Reaction of Escherichia coli cytochrome bo3 with substoichiometric ubiquinol-2: a freeze-quench electron paramagnetic resonance investigation.
The reaction of the quinol oxidase cytochrome bo3 from Escherichia coli with ubiquinol-2 (UQ2H2) was carried out using substoichiometric (0.5 equiv) amounts of substrate. Reactions were monitored through the use of freeze-quench EPR spectroscopy. Under 1 atm of argon, semiquinone was formed at the QB site of the enzyme with a formation rate constant of 140 s-1; the QB semiquinone EPR signal decayed with a rate constant of about 5 s-1. Heme b and CuB were reduced within the 10-ms dead time of the freeze-quench experiment and remained at a constant level of reduction over the 1-s time course of the experiment. Quantitation of the reduction levels of QB and heme b during this reaction yielded a reduction potential of 30-60 mV for heme b. Under a dioxygen atmosphere, the rates of semiquinone formation and its subsequent decay were not altered significantly. However, accurate quantitation of the EPR signals for heme b and heme o3 could not be made, due to interference from dioxygen. In the reaction between the QB-depleted enzyme and UQ2H2 under substoichiometric conditions, there was no observable change in the EPR spectra of the enzyme over the time course of the reaction, suggesting an electron transfer from heme b to the binuclear site in the absence of QB which occurs within the dead time of the freeze-quench apparatus. Analysis of the thermodynamics and kinetics of electron transfers in this enzyme suggests that a Q-cycle mechanism for proton translocation is more likely than a cytochrome c oxidase-type ion-pump mechanism.
Schultz
B E
BE
Division of Chemistry and Chemical Engineering, Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125, USA.
Edmondson
D E
DE
Chan
S I
SI
D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D016623:Ion Pumps / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013816:Thermodynamics; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D016623:Ion Pumps; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; C003741:ubiquinol