Evidence for a proposed intermediate redox state in the CO/CO2 active site of acetyl-CoA synthase (carbon monoxide dehydrogenase) from Clostridium thermoaceticum

When samples of the enzyme in the C-red1 State were reduced with Ti3+ 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 Ti3+ citrate, while Cred2 formed after 60 min. Ti3+ 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. Ti3+ citrate had a different effect on enzyme in a CO2 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 Bred. The saturation properties of Bred 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. Sat:! was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO2. 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.