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

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.

Fostering responsible research with genome editing technologies: a European perspective.

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.

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

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.

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

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.

Rubredoxin-related maturation factor guarantees metal cofactor integrity during aerobic biosynthesis of membrane-bound [NiFe] hydrogenase.

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.

Coping with anoxia: a comprehensive proteomic and transcriptomic survey of denitrification.

Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.

A universal scaffold for synthesis of the Fe(CN)2(CO) moiety of [NiFe] hydrogenase.

Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN(-)) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN(-) ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)(2)(CO) entity of [NiFe] hydrogenase.

Novel, oxygen-insensitive group 5 [NiFe]-hydrogenase in Ralstonia eutropha.

Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 µmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 µM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.

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

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.

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.

Autotrophic production of stable-isotope-labeled arginine in Ralstonia eutropha strain H16.

With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong P(cbbL) promoter of the R. eutropha H16 cbb(c) operon (coding for autotrophic CO(2) fixation). In batch cultures growing on H(2) and CO(2) as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with (13)CO(2) and (15)NH(4)Cl, and the (13)C/(15)N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [(13)C/(15)N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.

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

In biology, rapid oxidation and evolution of H(2) 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 O(2). The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H(2) as an energy source with O(2) as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an "O(2)-tolerant" hydrogenase. The mechanism of O(2) tolerance of Re MBH has been probed by measuring H(2) oxidation activity in the presence of O(2) over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O(2) and subsequent reactivation of the active site. Most significantly, O(2) tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H(2) affinity or the kinetics of O(2) attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O(2) tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases.

The maturation factors HoxR and HoxT contribute to oxygen tolerance of membrane-bound [NiFe] hydrogenase in Ralstonia eutropha H16.

The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O(2) and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O(2) levels. MBH-driven growth on H(2) is inhibited or retarded at high O(2) partial pressure (pO(2)) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO(2). Lack of hoxR and hoxT can phenotypically be restored by providing O(2)-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions.

Complete genome sequence of the type strain Cupriavidus necator N-1.

Here we announce the complete genome sequence of the copper-resistant bacterium Cupriavidus necator N-1, the type strain of the genus Cupriavidus. The genome consists of two chromosomes and two circular plasmids. Based on genome comparison, the chromosomes of C. necator N-1 share a high degree of similarity with the two chromosomal replicons of the bioplastic-producing hydrogen bacterium Ralstonia eutropha H16. The two strains differ in their plasmids and the presence of hydrogenase genes, which are absent in strain N-1.

A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16.

The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.

[NiFe] and [FeS] cofactors in the membrane-bound hydrogenase of Ralstonia eutropha investigated by X-ray absorption spectroscopy: insights into O(2)-tolerant H(2) cleavage.

Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H(2)) in the presence of dioxygen (O(2)) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O(2)-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O(2)-sensitive periplasmic hydrogenase (PH) of Desulfovibrio gigas. Fe-XAS indicated an unusual complement of iron-sulfur centers in the MBH, likely based on a specific structure of the FeS cluster proximal to the active site. This cluster is a [4Fe4S] cubane in PH. For MBH, it comprises less than ~2.7 Å Fe-Fe distances and additional longer vectors of ≥3.4 Å, consistent with an Fe trimer with a more isolated Fe ion. Ni-XAS indicated a similar architecture of the [NiFe] site in MBH and PH, featuring Ni coordination by four thiolates of conserved cysteines, i.e., in the fully reduced state (Ni-SR). For oxidized states, short Ni-µO bonds due to Ni-Fe bridging oxygen species were detected in the Ni-B state of the MBH and in the Ni-A state of the PH. Furthermore, a bridging sulfenate (CysSO) is suggested for an inactive state (Ni(ia)-S) of the MBH. We propose that the O(2) tolerance of the MBH is mainly based on a dedicated electron donation from a modified proximal FeS cluster to the active site, which may favor formation of the rapidly reactivated Ni-B state instead of the slowly reactivated Ni-A state. Thereby, the catalytic activity of the MBH is facilitated in the presence of both H(2) and O(2).

Analyses of soluble and membrane proteomes of Ralstonia eutropha H16 reveal major changes in the protein complement in adaptation to lithoautotrophy.

The soil-dwelling lithoautotrophic bacterium Ralstonia eutropha H16 utilizes hydrogen as the key source of energy during aerobic growth on hydrogen and carbon dioxide. We examined the soluble and membrane protein complements of lithoautotrophically grown cells and compared them to the protein complements of cells grown organoheterotrophically on succinate. (14)N/(15)N-based inverse metabolic labeling in combination with GeLC-MS led to the identification of 1452 proteins, 1174 of which could be quantitated. Far more proteins were found to be more abundant in the lithoautotrophically than in the organoheterotrophically grown cells. In addition to the induction of the key enzymes of hydrogen oxidation and carbon dioxide fixation, we observed several characteristic alterations in the proteome correlated with lithoautotrophic growth. (I) Genes for three terminal oxidases were upregulated. (II) NAD(P) transhydrogenase and enzymes for the accumulation of poly(3-hydroxybutyrate) (PHB) showed increased protein abundance. (III) Lithoautotrophically grown cells were equipped with an enhanced inventory of transport systems. (IV) The expression of cell surface appendages involved in cell movement was markedly increased, while proteins involved in cell adhesion were decreased. Our data show that the hydrogen-based lifestyle of R. eutropha H16 relies on an extensive protein repertoire adapting the organism to the alternative energy and carbon sources.

Protein-protein complex formation affects the Ni-Fe and Fe-S centers in the H2-sensing regulatory hydrogenase from Ralstonia eutropha H16.

The regulatory Ni-Fe hydrogenase (RH) from the H(2)-oxidizing bacterium Ralstonia eutropha functions as an oxygen-resistant hydrogen sensor, which is composed of the large, active-site-containing HoxC subunit and the small subunit HoxB carrying Fe-S clusters. In vivo, the HoxBC subunits form a dimer designated as RH(wt). The RH(wt) protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH(wt) (RH(wt)-HoxJ(wt)), located in the cytoplasm. In this study, we used X-ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni-Fe active site and the Fe-S clusters. Aside from the RH(wt) protein and the RH(wt)-HoxJ(wt) complex, we investigated the RH(stop) protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RH(wt)-HoxJ(Deltakinase), in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H(2), leading to the formation of the EPR-detectable Ni(III)-C state of the active site and to the reduction of Fe-S clusters detectable by XAS, thus corroborating that H(2) cleavage is independent of the presence of the HoxJ protein. In RH(stop), presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH(stop) differed from that in RH(wt) and the RH(wt)-HoxJ complexes, in which additional Ni--O bonds were detected by XAS. The Ni--O bonds caused only very minor changes of the EPR g-values of the Ni-C and Ni-L states and of the IR vibrational frequencies of the diatomic CN(-) and CO ligands at the active-site Fe ion. Both one Fe-S cluster in HoxB and an oxygen-rich Ni coordination seem to be stabilized by RH dimerization involving the C-terminus of HoxB and by complex formation with HoxJ.

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

[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.