EXAFS, EPR, AND ELECTRONIC ABSORPTION SPECTROSCOPIC STUDY OF THE ALPHA-METALLOSUBUNIT OF CO DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM

The alpha metallosubunit of carbon monoxide dehydrogenase from Clostri dium thermoaceticum was isolated by subjecting native enzyme to low co ncentrations of the detergent sodium dodecyl sulfate, followed by anae robic preparative native polyacrylamide gel electrophoresis. The isola ted alpha subunit absorbs in the 400 nm region and contains one Ni and four Fe ions. The irons are organized into an [Fe4S4](2+/1+) cluster, the reduced form of which exhibits EPR features between g = 6 and 3, and a weak (0.1 spin/alpha) g(av) = 1.94 signal. The reduced cluster a ppears to exist in an S = 3/2 : S = 1/2 spin-state mixture and to be p redominantly S = 3/2 at 10 K. The Ni center is EPR silent and presumab ly Ni(II). X-ray absorption edge and EXAFS spectra reveal that the Ni center has a distorted square-planar geometry with two S donors at 2.1 9 degrees and two N/O donors at 1.89 Angstrom. Comparison of the Ni ed ge spectrum with those of structurally characterized Ni(II)N2S2 model compounds suggests a D-2d distortion with a dihedral angle of about 20 -30 degrees. The Ni center does not appear to be incorporated into the cluster, and it may or may not be bridged to the cluster. These cente rs may be decompositional relatives of the A-cluster, the active site for acetyl-coenzyme A synthesis.