title: Evidence for a proposed intermediate redox state in the CO/CO(2) active site of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium thermoaceticum.
abstract: When samples of the enzyme in the C(red1) state were reduced with Ti(3+) citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded C(red2). The EPR-silent state formed within 1 min of adding Ti(3+) citrate, while C(red2) formed after 60 min. Ti(3+) citrate appeared to slow the rate by which C(red2) formed from C(red1) and stabilize the C-cluster in the previously proposed C(int) state. This is the first strong evidence for C(int), and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti(3+) citrate had a different effect on enzyme in a CO(2) atmosphere; it shifted reduction potentials of metal centers (relative to those obtained using CO) and did not stabilize C(int). Different redox behavior was also observed when methyl viologen and benzyl viologen were used as reductants. This variability was exploited to prepare enzyme samples in which EPR from C(red2) was present without interfering signals from B(red). The saturation properties of B(red) depended upon the redox state of the enzyme. Three saturation "modes", called Sat1-Sat3, were observed. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2. 04 and 1.90 resonances, and was observed in samples poised at slightly negative potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO(2). Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was observed at the most negative potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.
authors.affiliation: Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77842, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Fraser
authors.forename: P A
authors.initials: PA
authors.lastname: Lindahl
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D001594:Benzyl Viologen / Q000737:chemistry; D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D009097:Multienzyme Complexes / Q000737:chemistry*; D010084:Oxidation-Reduction; D010269:Paraquat / Q000737:chemistry; D011487:Protein Conformation; D014025:Titanium / Q000737:chemistry; D017186:Titrimetry
chemical_list: D009097:Multienzyme Complexes; D001594:Benzyl Viologen; D002245:Carbon Dioxide; D002248:Carbon Monoxide; D014025:Titanium; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D010269:Paraquat