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

When samples of the enzyme in the C-red1 State were reduced with Ti3+ citra te, the C-cluster stabilized in an EPR-silent state. Subsequent treatment w ith CO or dithionite yielded C-red2 The EPR-silent state formed within 1 mi n of adding Ti3+ citrate, while Cred2 formed after 60 min. Ti3+ citrate app eared 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 i ts existence. This mechanism is analogous to those used by flavins and hydr ogenases to convert between n = 2 and n = 1 processes. Ti3+ citrate had a d ifferent effect on enzyme in a CO2 atmosphere; it shifted reduction potenti als of metal centers (relative to those obtained using CO) and did not stab ilize C-int. Different redox behavior was also observed when methyl viologe n and benzyl viologen were used as reductants. This variability was exploit ed to prepare enzyme samples in which EPR from C-red2 was present without i nterfering signals from Bred. The saturation properties of Bred depended up on the redox state of the enzyme. Three saturation "modes", called Sat1-Sat 3, 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 poi sed at slightly negative potentials. Sat:! was characterized by weak intens ity from all three resonances, and was strictly associated with intermediat e redox states and the presence of CO2. Sat3 was characterized by strong br oad resonances with normalized intensities essentially unchanged relative t o nonsaturating conditions, and was observed at the most negative potential s. Each mode probably reflects different spatial relationships among magnet ic components in the enzyme.