Stoichiometric CO reductive titrations of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium...

Acetyl-CoA synthase (carbon monoxide dehydrogenase) from Clostridium thermoaceticum was stoichiometrically titrated under CO(2) with CO and thionin. Resulting EPR titration curves were simulated assuming different redox descriptions of the enzyme. Samples began slightly reduced, with approximately 20% of the C-cluster reduced to the C(red1) state. With increasing CO intensities, the intensity of C(red1) increased to a maximum, and then declined as the intensities of C(red2), B(red), and A(red)-CO increased and plateaued. Simulations revealed that only a fraction of the A-, B-, and C-centers in the enzyme were redox-active and that, within the probed potentials (-0.2 to -0.45 V), there are probably no other redox sites in the enzyme. Oxidative processes occurred at higher potentials, but they are catalytically irrelevant. Additional low-potential redox sites may be present, but this could not be assessed from the titrations. Best-fit E degrees (C)()ox(/C)()red1 = -0.12 +/- 0.04 V, 0.1 V less negative than under Ar. Titrated samples accepted from 3.5 to 5.0 equiv/alphabeta in accordance with the intensity of the sample's EPR signals. A similar titration under Ar revealed a different pattern of reduction. The intensity of B(red) increased first; then C(red1) converted to C(red2), and then A(red)-CO developed. The sample accepted approximately 4 equiv/alphabeta. The heterogeneity in the enzyme is summarized as follows. About forty percent of A(ox) can be reduced by one electron and bound with CO, yielding A(red)-CO. The remaining A(ox) cannot be reduced. All B(ox) can be reduced, most ( approximately 65%) to the S = (1)/(2) state and the remainder to the S = (3)/(2) state. About 40% of C(ox) are reduced by one electron to C(red1) and then by two more electrons to C(red2). The remaining C(ox) clusters are reduced by one electron to an S = (3)/(2) form. Possible origins of this heterogeneity are discussed.