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 QB site of the enzyme with a formation rateconstant of 140 s-1; the QBsemiquinone EPRsignal decayed with a rate constant of about 5s-1. Heme b andCuB 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 ofQB 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 QB-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 QB 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.