. The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.

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 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 tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD؉ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H 2 -oxidizing activity. The enzyme hydrogenase catalyzes the reversible cleavage of H 2 into 2 H ϩ ions and 2 electrons. Hydrogenases are found in almost all phylogenetic lines of prokaryotes and in a few unicellular eukaryotes. The physiological function of a given hydrogenase is dependent on its cellular location and its capacity to interact with various redox partners. Based on their metal contents, two major classes are distinguished: the [NiFe] and Fe-only hydrogenases. Fe-only hydrogenases are extremely sensitive to O 2 and so far have been found only in obligate anaerobes, where they occur either as a monomer in the cytoplasm or as a heterodimer in the periplasm (1, 45). [NiFe] hydrogenases are composed of at least two heterologous polypeptides, a large subunit of approximately 60 kDa and a small subunit that is relatively diverse in size and cofactor composition (40).Crystallographic data are available for both classes of hydrogenases (12,15,16,25,27,42 Electron paramagnetic resonance (EPR) spectroscopy and Xray absorption spectroscopy (XAS) show that H 2 activation occurs close to the nickel atom, suggesting a change in its oxidation state during the catalytic cycle (14,26,39).Mutant proteins are useful tools for mechanistic studies. A complex protein-assisted maturation pathway is involved in the assembly of the [NiFe] active site (7,17,19). Almost no information is available on these posttranslational processes in strictly anaerobic organisms, whose hydrogenase structure is well defined. On the other hand, elaborate efforts to crystallize [NiFe] hydrogenases from aerobic organisms, for which hydrogenase biosynthesis has been extensively studied, have not yet been successful.More than 100 hydrogenase gene sequences are now available in the database. Inspection of these sequences shows that despite functional and structural diversity, core signatures are highly conserved in the catalytic subunit, in particular in the environment of the [NiFe] active site (3,43). Therefore, analysis of mutant proteins, in correlation with the available atomic structures, should provide further insight into the reaction mechanism of [NiFe] hydrogenases.In this study we selected the cytoplasmic [NiFe] hydrogenase (soluble hydrogenase [SH]) of Ralstonia eutropha as a model. This hydrogenase consists of a heterodimeric hydrogenase module together with a flavin mononucleotide (FMN)-containing iron-sulfur protein, in which the capacity to couple H 2 oxidation with the redu...