effect transistors (FETs), with thickness ranging from few to dozens of layers, are demonstrated on 20 nm thick HfO 2 /Si substrates. A small threshold voltage of -0.25 V, high on/off current ratio of up to ∼10 7 , small subthreshold swing of 116 mV/dec, and electron carrier mobility of 6.02 cm 2 /V·s are obtained for the two-layer ReS 2 FETs. Low-frequency noise characteristics in ReS 2 FETs are analyzed for the first time, and it is found that the carrier number fluctuation mechanism well describes the flicker (1/f) noise of ReS 2 FETs with different thicknesses. pH sensing using a two-layer ReS 2 FET with HfO 2 as a sensing oxide is then demonstrated with a voltage sensitivity of 54.8 mV/pH and a current sensitivity of 126. The noise characteristics of the ReS 2 FET-based pH sensors are also examined, and a corresponding detection limit of 0.0132 pH is obtained. Our studies suggest the high potential of ReS 2 for future low-power nanoelectronics and biosensor applications.

Synthesis of ZnO thin film by sol-gel spin coating technique for H2S gas sensing application

NASA Astrophysics Data System (ADS)

Nimbalkar, Amol R.; Patil, Maruti G.

2017-12-01

In this present work, zinc oxide (ZnO) thin film synthesized by a simple sol-gel spin coating technique. The structural, morphology, compositional, microstructural, optical, electrical and gas sensing properties of the film were studied by using XRD, FESEM, EDS, XPS, HRTEM, Raman, FTIR and UV-vis techniques. The ZnO thin film shows hexagonal wurtzite structure with a porous structured morphology. Gas sensing performance of synthesized ZnO thin film was tested initially for H2S gas at different operating temperatures as well as concentrations. The maximum gas response is achieved towards H2S gas at 300 °C operating temperature, at 100 ppm gas concentration as compared to other gases like CH3OH, Cl2, NH3, LPG, CH3COCH3, and C2H5OH with a good stability.

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

Silicon photonic dual-gas sensor for H2 and CO2 detection.

PubMed

Mi, Guangcan; Horvath, Cameron; Van, Vien

2017-07-10

We report a silicon photonic dual-gas sensor based on a wavelength-multiplexed microring resonator array for simultaneous detection of H 2 and CO 2 gases. The sensor uses Pd as the sensing layer for H 2 gas and a novel functional material based on the Polyhexamethylene Biguanide (PHMB) polymer for CO 2 gas sensing. Gas sensing experiments showed that the PHMB-functionalized microring exhibited high sensitivity to CO 2 gas and excellent selectivity against H 2 . However, the Pd-functionalized microring was found to exhibit sensitivity to both H 2 and CO 2 gases, rendering it ineffective for detecting H 2 in a gas mixture containing CO 2 . We show that the dual-gas sensing scheme can allow for accurate measurement of H 2 concentration in the presence of CO 2 by accounting for the cross-sensitivity of Pd to the latter.

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

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.

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.

Platelike WO3 sensitized with CdS quantum dots heterostructures for photoelectrochemical dynamic sensing of H2O2 based on enzymatic etching.

PubMed

Wang, Yanhu; Gao, Chaomin; Ge, Shenguang; Yu, Jinghua; Yan, Mei

2016-11-15

A platelike tungsten trioxide (WO3) sensitized with CdS quantum dots (QDs) heterojunction is developed for solar-driven, real-time, and selective photoelectrochemical (PEC) sensing of H2O2 in the living cells. The structure is synthesized by hydrothermally growing platelike WO3 on fluorine doped tin oxide (FTO) and subsequently sensitized with CdS QDs. The as-prepared WO3-CdS QDs heterojunction achieve significant photocurrent enhancement, which is remarkably beneficial for light absorption and charge carrier separation. Based on the enzymatic etching of CdS QDs enables the activation of quenching the charge transfer efficiency, thus leading to sensitive PEC recording of H2O2 level in buffer and cellular environments. The results indicated that the proposed method will pave the way for the development of excellent PEC sensing platform with the quantum dot sensitization. This study could also provide a new train of thought on designing of self-operating photoanode in PEC sensing, promoting the application of semiconductor nanomaterials in photoelectrochemistry. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

Immobilization of hydrogenase on carbon nanotube polyelectrolytes as heterogeneous catalysts for electrocatalytic interconversion of protons and hydrogen

