The mechanism of the dioxygen (O2) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O2-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (ΔUbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the FI intermediate possessing ferryl-oxo heme o, cupric CuB, and ferric heme b is converted to the FII intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the FII intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and CuB and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.
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