In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected].
The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected].
Laboratory for Nitrogen Fixation, Oregon State University, Corvallis 97331-2902.
L A
LA
Sayavedra-Soto
D J
DJ
Arp
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics*; D001594:Benzyl Viologen / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003903:Deuterium / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism*; D008751:Methylene Blue / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype
D001594:Benzyl Viologen; D006859:Hydrogen; D003903:Deuterium; D006864:Hydrogenase; D003545:Cysteine; D010100:Oxygen; D008751:Methylene Blue