Fe-only Hydrogenase Research Articles

19] Fe-only hydrogenase from Thermotoga maritirria

Hydrogenases catalyze the reversible oxidation of hydrogen gas (H2). The Fe-hydrogenases contain iron as the only metal and this is present in the form of both conventional and novel iron-sulfur dusters. This type of hydrogenase is found in anaerobic bacteria and in the hydrogenosomes of some unicellular, anaerobic eukaryotes. To date Fe-only hydrogenases have not been identified in archaea. This chapter focuses on the Fe-hydrogenase from the hyperthermophilic bacterium, Thermotoga maritima . This strictly anaerobic, hyperthermophilic bacterium grows optimally at 80 ° using sugars or peptides as a carbon and energy source. Herein are described the methods used to grow T. maritima and to purify and characterize its Fe-hydrogenase. So far, this is the only known hyperthermophilic example of this type of enzyme.

Reversible carbon monoxide binding and inhibition at the active site of the Fe-only hydrogenase.

Carbon monoxide binding and inhibition have been investigated by electron paramagnetic resonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. Simulation of the EPR spectrum of the as-isolated state indicates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clusters. Addition of carbon monoxide to CpI in the presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristic axial EPR signal [g(eff(1)), g(eff(2)), and g(eff(3)) = 2.0725, 2.0061, and 2.0061, respectively] was observed. Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resulted in samples that exhibited the native oxidized EPR signature that could be converted to the reduced form upon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopically defined states of CpI and those observed structurally by X-ray crystallography, we have examined the CpI crystals using EPR spectroscopy. EPR spectra of the crystals in the CO-bound state exhibit the previously described axial signal associated with CO binding. The results indicate that the addition of carbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized by loss of enzyme activity and the distinctive axial EPR signal.

Carbon Monoxide and Cyanide Ligands in a Classical Organometallic Complex Model for Fe-Only Hydrogenase

The FeI organometallic complex [(μ-SCH2CH2CH2S)Fe2(CO)6] provides a structural model for the cyano-carbonyl diiron site of Fe-only hydrogenase as characterized by X-ray crystallography (the picture shows the structure (black) of the model overlaid with that of the Fe–Fe dimetallic site in the hydrogenase isolated from Desulfovibrio desulfuricans). Cyanide substitution of CO occurs readily and provides spectroscopic references for the active site.

Carbon Monoxide and Cyanide Ligands in a Classical Organometallic Complex Model for Fe-Only Hydrogenase

The Fe(I) organometallic complex [(µ-SCH(2)CH(2)CH(2)S)Fe(2)(CO)(6)] provides a structural model for the cyano-carbonyl diiron site of Fe-only hydrogenase as characterized by X-ray crystallography (the picture shows the structure (black) of the model overlaid with that of the Fe-Fe dimetallic site in the hydrogenase isolated from Desulfovibrio desulfuricans). Cyanide substitution of CO occurs readily and provides spectroscopic references for the active site.

Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum.

A site for the binding of exogenously added carbon monoxide has been identified at the active site of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. The binding and inhibition of carbon monoxide have been exploited in biochemical and spectroscopic studies to gain mechanistic insights. In the present study, we have taken advantage of the ability to generate an irreversibly carbon monoxide bound state of CpI. The crystallization and structural characterization of CpI inhibited in the presence of carbon monoxide indicates the addition of a single molecule of carbon monoxide. The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site of carbon monoxide binding at the active site of CpI. Carbon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule in the as crystallized native state of CpI that has been previously suggested to be a potential site of reversible hydrogen oxidation. Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), providing a rational potential mechanism for inhibition of reversible hydrogen oxidation at the active site of CpI.

Structural variability of the active site of Fe-only hydrogenase and its hydrogenated forms

Density functional calculations show that the (cys-S)- (CO)(CN)FeS2(µ-CO)Fe(CO)(CN) active site of the Fe-only hydrogenase from Clostridium pasteurianum is redox ambivalent and stereochemically flexible at the CO and S bridges, and at the Fe atoms, and with bound hydrogen: the fundamentals of probable mechanisms are revealed.