NASA Astrophysics Data System (ADS)

Liu, Jiang; Wu, Wen-Jie; Fang, Fang; Zorin, Nikolay A.; Chen, Meng; Qian, Dong-Jin

2016-08-01

Immobilization of active enzymes on the surfaces of electrodes and nanomaterials is important in the fields of bioscience, and biotechnology. In this study, we investigated electrocatalytic properties of the interconversion of protons and hydrogen by means of hydrogenase (H2ase)-functionalized carbon nanotube polyelectrolyte composites. Multiwalled carbon nanotube polyelectrolytes (MWNT-PEs) were synthesized through a diazonium and an addition reaction with poly(4-vinylpyridine) (P4VP), followed by another addition reaction with either methyl iodide (CH3I) or N-methyl- N'-benzyl bromide bipyridinium (VBenBr) to produce MWNT-P4VPMe or MWNT-P4VPBenV polyelectrolytes, respectively. The MWNT-PE@H2ase bio-nanocomposites were then prepared by means of MWNT-PEs as substrates to bind with H2ase. The redox current density of the MWNT-PE@H2ase-modified electrodes increased with a decrease in pH values of the Ar-saturated electrolyte solution owing to the catalytic reduction of protons (H2 production); further, it increased with the increasing pH values of the H2-saturated solution owing to the catalytic oxidation of hydrogen. The reversible color change between blue-colored and colorless viologen (catalyzed by the MWNT-PE@H2ase bio-nanocomposites) suggested that they may be developed as nano-biosensors for molecular H2. The as-synthesized bio-nanocomposites showed strong long-term stability and high bioactivity.

Redox sensing molecular mechanism of an iron metabolism regulatory protein FBXL5.

PubMed

Wei, Yaozhu; Yuan, Hong; Xu, Pengbiao; Tan, Xiangshi

2017-02-15

FBXL5 is a subunit of the SCF FBXL5 ubiquitin ligase complex that targets the proteasomal degradation of iron regulatory protein IRP2, which is an important regulator in iron metabolism. The degradation of FBXL5 itself is regulated in an iron- and oxygen-responsive manner through its diiron center containing Hr-like domain. Although the crystal structure of the Hr-like domain of FBXL5 and its degradation based on iron/oxygen sensing has been reported, the redox sensing molecular mechanism is still not clear. Herein the redox properties of FBXL5 were investigated via EPR, direct electrochemistry, SRCD, fluorescence emission spectroscopy, and redox kinetics. The results indicated that the conformation and function of FBXL5 are tuned by the redox states of the diiron center. The redox reactions of the diiron center are accompanied with conformational changes and iron release, which are associated with FBXL5 stability and degradation. These results provide insights into the redox sensing mechanism by which FBXL5 can serve as an iron metabolism regulator within mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

One-pot preparation of PEDOT:PSS-reduced graphene decorated with Au nanoparticles for enzymatic electrochemical sensing of H2O2

NASA Astrophysics Data System (ADS)

Mercante, Luiza A.; Facure, Murilo H. M.; Sanfelice, Rafaela C.; Migliorini, Fernanda L.; Mattoso, Luiz H. C.; Correa, Daniel S.

2017-06-01

The development of novel graphene-based nanocomposites is a hotspot in materials science due to their unique optical, electronic, thermal, mechanical and catalytic properties for varied applications. The present work reports on the development of a graphene-based ternary nanocomposite of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), reduced graphene oxide and gold nanoparticles (PEDOT:PSS-rGO-AuNPs) for the detection of hydrogen peroxide (H2O2). The hybrid nanocomposite showed superior electrochemical properties and higher stability compared to each individual component as electrode materials, showing a synergistic effect between PEDOT, rGO and AuNPs. The nanocomposite was obtained via a facile one-step approach and was assembly with horseradish peroxidase (HRP). The PEDOT:PSS-rGO-AuNPs-HRP modified electrode has been used for the amperometric detection of H2O2 and exhibited a high sensitivity of up to 677 μA mM-1 cm-2, with a wide linear range from 5 to 400 μM and a low detection limit of 0.08 μM (S/N = 3). This developed enzymatic biosensor showed to be highly stable and unresponsive to potentially interfering substances, and it could be used for sensing H2O2 in real samples, including tap water and bovine milk samples. These enhanced sensing performance could be ascribed to the intimate contact of AuNPs onto the rough surface of the PEDOT:PSS-rGO nanocomposite, which has a high electrical conductivity and large surface area, providing it as an excellent substrate for the growth and support of nanoparticles. The method developed in this work opens up a general route to prepare a wide range of graphene-based hybrid nanocomposite films with multiple functions including sensing and biosensing.

