CHARACTERIZATION OF THE NI-FE-C COMPLEX FORMED BY REACTION OF CARBON-MONOXIDE WITH THE CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM BY Q-BAND ENDOR

Q-Band ENDOR studies on carbon monoxide dehydrogenase (CODH) from the acetogenic bacterium Clostridium thermoaceticum provided unambiguous evidence that the reaction of CO with CODH produces a novel metal center that includes at least one nickel, at least three iron sites, and the carbon of one CO. The Fe-57 hyperfine couplings determined by ENDOR are similar to the values used in simulation of the Mossbauer spectra [Lindahl et al. (1989) J. Biol. Chem. 265, 3880-3888]. EPR simulation using these A(Fe) values is equally good for a 4Fe or a 3Fe center. The C-13 ENDOR data are consistent with the binding of a carbon atom to either the Ni or the Fe component of the spin-coupled cluster. The C-13 hyperfine couplings are similar to those determined earlier for the CO-bound form of the H cluster of the Clostridium pasteurianum hydrogenase, proposed to be the active site of hydrogen activation [Telser et al. (1987) J. Biol. Chem. 262, 6589-5694]. The Ni-61 ENDOR data are the first nickel ENDOR recorded for an enzyme. The EPR simulation using the ENDOR-derived hyperfine values for Ni-61 is consistent with a single nickel site in the Ni-Fe-C complex. On the basis of our results and the Mossbauer data [Lindahl et al. (1989) J. Biol. Chem. 265, 3880-3888], we propose the stoichiometry of the components of the Ni-Fe-C complex to be Ni1Fe3-4S greater-than-or-equal-to 4C1, with four acid-labile sulfides.