Micro-environment of Fe-Cu binuclear center in Cytoobrome Oxidase Studied by Electron Paramagnetic Resonance Spectroscopy

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Cytochrome bo-type ubiquinol oxidase from Eschenchia coli is a member of heme-copper terminal oxidase superfamily. It contains four redox centers, heme b, heme o, Cu_B, and ubiquinol-8(Q_H). Heme o and Cu_B constitute a binuclear center and reduce dioxygen to water. To elucidate the mechanism of dioxygen reduction, it is inevitable to detennine the electronic states of the redox centers in the reaction intermediates.1. The fully oxidized cytochrome bo contains a spin-coupled pair of high-spin heme o (S_<Fe>=5/2) and Cu_B (S_<Cu>=1/2) with an effective spin of S'=2. Integer-spin EPR signals at g=3.6 were investigated by a newly developed bimodal X-band cavity. No signal was observed with the parallel mode cavity due to its low sensitlvty.2. P intermediate can be obtained by addition of low concentrations of hydrogen peroxide to the oxidized enzyme (pulsed form). The reaction of fully oxidized cytochrome bo with hydrogen peroxide was studied by EPR. In the presence of 1 molar equiv of hydrogen peroxide, two EPR signals were observed at g〜2 typical of organic radicals. The broader signal might be a cross-linked tyrosine radical that is magnetically coupled to Cu_B, but it is uncertain at this moment. It would seem quite possible that this radical is an intermediate in the mechanism of dioxygen reduction.3. The reaction of fully reduced cytochrome be with dioxygen was investigated by the direct mixing method with EPR spectroscopy combined with the rapid-freeze quenching device. While no EPR signal derived from the binuclear center and heme b was observed in the time domain from 0.2 to 1 ms, the signals derived from the ferric hydroxide-bound form of heme o and the oxidized form of heme b were simultaneously observed after 1 ms. We interpret that the enzyme is in the F and hydroxy intermediates at 0.2 and 1 ms, respectively.