UC Riverside

Carbon monoxide dehydrogenase from Oligotropha carboxidovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are passed intramolecularly through two [2Fe-2S] clusters and finally to a FAD cofactor. From FAD the electrons are ultimately passed to the electron transport chain of the Gram-negative organism.

In the current study we have examined a variety of aspects of this enzyme in the oxidative- and reductive-half reactions and propose mechanisms for the oxidation of carbon monoxide and the proximal electron acceptor of the enzyme.

First, we have identified the proximal acceptor of reducing equivalents. We have found CO dehydrogenase passes electrons directly to the quinone pool without using a cytochrome as an intermediary as had previously been proposed. This establishes a new category of redox-partner for the xanthine oxidase family of enzymes.

Next, we examined the active site and find silver can be replaced for the active site copper. Cyanide effectively removes the copper and a Ag(I)-thiourea solution can reactive the enzyme, albeit at a lower turnover rate. The silver reconstitution can be verified by EPR, evident by the lack of coupling to the copper I=3/2 nucleus and in its place the sliver I=1/2 nucleus. This altered but active form of the protein is used to compare and contrast with the native copper- containing enzyme to develop a mechanism for CO oxidation.

We then examined the EPR of CO dehydrogenase reduced by CO by electron nuclear double resonance spectroscopy (ENDOR). The 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, a_iso = +148 MHz. 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 leads us to conclude that the intermediate contains a partially reduced, Mo(V)/Cu(I), center with CO bound at the copper.

We next further characterized the kinetics and mechanisms of hydrogenase activity previously reported and find CO dehydrogenase effectively catalyzes H₂ oxidation to protons. This activity is found to be independent of pH and does not appear to be reversible. A new EPR signal was found and is attributed to the H₂ bound state with the molybdenum in an oxidation state, Mo(V), that prevents further catalysis.

Finally, we have examined the inhibition of the enzyme by n-butylisonitrile and bicarbonate. We find that n-butylisonitrile reduces the and irreversibly inhibits the enzyme as is suggested by the crystal structure and computational studies previously reported. Bicarbonate acts as an uncompetitive inhibitor, reducing v_max and K_m, while also producing a new EPR signal of the bicarbonate complex.