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 ^63,65Cu 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 ¹³CO, coupling to the ¹³C 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.