The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16.
The H(2)-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H(2)-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H(2) sensing and H(2) oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.
Winter
Gordon
G
Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestr. 117, 10115 Berlin, Germany.
Buhrke
Thorsten
T
Jones
Anne K
AK
Friedrich
Bärbel
B
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020118:Cupriavidus necator / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics / Q000502:physiology*; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D015151:Immunoblotting; D009532:Nickel / Q000378:metabolism; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000378:metabolism
D001426:Bacterial Proteins; C108874:HypC protein, Bacteria; D021122:Protein Subunits; D009532:Nickel; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine