New Insights into [FeFe] Hydrogenase Activation and Maturase Function

[FeFe] hydrogenases catalyze H₂ production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN^–), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN^– ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN^– ligands that is a precursor to the H-cluster [2Fe]_H subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe]_H subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.     

Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine

[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.     

Biochemical Analysis of the Interactions between the Proteins Involved in the [FeFe]-Hydrogenase Maturation Process

[FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.     

An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni–Fe site of the soluble NAD⁺-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI₂) with aerobic specific H₂–NAD⁺ activities of 150–185 μmol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.     

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.     

Tyrosine,Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation

Background

[FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H₂. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated.

Principal Findings

We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation.

Significance

The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.     

Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met(415) and CrHydA1Lys(228) resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet(353) (CrHydA1Met(223)) and CpICys(299) (CrHydA1Cys(169)) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H(trans) state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM(353) (CrHydA1M(223)) to leucine led to a strong decrease in turnover without affecting the K(m) value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.     

Carbamoylphosphate serves as the source of CN(-), but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha

Within the catalytic centre of [NiFe]-hydrogenases one carbonyl and two cyanide ligands are covalently attached to the iron. To identify the metabolic origins of these ligands, the regulatory [NiFe] hydrogenase in conjunction with the indigenous Hyp maturation proteins of Ralstonia eutropha H16 were heterologously overproduced in E. coli grown in the presence of L-[ureido-(13)C] citrulline and NaH(13)CO(3). Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO(2) and carbamoylphosphate. Incorporation of label from (13)CO exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis.     

Cyanide inactivation of hydrogenase from Azotobacter vinelandii.

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.     

Infrared studies of the CO-inhibited form of the Fe-only hydrogenase from Clostridium pasteurianum I: examination of its light sensitivity at cryogenic temperatures

Infrared spectroscopy has been used to examine the oxidized and CO-inhibited forms of Fe-only hydrogenase I from Clostridium pasteurianum. For the oxidized enzyme, five bands are detected in the infrared spectral region between 2100 and 1800 cm(-1). The pattern of infrared bands is consistent with the presence of two terminally coordinated carbon monoxide molecules, two terminally coordinated cyanide molecules, and one bridging carbon monoxide molecule, ligated to the Fe atoms of the active site [2Fe] subcluster. Infrared spectra of the carbon monoxide-inhibited state, prepared using both natural abundance CO and 13CO, indicate that the two terminally coordinated CO ligands that are intrinsic to the enzyme are coordinated to different Fe atoms of the active site [2Fe] subcluster. Irradiation of the CO-inhibited state at cryogenic temperatures gives rise to two species with dramatically different infrared spectra. The first species has an infrared spectrum identical to the spectrum of the oxidized enzyme, and can be assigned as arising from the photolysis of the exogenous CO from the active site. This species, which has been observed in X-ray crystallographic measurements [Lemon, B. J., and Peters, J. W. (2000) J. Am. Chem. Soc. 122, 3793], decays above 150 K. The second light-induced species decays above 80 K and is characterized by loss of the infrared band associated with the Fe bridging CO at 1809 cm(-1). Potential models for the second photolysis event are discussed.     

Specific methylation of the CpG-rich domains in the promoter of the human tissue transglutaminase gene

The presence of a [Fe]-hydrogenase in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2 has been demonstrated by immunocytochemistry, subcellular fractionation, Western-blotting and measurements of hydrogenase activity in the presence of various concentrations of carbon monoxide (CO). Since the hydrogenosomal hydrogenase activity can be inhibited nearly completely by low concentrations of CO, it is likely that the [Fe]-hydrogenase is responsible for at least 90% of the hydrogen production in isolated hydrogenosomes. Most likely, this hydrogenase is encoded by the gene hydL2 that exhibits all the motifs that are characteristic of [Fe]-hydrogenases. The open reading frame starts with an N-terminal extension of 38 amino acids that has the potential to function as a hydrogenosomal targeting signal. The downstream sequences encode an enzyme of a calculated molecular mass of 66.4 kDa that perfectly matches the molecular mass of the mature hydrogenase in the hydrogenosome. Phylogenetic analysis revealed that the hydrogenase of Neocallimastix sp. L2. clusters together with similar ('long-type') [Fe]-hydrogenases from Trichomonas vaginalis, Nyctotherus ovalis, Desulfovibrio vulgaris and Thermotoga maritima. Phylogenetic analysis based on the H-cluster - the only module of [Fe]-hydrogenases that is shared by all types of [Fe]-hydrogenases and hydrogenase-like proteins - revealed a monophyly of all hydrogenase-like proteins of the aerobic eukaryotes. Our analysis suggests that the evolution of the various [Fe]-hydrogenases and hydrogenase-like proteins occurred by a differential loss of Fe-S clusters in the N-terminal part of the [Fe]-hydrogenase.     

Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.     

The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different

The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in [NiFe]-hydrogenases are different.     