Hydrogen evolution in [NiFe] hydrogenases and related biomimetic systems: similarities and differences.

PubMed

Das, Ranjita; Neese, Frank; van Gastel, Maurice

2016-09-21

In this work, a detailed quantum chemical study of the mechanism of [Ni(bdt)(dppf)] (Ni(II)L) catalyzed hydrogen formation [A. Gan, T. L. Groy, P. Tarakeshwar, S. K. S. Mazinani, J. Shearer, V. Mujica and A. K. Jones, J. Am. Chem. Soc., 2015, 137, 1109-1115] following an electro-chemical-electro-chemical (ECEC) pathway is reported. The complex exclusively catalyzes the reduction of protons to molecular hydrogen. The calculations suggest that the first one-electron reduction of the [Ni(II)L] catalyst is the rate limiting step of the catalytic cycle and hence, the buildup of detectable reaction intermediates is not expected. The catalytic activity of the [Ni(II)L] complex is facilitated by the flexibility of the ligand system, which allows the ligand framework to adapt to changes in the Ni oxidation state over the course of the reaction. Additionally, a comparison is made with the catalytic activity of [NiFe] hydrogenase. It is argued that the directionality of the reversible hydrogen formation reaction is controlled by the ligand field of the nickel ion and the possibility for side-on (η(2)) binding of H2: if the ligand framework does not allow for η(2) binding of H2, as is the case for [Ni(II)L], the catalyst irreversibly reduces protons. If the ligand field allows η(2) binding of H2, the catalyst can in principle work reversibly. The conditions for η(2) binding are discussed.

An iron-iron hydrogenase mimic with appended electron reservoir for efficient proton reduction in aqueous media

PubMed Central

Becker, René; Amirjalayer, Saeed; Li, Ping; Woutersen, Sander; Reek, Joost N. H.

2016-01-01

The transition from a fossil-based economy to a hydrogen-based economy requires cheap and abundant, yet stable and efficient, hydrogen production catalysts. Nature shows the potential of iron-based catalysts such as the iron-iron hydrogenase (H2ase) enzyme, which catalyzes hydrogen evolution at rates similar to platinum with low overpotential. However, existing synthetic H2ase mimics generally suffer from low efficiency and oxygen sensitivity and generally operate in organic solvents. We report on a synthetic H2ase mimic that contains a redox-active phosphole ligand as an electron reservoir, a feature that is also crucial for the working of the natural enzyme. Using a combination of (spectro)electrochemistry and time-resolved infrared spectroscopy, we elucidate the unique redox behavior of the catalyst. We find that the electron reservoir actively partakes in the reduction of protons and that its electron-rich redox states are stabilized through ligand protonation. In dilute sulfuric acid, the catalyst has a turnover frequency of 7.0 × 104 s−1 at an overpotential of 0.66 V. This catalyst is tolerant to the presence of oxygen, thereby paving the way for a new generation of synthetic H2ase mimics that combine the benefits of the enzyme with synthetic versatility and improved stability. PMID:26844297

Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science.

PubMed

Urano, K; Tamaoki, N; Nomura, T

2012-01-01

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.

CuO-Decorated ZnO Hierarchical Nanostructures as Efficient and Established Sensing Materials for H2S Gas Sensors

PubMed Central

Vuong, Nguyen Minh; Chinh, Nguyen Duc; Huy, Bui The; Lee, Yong-Ill

2016-01-01

Highly sensitive hydrogen sulfide (H2S) gas sensors were developed from CuO-decorated ZnO semiconducting hierarchical nanostructures. The ZnO hierarchical nanostructure was fabricated by an electrospinning method following hydrothermal and heat treatment. CuO decoration of ZnO hierarchical structures was carried out by a wet method. The H2S gas-sensing properties were examined at different working temperatures using various quantities of CuO as the variable. CuO decoration of the ZnO hierarchical structure was observed to promote sensitivity for H2S gas higher than 30 times at low working temperature (200 °C) compared with that in the nondecorated hierarchical structure. The sensing mechanism of the hybrid sensor structure is also discussed. The morphology and characteristics of the samples were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), UV-vis absorption, photoluminescence (PL), and electrical measurements. PMID:27231026

