TY - JOUR
T1 - The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16
AU - Winter, Gordon
AU - Buhrke, Thorsten
AU - Jones, Anne K.
AU - Friedrich, Bärbel
N1 - Funding Information:
Acknowledgements The authors thank A.L. DeLacey for measuring D2/H+ exchange activities. This work was funded by the Deutsche Forschungsgemeinschaft (SFB 498, TP C1) and the Fonds der Chemischen Industrie. AKJ thanks the Alexander von Humboldt Foundation for a post-doctoral fellowship.
PY - 2004/10
Y1 - 2004/10
N2 - The H2-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 H2 sensing and H2 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.
AB - The H2-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 H2 sensing and H2 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.
KW - H sensor
KW - Metal center assembly
KW - Nickel metabolism
KW - Ralstonia eutropha
KW - [NiFe] hydrogenase
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U2 - 10.1007/s00203-004-0680-6
DO - 10.1007/s00203-004-0680-6
M3 - Article
C2 - 15340794
AN - SCOPUS:5444225517
SN - 0302-8933
VL - 182
SP - 138
EP - 146
JO - Archives of Microbiology
JF - Archives of Microbiology
IS - 2-3
ER -