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Spectroscopic and Mechanistic Studies of the Mo/Cu Carbon Monoxide Dehydrogenase From Oligotropha carboxidovorans

  • Author(s): Dingwall, Stephanie
  • Advisor(s): Hille, Russ
  • et al.
Abstract

The molybdenum- and copper-containing enzyme carbon monoxide dehydrogenase from Oligotropha carboxidovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, bioremediating about 400 million tons of CO from the atmosphere annually. During catalysis, the substrate is oxidized at a binuclear metal center containing Mo and Cu, with electrons passed via two [2Fe-2S] clusters to a FAD cofactor before ultimately being transferred to the quinone pool of the electron transport chain. Our studies have examined different aspects of catalysis, from the nature of the electron flow through the system to an examination of the binuclear center during enzyme turnover.

First, we have identified the formation of the FADH● semiquinone species during catalysis. This is the first confirmed appearance of the neutral radical species in CO dehydrogenase, revealed from enzyme-monitored turnover, quench flow, and reductive titration experiments.

Next, we have determined that pH effects in CO dehydrogenase are unique to the enzyme and associated with its FAD. pH jump and reductive titration experiments at pH 6 and 10 reveal pH-dependent UV/visible spectra. Upon covalent modification of the flavin by diphenyliodonium chloride, which leads to its covalent modification and inactivation at the FAD, spectral differences at the two pH extremes are abolished. Similar experiments involving xanthine oxidase and xanthine dehydrogenase show no pH-dependent spectral differences, implying that the pH effects are unique to CO dehydrogenase.

Lastly, electron nuclear double resonance (ENDOR) experiments have been performed to further characterize the binuclear center of the partially-reduced enzyme. ENDOR data of 12C and 13C bicarbonate-bound enzyme reveal that bicarbonate is bound to the copper, rather than the molybdenum of the binuclear center, and so is unlikely to be an intermediate during catalysis. Analysis of 16O and 17O Mims ENDOR indicate that the equatorial ligand in the molybdenum coordination sphere is not a Mo=O but Mo-OH, and is catalytically-labile, being incorporated into the product CO2 and regenerated from solvent in the course of each catalytic sequence.

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