Reaction of Escherichia coli Cytochrome bo3 with Substoichiometric Ubiquinol-2: A Freeze-Quench Electron Paramagnetic Resonance Investigation

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• 作者：Brian E. Schultz ; Dale E. Edmondson ; and Sunney I. Chan
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：March 24, 1998
• 年：1998
• 卷：37
• 期：12
• 页码：4160 - 4168
• 全文大小：139K
• 年卷期：v.37,no.12(March 24, 1998)
• ISSN：1520-4995

文摘

The reaction of the quinol oxidase cytochromebo₃ from Escherichia coli withubiquinol-2(UQ₂H₂) was carried out using substoichiometric(0.5 equiv) amounts of substrate. Reactions weremonitored through the use of freeze-quench EPR spectroscopy. Under1 atm of argon, semiquinone wasformed at the Q_B site of the enzyme with a formation rateconstant of 140 s^-1; the Q_Bsemiquinone EPRsignal decayed with a rate constant of about 5s^-1. Heme b andCu_B were reduced within the 10-ms deadtime of the freeze-quench experiment and remained at a constant levelof reduction over the 1-s timecourse of the experiment. Quantitation of the reduction levels ofQ_B and heme b during this reactionyielded a reduction potential of 30-60 mV for heme b.Under a dioxygen atmosphere, the rates ofsemiquinone formation and its subsequent decay were not alteredsignificantly. However, accuratequantitation of the EPR signals for heme b and hemeo₃ could not be made, due to interferencefromdioxygen. In the reaction between the Q_B-depletedenzyme and UQ₂H₂ under substoichiometricconditions,there was no observable change in the EPR spectra of the enzyme overthe time course of the reaction,suggesting an electron transfer from heme b to the binuclearsite in the absence of Q_B which occurswithin the dead time of the freeze-quench apparatus. Analysis ofthe thermodynamics and kinetics ofelectron transfers in this enzyme suggests that a Q-cycle mechanism forproton translocation is morelikely than a cytochrome c oxidase-type ion-pumpmechanism.