Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases
OAK-B135 Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was ''rescued'' by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also ''rescued'' by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster. Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.
- Research Organization:
- Texas A& M University, College Station, TX (US)
- Sponsoring Organization:
- Office of Energy Research; basic biological sciences (US)
- DOE Contract Number:
- FG03-01ER15177
- OSTI ID:
- 826210
- Report Number(s):
- DOE/ER/15177-5; TRN: US0403828
- Journal Information:
- Biochemistry, Vol. 43, Issue 19; Other Information: Published in Biochemistry, Volume 43, No.19; DOI 10.1021/bi036062u; PBD: 18 May 2004
- Country of Publication:
- United States
- Language:
- English
Similar Records
Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation
Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue