Stoichiometric CO reductive titrations of acetyl-CoA synthase (carbon monoxide dehydrogenase) from Clostridium thermoaceticum

Acetyl-CoA synthase (carbon monoxide dehydrogenase) from Clostridium thermo aceticum was stoichiometrically titrated under CO2 with CO and thionin, Res ulting EPR titration curves were simulated assuming different redox descrip tions of the enzyme. Samples began slightly reduced, with similar to 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 i ntensities of C-red2, B-red, and A(red)-CO increased and plateaued. Simulat ions revealed that only a fraction of the A-, B-, and C-centers in the enzy me 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 proces ses occurred at higher potentials, but they are catalytically irrelevant. A dditional low-potential redox sites may be present, but this could not be a ssessed from the titrations. Best-fit E degrees(Cox/Cred1) = -0.12 +/- 0.04 V, 0.1 V less negative than under Ar. Titrated samples accepted from 3.5 t o 5.0 equiv/alpha beta in accordance with the intensity of the sample's EPR signals. A similar titration under Ar revealed a different pattern of redu ction. The intensity of Bred increased first; then C-red1 converted to C-re d2, and then A(red)-CO developed. The sample accepted similar to 4 equiv/al pha beta. The heterogeneity in the enzyme is summarized as follows. About f orty percent of A(ox) can be reduced by one electron and bound with CO, yie lding A(red)-CO, The remaining A(ox) cannot be reduced. All B-ox can be red uced, most (similar to 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 t hen 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.