Mössbauer study of CO dehydrogenase from Clostridium thermoaceticum.

We have studied with Mössbauer spectroscopy the metal clusters of CO dehydrogenase from Clostridium thermoaceticum. At potentials greater than -200 mV, all of the approximately 12 irons reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials a variety of components are observed. About 40% of the Fe appears to belong to one [Fe4S4]1+ cluster. We have also observed the Mössbauer spectrum (approximately 18% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65. Also present is a doublet (9% of Fe) with delta EQ = 2.90 mm/s and delta = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component seems to interact with a nickel site to form an EPR-silent complex with half-integral electronic spin. We have also characterized the iron environments of the S = 1/2 NiFeC complex. This complex contributes approximately 20% of the total Mössbauer absorption when the EPR signal has approximately 0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, we speculate that the NiFeC complex may consist of a nickel site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the Mössbauer and EPR data, taken together, force us to conclude that current preparations, while homogeneous according to purifications standards, are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compositions in this enzyme premature.