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

H2O2 sensing using HRP modified catalyst-free ZnO nanorods synthesized by RF sputtering

NASA Astrophysics Data System (ADS)

Srivastava, Amit; Kumar, Naresh; Singh, Priti; Singh, Sunil Kumar

2017-06-01

Catalyst-free ( 00 l) oriented ZnO nanorods (NRs) -based biosensor for the H2O2 sensing has been reported. The (002) oriented ZnO NRs as confirmed by X-ray diffraction were successfully grown on indium tin oxide (ITO) coated glass substrate by radio frequency (RF) sputtering technique without using any catalyst. Horseradish peroxidase (HRP) enzyme was immobilized on ZnO NRs by physical adsorption technique to prepare the biosensor. In this HRP/ZnO NR/ITO bioelectrode, nafion solution was added to form a tight membrane on surface. The prepared bioelectrode has been used for biosensing measurements by electrochemical analyzer. The electrochemical studies reveal that the prepared HRP/ZnO NR/ITO biosensor is highly sensitive to the detection of H2O2 over a linear range of 0.250-10 μM. The ZnO NR-based biosensor showed lower value of detection limit (0.125 μM) and higher sensitivity (13.40 µA/µM cm2) towards H2O2. The observed value of higher sensitivity attributed to larger surface area of ZnO nanostructure for effective loading of HRP besides its high electron communication capability. In addition, the biosensor also shows lower value of enzyme's kinetic parameter (Michaelis-Menten constant, K m) of 0.262 μM which indicates enhanced enzyme affinity of HRP to H2O2. The reported biosensor may be useful for various applications in biosensing, clinical, food, and beverage industry.

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.

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.

The Dynamics and Regulatory Mechanism of Pronuclear H3k9me2 Asymmetry in Mouse Zygotes

PubMed Central

Ma, Xue-Shan; Chao, Shi-Bin; Huang, Xian-Ju; Lin, Fei; Qin, Ling; Wang, Xu-Guang; Meng, Tie-Gang; Zhu, Cheng-Cheng; Schatten, Heide; Liu, Hong-Lin; Sun, Qing-Yuan

2015-01-01

H3K9 methylation is an important histone modification that is correlated with gene transcription repression. The asymmetric H3K9 dimethylation (H3K9me2) pattern between paternal and maternal genomes is generated soon after fertilization. In the present study, we carefully determined the dynamics of H3K9me2 changes in mouse zygotes, and investigated the regulatory mechanisms. The results indicated that histone methyltransferase G9a, but not GLP, was involved in the regulation of asymmetric H3K9me2, and G9a was the methyltransferase that induced the appearance of H3K9me2 in the male pronucleus of the zygote treated with cycloheximide. We found that there were two distinct mechanisms that regulate H3K9me2 in the male pronucleus. Before 8 h of in vitro fertilization (IVF), a mechanism exists that inhibits the association of G9a with the H3K9 sites. After 10 h of IVF the inhibition of G9a activity depends on yet unknown novel protein(s) synthesis. The two mechanisms of transfer take place between 8–10 h of IVF, and the novel protein failed to inhibit G9a activity in time, resulting in the appearance of a low level de novo H3K9me2 in the male pronucleus. PMID:26639638

Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1 ▿

PubMed Central

Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.; Heald, Steve; Dohnalkova, Alice C.; Sybirna, Kateryna; Bottin, Hervé; Squier, Thomas C.; Zachara, John M.; Fredrickson, James K.

2011-01-01

Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that has been implicated in H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H2ase in trans restored the mutant's ability to produce H2 at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H2ase coupled H2 oxidation to reduction of Tc(VII)O4− and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated reduction of Tc(VII)O4− but not methyl viologen. Under the conditions tested, all Tc(VII)O4− used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O4− was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2·nH2O, which was also the product of Tc(VII)O4− reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H2ase catalyzes Tc(VII)O4− reduction directly by coupling to H2 oxidation. PMID:21724888