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