Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy.
The mechanism of the dioxygen (O(2)) 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 O(2)-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 (Delta UbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F(I) intermediate possessing ferryl-oxo heme o, cupric Cu(B), and ferric heme b is converted to the F(II) intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F(II) 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 Cu(B) and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japan.
Koji
K
Matsuura
Shiro
S
Yoshioka
Satoshi
S
Takahashi
Koichiro
K
Ishimori
Tatsushi
T
Mogi
Hiroshi
H
Hori
Isao
I
Morishima
D001665:Binding Sites; D003067:Coenzymes; D003573:Cytochrome b Group / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005609:Free Radicals / Q000737:chemistry; D005615:Freezing; D006418:Heme / Q000737:chemistry; D036103:Nanotechnology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D013053:Spectrophotometry; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry
D003067:Coenzymes; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D005609:Free Radicals; D014451:Ubiquinone; C069808:heme O; D006418:Heme; C017168:cytochrome bo, E coli; C030778:ubiquinone 8; D010088:Oxidoreductases; C020094:duroquinol oxidase; C024989:coenzyme Q10; D010100:Oxygen