The Mechanism of Cytochrome c Oxidase-Catalyzed Dioxygen Reduction

Citation

Witt, Stephan N. (1988) The Mechanism of Cytochrome c Oxidase-Catalyzed Dioxygen Reduction. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/h1m5-ms09. https://resolver.caltech.edu/CaltechTHESIS:03072013-140610712

Abstract

Cytochrome c oxidase plays a primary role in energy transduction in most organisms. Free-energy is conserved by the oxidase in the form of a sizeable electrochemical hydrogen ion gradient across the inner mitochondrial membrane, which is generated during cytochrome c oxidase-catalyzed dioxygen reduction. In order to understand the energetics of cytochrome c oxidase-dependent energy conservation, the mechanism of cytochrome c oxidase-catalyzed dioxygen reduction must be understood in complete detail.

We investigated the kinetics and mechanism of cytochrome c oxidase-catalyzed dioxygen reduction at low temperatures (180-210 K) in order to determine the kinetic barriers to intramolecular electron transfer and to elucidate the structural details of the dioxygen intermediates which form at the dioxygen reduction site during a single turnover. Our results show that electron transfer to the dioxygen reduction site is possible from either of the low-potential metal centers. Upon transfer of the third electron to the dioxygen reduction site at low temperatures two reactive dioxygen intermediates form in sequence at the dioxygen reduction site. In both intermediates, dioxygen is at the three-electron level of reduction. The kinetics of the conversion between the two reactive intermediates were examined in detail. The conversion between these two reactive species is highly activated, although en tropically assisted. Our results suggest that the cleavage of the dioxygen bond in the first three-electron-reduced intermediate results in the formation of the second three-electron-reduced intermediate. By studying the reoxidation of partially reduced cytochrome c oxidase at low temperatures, we also demonstrate that the rate of electron transfer within the oxidase is dependent on the nature of the trapped dioxygen intermediate at the dioxygen reduction site. Finally, the method used for the low-temperature kinetic experiments utilizes carbon monoxide as an inhibitor. Our investigation of the reoxidation of fully reduced cytochrome c oxidase reveals that carbon monoxide is not an innocent spectator molecule over the course of the reoxidation of the oxidase. Specifically, the dissociation of carbon monoxide from the binuclear dioxygen reduction site is the rate-limiting step at low temperatures; and the second three-electron-reduced intermediate is thought to oxidize carbon monoxide to carbon dioxide at low temperature to produce a novel, readily detectable state of the enzyme.

We discovered that reactive partially reduced states of dioxygen may also be trapped at the dioxygen reduction site of cytochrome c oxidase at more ambient conditions (273-298 K) under a variety of experimental conditions. Combined chemical and spectroscopic results show that a reactive intermediate is produced at the dioxygen reduction site of cytochrome c oxidase when partially reduced cytochrome c oxidase is reoxdized with dioxygen and upon treatment of the oxidase with excess hydrogen peroxide. The trapped intermediate exhibits identical reactivity with carbon monoxide as the second three-electron-reduced intermediate which formed upon reoxidation of the fully reduced enzyme at low temperature. These results demonstrate that the same reactive three-electron-reduced dioxygen intermediate may be trapped at the dioxygen reduction site of cytochrome c oxidase either upon intramolecular electron transfer of the third electron to the peroxidic adduct, which is trapped at the binuclear site, or via an intermolecular reaction between the peroxidic adduct and hydrogen peroxide. This latter reaction is thought to involve the reduction of the enzymatic peroxidic adduct via a direct one-electron transfer from hydrogen peroxide. Our combined chemical and spectroscopic results are consistent with a binuclear dioxygen reduction site composed of a ferryl/cupric couple for the second three-electron-reduced dioxygen intermediate. Finally, we exploited the unique reaction between the second three-electron-reduced intermediate and carbon monoxide to probe the coordination sphere of the copper center at the reactive dioxygen reduction site of cytochrome c oxidase.

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