Recent research on hydrogenases has been notably motivated by a desire to utilize these remarkable hydrogen oxidation catalysts in biotechnological applications. Progress in the development of such applications is substantially hindered by the oxygen sensitivity of the majority of hydrogenases. This problem tends to inspire the study of organisms such as Ralstonia eutropha H16 that produce oxygen-tolerant [NiFe]-hydrogenases. R. eutropha H16 serves as an excellent model system in that it produces three distinct [NiFe]-hydrogenases that each serve unique physiological roles: a membrane-bound hydrogenase (MBH) coupled to the respiratory chain, a cytoplasmic, soluble hydrogenase (SH) able to generate reducing equivalents by reducing NAD⁺ at the expense of hydrogen, and a regulatory hydrogenase (RH) which acts in a signal transduction cascade to control hydrogenase gene transcription. This review will present recent results regarding the biosynthesis, regulation, structure, activity, and spectroscopy of these enzymes. This information will be discussed in light of the question how do organisms adapt the prototypical [NiFe]-hydrogenase system to function in the presence of oxygen.

Infrared spectra of (15)N-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(micro-RS)(2)Fe(CN)(3)(CO) centre (R=Cys) and that H(2) 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.

Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni-Fe site of the soluble NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI(2)) with aerobic specific H(2)-NAD(+) activities of 150-185 mumol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD ؉ at the expense of H 2 . We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI 2 . Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I). Hydrogenases (reaction H 2 7 HϪ ϩ H ϩ 7 2H ϩ ϩ 2e Ϫ ) are the key enzymes in the H 2 metabolism of many microorganisms. All hydrogenases are metalloenzymes. Presently, three main classes are known. Although these classes are phylogenetically unrelated (16,76,77), it is most amazing to note that the active sites of hydrogenases have two properties in common: (i) all contain Fe and most contain Ni as well, and (ii) all contain CO as ligand to Fe and most contain CN as ligand as well. Most enzymes belong to the class of [NiFe]-hydrogenases, which have a (CysS) 2 Ni(-ЈOЈ)(-CysS) 2 Fe(CN) 2 (CO) active site in the aerobically isolated form (5,6,26,50,78,79). Very recent crystallographic studies indicated that the oxygen species in the ЈOЈ bridge can be a di-oxo species (peroxide) or a mono-oxy species (hydroxide) (A. Volbeda, personal communication). When the oxygen bridge is present, the enzymes are inactive. Reduction with H 2 removes this ligand and replaces it with a hydride, resulting in active enzymes (11,22,64 (45,46,49,51,74). Also, here, the ЈOЈ species is present only in the inactive state of these enzymes. The [Fe]-hydrogenases form the third class and contain a Fe(CO) 2 group bound to an organic cofactor (38,39,62). No crystal structure of a member of this class is available yet.The facultative chemolithoautotrophic proteobacterium Ralstonia eutropha H16 (Table 1) (formerly Alcaligenes eutrophus H16 [18]) is able to use hydrogen as the sole energy source in an oxic environment. Energy-yielding H 2 oxidation in this bacterium is catalyzed by two [NiFe]-hydrogenases: (i) a membrane-bound enzyme (MBH) which is associated with the respiratory chain via a b-type cy...

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 HypXnegative 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 H 2 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. 2 from the ␤-proteobacterium Ralstonia eutropha H16. This is a heterotetrameric enzyme with subunits HoxF (67 kDa), HoxH (55 kDa), HoxU (26 kDa), and HoxY (23 kDa) (9, 10). The enzyme comprises two functionally different heterodimeric complexes, which have been separated and characterized (9, 11). The HoxFU dimer constitutes an enzyme module, termed NADH dehydrogenase or diaphorase, involved in the reduction of NAD Fig. 1A. The bridging oxygen ligand is removed upon reduction, whereby these enzymes become activated (17,24).The SH of R. eutropha belongs to a subclass of [NiFe]-hydrogenases where the polypeptide of the small hydrogenase subunit (HoxY) ends shortly after the position of the fourth Cys residue coordinating the proximal cluster. The large hydrogenase subunit (HoxH) of the SH contains the four Cys residues, conserved in all [NiFe]-hydrogenases, for the binding of the

The soluble, cytoplasmic NAD + -reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H 2 -NAD + reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity -the reduction of NAD + by H 2 . This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. [14,15]. The SH of R. eutropha belongs to a subclass of [NiFe]-hydrogenases where the polypeptide of the small hydrogenase subunit ends shortly after the position of the fourth Cys residue co-ordinating the proximal cluster [4]. The large HoxH subunit in the SH contains all conserved amino acid residues for binding of the Ni-Fe site [23,24]. Hence, the amino acid sequence suggests that the hydrogenase module in this enzyme only contains the Ni-Fe site and the proximal cluster as prosthetic groups. Fourier-transform infrared (FTIR) studies on the SH indicated that the Ni-Fe site contains two more CN ligands than the active site in standard hydrogenases, and is a (RS) 2 (CN)Ni(l-RS) 2 Fe(CN) 3 (CO) centre [25]. In contrast to standard hydrogenases, the SH is not sensitive towards oxygen and carbon monoxide and shows no redox changes of the Ni-Fe site. The Fe-S clusters in the HoxFUY subunits and the flavin in the HoxF subunit are all considered to be functional in the intramolecular electron transfer during the H 2 -NAD + reaction. It was shown recently that the protein content of SH preparations is considerably overestimated by the routine colourimetric protein-determination methods. This led to the finding that the SH contains two FMN groups and one NADH-reducible [2Fe-2S] cluster [26]. In the present paper we have investigated the possible role of the two FMN Correspondence to S. P. J. Albracht,

The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.