The Soluble NAD⁺-Reducing [NiFe]-Hydrogenase fromRalstonia eutrophaH16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

Abstract

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...

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

“…Once thought to be confined to anaerobic archaea , these enzymes actually occur in at least 27 bacterial and archaeal phyla, among them multiple representatives of the 'MDM' (Rinke et al, 2013) (Figure 3). In addition to the versatile group 3d [NiFe]-hydrogenases (Burgdorf et al, 2005), these oxygen-tolerant enzymes are proposed to serve as redox valves that interconvert electrons between NAD(P)H and H 2 depending on the availability of exogenous electron acceptors . These enzymes are also abundant at the metagenome level, constituting dominant groups in aerated soils and hot spring ecosystems (Figure 4).…”

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…All of the NAD(P)H-dependent hydrogenases are multimeric enzyme complexes [135], including a subunit with a flavin moiety which carries out the oxidation/reduction reaction of the nicotinamide cofactor. The best studied member is the bidirectional soluble [NiFe]-hydrogenase of Cupriavidus necator H16 [136], which oxidizes or produces H 2 in response to changes in cytoplasmic reduction states.…”

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

“…In R. eutropha, the purified Hox complex is Hox-FUYHI 2 , although HoxI (not present in other characterized Hox hydrogenases) dissociates easily upon more stringent purification conditions (which is why the complex was initially characterized as HoxFUYH) (16). Other purifications in R. eutropha utilizing gene knockouts and ectopic expression demonstrate that HoxYH and HoxFU subcomplexes can be isolated, although these subcomplexes were reportedly unstable (13).…”

“…The [FeS] cluster-containing subunits HoxF, HoxU, and, in some organisms, HoxE form the diaphorase moiety (13,14) that catalyzes the oxidation/reduction of NAD(P)H/ NAD(P) ϩ (via FMN and NAD binding sites in HoxF) coupled to the hydrogenase moiety (HoxYH) (10,(12)(13)(14)(15). The R. eutropha Hox hydrogenase does not contain HoxE and instead harbors an unrelated fifth subunit, HoxI, which functions in linkage to NADPH (16).…”