In vitro activation of [FeFe] hydrogenase: new insights into hydrogenase maturation

The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with addition of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for nickel and a three-iron center in the hydrogenase of Desulfovibrio desulfuricans

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and M?ssbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the M?ssbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the M?ssbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and M?ssbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.     

Interaction of potassium cyanide with the [Ni-4Fe-5S] active site cluster of CO dehydrogenase from Carboxydothermus hydrogenoformans

The Ni-Fe carbon monoxide (CO) dehydrogenase II (CODHII(Ch)) from the anaerobic CO-utilizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO, presumably at the Ni-(micro(2)S)-Fe1 subsite of the [Ni-4S-5S] cluster in the active site. The CO oxidation mechanism proposed on the basis of several CODHII(Ch) crystal structures involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. To understand how CO interacts with the active site, we have studied the reactivity of the cluster with potassium cyanide and analyzed the resulting type of nickel coordination by x-ray absorption spectroscopy. Cyanide acts as a competitive inhibitor of reduced CODHII(Ch) with respect to the substrate CO and is therefore expected to mimic the substrate. It inhibits the enzyme reversibly, forming a nickel cyanide. In this reaction, one of the four square-planar sulfur ligands of nickel is replaced by the carbon atom of cyanide, suggesting removal of the micro(2)S from the Ni-(micro(2)S)-Fe1 subsite. Upon reactivation of the inhibited enzyme, cyanide is released, and the square-planar coordination of nickel by 4S ligands is recovered, which includes the reformation of the Ni-(micro(2)S)-Fe1 bridge. The results are summarized in a model of the CO oxidation mechanism at the [Ni-4Fe-5S] active site cluster of CODHII(Ch) from C. hydrogenoformans.     

Inhibition of biocatalysis in [Fe-Fe] hydrogenase by oxygen: molecular dynamics and density functional theory calculations

Designing O(2)-tolerant hydrogenases is a major challenge in applying [Fe-Fe]H(2)ases for H(2) production. The inhibition involves transport of oxygen through the enzyme to the H-cluster, followed by binding and subsequent deactivation of the active site. To explore the nature of the oxygen diffusion channel for the hydrogenases from Desulfovibrio desulfuricans (Dd) and Clostridium pasteurianum (Cp), empirical molecular dynamics simulations were performed. The dynamic nature of the oxygen pathways in Dd and Cp was elucidated, and insight is provided, in part, into the experimental observation on the difference of oxygen inhibition in Dd and the hydrogenase from Clostridium acetobutylicum (Ca, assumed homologous to Cp). Further, to gain an understanding of the mechanism of oxygen inhibition of the [Fe-Fe]H(2)ase, density functional theory calculations of model compounds composed of the H-cluster and proximate amino acids are reported. Confirmation of the experimentally based suppositions on inactivation by oxygen at the [2Fe](H) domain is provided, validating the model compounds used and oxidation state assumptions, further explaining the mode of damage. This unified approach provides insight into oxygen diffusion in the enzyme, followed by deactivation at the H-cluster.     

The soluble [NiFe]-hydrogenase from<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> contains four cyanides in its active site,one of which is responsible for the insensitivity towards oxygen

Infrared spectra of ¹⁵N-enriched preparations of the soluble cytoplasmic NAD⁺-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)₂(CN)Ni(-RS)₂Fe(CN)₃(CO) centre (R=Cys) and that H₂ activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.Abbreviations BV benzyl viologen - DCIP 2,6-dichlorophenol-indophenol - EXAFS extended X-ray absorption fine structure - FTIR Fourier transform infrared - MV methyl viologen - SH soluble NAD⁺-reducing hydrogenase - XAS X-ray absorption spectroscopy     

Probing the origin of the metabolic precursor of the CO ligand in the catalytic center of [NiFe] hydrogenase

The O(2)-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H(2) conversion in the presence of ambient O(2). Oxygen represents not only a challenge for catalysis but also for the complex assembling process of the [NiFe] active site. Apart from nickel and iron, the catalytic center contains unusual diatomic ligands, namely two cyanides (CN(-)) and one carbon monoxide (CO), which are coordinated to the iron. One of the open questions of the maturation process concerns the origin and biosynthesis of the CO group. Isotope labeling in combination with infrared spectroscopy revealed that externally supplied gaseous (13)CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding (13)CO titration experiments showed that a concentration 130-fold higher than ambient CO (0.1 ppmv) caused a 50% labeling of the carbonyl ligand in the [NiFe] hydrogenase, leading to the conclusion that the carbonyl ligand originates from an intracellular metabolite. A novel setup allowed us to the study effects of CO depletion on maturation in vivo. Upon induction of CO depletion by addition of the CO scavenger PdCl(2), cells cultivated on H(2), CO(2), and O(2) showed severe growth retardation at low cell concentrations, which was on the basis of partially arrested hydrogenase maturation, leading to reduced hydrogenase activity. This suggests gaseous CO as a metabolic precursor under these conditions. The addition of PdCl(2) to cells cultivated heterotrophically on organic substrates had no effect on hydrogenase maturation. These results indicate at least two different pathways for biosynthesis of the CO ligand of [NiFe] hydrogenase.