13C and 6365Cu ENDOR studies of CO dehydrogenase from Oligotropha carboxidovorans. Experimental evidence in support of a copper-carbonyl intermediate

摘要

We report here an ENDOR study of an S = ½ 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 exhibits strong and almost entirely isotropic coupling, show that this coupling atypically has a positive sign, aiso = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using ¹³CO, coupling to the ¹³C is observed, with aiso = +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.