Tanja Burgdorf

[NiFe]-Hydrogenases of Ralstonia eutropha H16: Modular Enzymes for Oxygen-Tolerant Biological Hydrogen Oxidation

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.

The soluble [NiFe]-hydrogenase from Ralstonia eutropha 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(μ-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.

Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

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. In one of these isolates (HoxH I64A), H(2) binding was impaired. Class II mutants revealed a high D(2)/H(+) exchange rate relative to a low H(2)-oxidizing activity. A representative (HoxH H16L) displayed D(2)/H(+) exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O(2)-sensitive growth on hydrogen due to an O(2)-sensitive SH protein.

A hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in Ralstonia eutropha.

H(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. Thus aerobic H(2)-oxidizing organisms receive H(2) only occasionally and in limited amounts. Metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of H(2). The proteobacterium Ralstonia eutropha strain H16 that harbours three [NiFe] hydrogenases perfectly meets these demands. The unusual biochemical and structural properties of the hydrogenases are described, including the strategies that confer O(2) tolerance to the NAD-reducing soluble hydrogenase and the H(2)-sensing regulatory hydrogenase. The regulatory hydrogenase that forms a complex with a histidine protein kinase recognizes H(2) in the environment and transmits the signal to a response regulator, which in turn controls transcription of the hydrogenase genes.

Impact of alterations near the [NiFe] active site on the function of the H2 sensor from Ralstonia eutropha

In proteobacteria capable of H2 oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H2. The H2‐sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]‐hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni–Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H2‐oxidizing and D2/H+ exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para‐/ortho‐H2 conversion activity showed that these centres were still able to bind H2. This indicates that H2 binding at the active site is not sufficient for the regulatory function of H2 sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy‐linked [NiFe]‐hydrogenases. The respective RH mutant protein showed only traces of H2‐oxidizing activity, whereas its D2/H+‐exchange activity and H2‐sensing function were almost unaffected. H2‐dependent signal transduction in this mutant was less sensitive to oxygen than in the wild‐type strain. These results suggest that H2 turnover is not crucial for H2 sensing. It may even be detrimental for the function of the H2 sensor under high O2 concentrations.

Non-standard structures of the Ni-Fe cofactor in the regulatory and the NAD-reducing hydrogenases from Ralstonia eutropha.

Spectroscopy on two oxygen-insensitive Ni-Fe hydrogenases from Ralstonia eutropha (NAD-reducing, soluble hydrogenase; hydrogen sensor, regulatory hydrogenase) reveals non-standard catalytic behaviour and unique structures of their Ni-Fe cofactors. Possible mechanistic implications are briefly discussed.