A di-iron dithiolate possessing structural elements of the carbonyl/cyanide sub-site of the H-centre of Fe-only hydrogenase

The synthesis and characterisation of the first {2Fe2S}-cluster bearing both CO and CN ligands is described; the iron atoms are linked by the bridging 1,3-propanedithiolate unit that has been identified in the crystallographic structure of the {2Fe2S} sub-unit of the H-centre of the all-iron hydrogenase from Desulfovibrio desulfuricans.

X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution.

A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems.

Kinetics and interaction studies between cytochrome c3 and Fe-only hydrogenase fromDesulfovibrio vulgaris hildenborough

Hydrogenases from Desulfovibrio are found to catalyze hydrogen uptake with low potential multiheme cytochromes, such as cytochrome c3, acting as acceptors. The production of Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough was improved with respect to the growth phase and media to determine the best large-scale bacteria growth conditions. The interaction and electron transfer from Fe-only hydrogenase to multiheme cytochrome has been studied in detail by both BLAcore and steady-state measurements. The electron transfer between [Fe] hydrogenase and cytochrome c3 appears to be a cooperative phenomenon (h = 1.37). This behavior could be related to the conductivity properties of multihemic cytochromes. An apparent dissociation constant was determined (2 x 10(-7) M). The importance of the cooperativity for contrasting models proposed to describe the functional role of the hydrogenase/cytochrome c3 complex is discussed. Presently, the only determined structure is from [NiFe] hydrogenase and there are no obvious similarities between [NiFe] and [Fe] hydrogenase. Furthermore, no crystallographic data are available concerning [Fe] hydrogenase. The first results on crystallization and X-ray crystallography are reported.

Hydrogen metabolism in organisms with oxygenic photosynthesis: hydrogenases as important regulatory devices for a proper redox poising?

Abstract Three different hydrogenases and the nitrogenase putatively participate in the hydrogen metabolism of micro-organisms carrying out oxygenic photosynthesis. Hydrogenases either produce hydrogen or split hydrogen into protons and electrons depending on their redox partners, whereas the nitrogenase produces hydrogen unidirectionally as a byproduct during the reduction of nitrogen to ammonia. Hydrogenases are well-characterized enzymes on the enzymatic, structural and genetic level, especially in prokaryotic micro-organisms. They can be classified regarding the metal composition of their active site (Fe-only, NiFe or metal-free), their preferential direction of reaction (uptake only or bidirectional/reversible) and their in vivo electron donors or acceptors. The main physiological role of the uptake hydrogenase in cyanobacteria is probably recapturing the hydrogen produced by nitrogenase. The role of the bidirectional hydrogenase in phototrophs is still a matter of debate. Based on recent results which showed it to be of the NAD(P)-reducing type, a model for its physiological function is suggested. This model includes that this type of hydrogenase is linked to complex I of the respiratory electron-transport chain and might be an important electron valve during photosynthesis under rapidly changing light conditions. The existence of an Fe-only hydrogenase as well as an NiFe-hydrogenase in green algae is still enigmatic and is discussed as hydrogenases either participating in the production of hydrogen or during fermentation.

The operon for the Fe-hydrogenase in Desulfovibrio vulgaris (Hildenborough): mapping of the transcript and regulation of expression.

The genes for the subunits of the Fe-only hydrogenase from Desulfovibrio vulgaris are transcribed as a 1.9 kb mRNA; the operon contains no other genes besides those encoding the two subunits. The transcriptional start site of the operon was mapped. Determination of hydrogenase activity and hydrogenase mRNA levels indicates a growth-phase dependent regulation of hydrogenase expression at transcriptional level. However, it has not yet been possible to localize the sequences required for regulation and expression of the genes.

Resonance Raman studies of iron-only hydrogenases

The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy. The results indicate the presence of ferredoxin-like [4Fe-4S]2+,+ and [2Fe-2S]2+,+ clusters in both T. maritima hydrogenase and C. pasteurianum hydrogenase I, but only [4Fe-4S]2+,+ clusters in D. vulgaris hydrogenase. This necessitates a reevaluation of the iron-sulfur cluster composition of C. pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center [Macor, K. A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Biol. Chem. 262, 9945-9947] arise from an indigenous [2Fe-2S]2+ cluster. No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed. This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.

Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism

To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed.