Paramagnetic centers and acetyl-coenzyme A/CO exchange activity of carbon monoxide dehydrogenase from Methanothrix soehngenii.

Carbon monoxide (CO) dehydrogenase was purified, both aerobically and anaerobically, to apparent homogeneity from Methanothrix soehngenii. The enzyme contained 18 +/- 2 (n = 6) mol Fe/mol and 2.0 +/- 0.1 (n = 6) mol Ni/mol. Electron paramagnetic resonance (EPR) spectra of the aerobically purified CO dehydrogenase showed one sharp EPR signal at g = 2.014 with several characteristics of a 3Fe-4S]1+ cluster. The integrated intensity of this signal was low, 0.03 S = 1/2 spin/alpha beta dimer. The 3Fe spectrum was not affected by incubation with CO or acetyl-coenzyme A, but could be reduced by dithionite. The spectrum of the reduced, aerobically purified enzyme showed complex EPR spectra, which had several properties typical of two 4Fe-4S]1+ clusters, whose S = 1/2 spins weakly interacted by dipolar coupling. The integrated intensity was 0.1-0.2 spin/alpha beta dimer. The anaerobically isolated enzyme showed EPR spectra different from the reduced aerobically purified enzyme. Two major signals were apparent. One with g values of 2.05, 1.93 and 1.865, and an Em7.5 of -410 mV, which quantified to 0.9 S = 1/2 spin/alpha beta dimer. The other signal with g values of 1.997, 1.886 and 1.725, and an Em7.5 of -230 mV gave 0.1 spin/alpha beta dimer. When the enzyme was incubated with its physiological substrate acetyl-coenzyme A, these two major signals disappeared. Incubation of the enzyme under CO atmosphere resulted in a partial disappearance of the spectral component with g = 1.997, 1.886, 1.725. Acetyl-coenzyme A/CO exchange activity, 35 nmol.min-1.mg-1 protein, which corresponded to 7 mol CO exchanged min-1 mol-1 enzyme, could be detected in anaerobic enzyme preparations, but was absent in aerobic preparations. Carbon dioxide also exchanged with C-1 of acetyl-coenzyme A, but at a much lower rate than CO and to a much lower extent.