~(13)C and ~(63,65)Cu 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 = 1/2 intermediate state trapped during reduction of the binudear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the ~(63,65)Cu 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 ~(13)CO, coupling to the ~(13)C 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.