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

Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany,¹ Instituto de Catalisis (CSIC), Campus Universidad Autonoma de Madrid, 28049 Madrid, Spain²

Received 20 May 2002/ Accepted 16 August 2002

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 H₂-oxidizing activity. In one of these isolates (HoxH I64A), H₂ binding was impaired. Class II mutants revealed a high D₂/H⁺ exchange rate relative to a low H₂-oxidizing activity. A representative (HoxH H16L) displayed D₂/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 O₂-sensitive growth on hydrogen due to an O₂-sensitive SH protein.

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