13C and 63,65Cu ENDOR studies of CO Dehydrogenase from Oligotropha carboxidovorans. Experimental Evidence in Support of a Copper-Carbonyl Intermediate

We report here an ENDOR study of an S = 1/2 intermediate state trapped during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the Cu-63,Cu-65 nuclei exhibits strong and almost entirely isotropic coupling to the unpaired electron, show that this coupling atypically has a positive sign, a(iso) = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using (CO)-C-13, coupling to the C-13 is observed, with a(iso) = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase, in conjunction with complementary computational studies, leads us to conclude that the intermediate contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme.