EXAFS, EPR, and Electronic Absorption Spectroscopic Study of the .alpha. Metallo Subunit of CO Dehydrogenase from Clostridium thermoaceticum

Abstract

The a metallosubunit of carbon monoxide dehydrogenase from Clostridium thennoaceticum was isolated by subjecting native enzyme to low concentrations of the detergent sodium dodecyl sulfate, followed by anaerobic preparative native polyacrylamide gel electrophoresis. The isolated a subunit absorbs in the 400 nm region and contains one Ni and four Fe ions. The irons are organized into an [Fe4S4I2+'l+ cluster, the reduced form of which exhibits EPR features between g = 6 and 3, and a weak (0.1 spin/a) gav = 1.94 signal. The reduced cluster appears to exist in an S = 3/2 : S = '/2 spin-state mixture and to be predominantly S = 3/2 at 10 K. The Ni center is EPR silent and presumably 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 2.19 A and two N/O donors at 1.89 A. Comparison of the Ni edge spectrum with those of structurally characterized Ni(II)N& model compounds suggests a DU distortion with a dihedral angle of about 20-30'. The Ni center does not appear to be incorporated into the cluster, and it may or may not be bridged to the cluster. These centers may be decompositional relatives of the A-cluster, the active site for acetyl-coenzyme A synthesis. JA950685G

Evidence for Intersubunit Communication during Acetyl-CoA Cleavage by the Multienzyme CO Dehydrogenase/Acetyl-CoA Synthase Complex from Methanosarcina thermophila

“…The pseudo NiFeC signal also is formed when N-bromosuccinimide-inactivated CODH/ACS from C. thermoaceticum is treated with CO (33) or when the C. thermoaceticum ␣ subunit, isolated after SDS treatment, is reacted with CO (34). The pseudo NiFeC species exhibits 61 Ni, 57 Fe, and 13 CO hyperfine interactions (4) and Mössbauer and UV-visible spectroscopic parameters that are very similar to those of the standard NiFeC species (27,43). Thus, the truncated ␤ subunit contains an altered form of Cluster A that is still able to bind CO.…”

“…Apparently, some nickel was lost during the purification. Both Clusters A (4,26,27) and C (15, 28) contain a NiFeS cluster. Since Cluster C is unarguably associated with the ␣ subunit (1, 13), these results suggest that the nickel in the ␤ subunit is a component of Cluster A, which is responsible for acetyl-CoA cleavage and synthesis.…”

Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli

“…CO oxidation and CO͞acetyl-CoA exchange activities were performed as described (38,43). The Ni and Fe contents of purified AcsAB were determined using atomic absorption spectrometry (12). EPR spectra of AcsAB were determined using a Bruker ESP300 spectrometer and an Oxford Instruments ER910A cryostat.…”

“…The one-and three-electron reduced C red1 and C red2 states exhibit EPR signals with g av ϭ 1.82 and 1.86, respectively. The A cluster is the active site for acetyl-CoA synthesis and is also composed of an Ni center bridged to an Fe 4 S 4 cluster (12)(13)(14)(15). The Ni ion of the A cluster is labile and can be removed easily by incubating ACS Ct in the chelator 1,10-phenanthroline.…”

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

“…Examples include nitrile hydratase [1][2][3][4][5][6][7][8][9][10] and the A-cluster of carbon monoxide dehydrogenase/acetyl CoA synthase [11][12][13][14][15][16][17][18][19][20][21][22][23]. Understanding the catalytic mechanisms of these enzymes can be greatly aided by the study of small-molecule analogs, or model complexes, which reproduce the active-site structure and/or the function of the enzyme.…”