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Reaction of Escherichia coli Cytochrome bo_3 with Substoichiometric Ubiquinol-2: A Freeze-Quench Electron Paramagnetic Resonance Investigation

Schultz, Brian E. and Edmondson, Dale E. and Chan, Sunney I. (1998) Reaction of Escherichia coli Cytochrome bo_3 with Substoichiometric Ubiquinol-2: A Freeze-Quench Electron Paramagnetic Resonance Investigation. Biochemistry, 37 (12). pp. 4160-4168. ISSN 0006-2960. doi:10.1021/bi971714y.

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The reaction of the quinol oxidase cytochrome bo_3 from Escherichia coli with ubiquinol-2 (UQ_2H_2) 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 Q_B site of the enzyme with a formation rate constant of 140 s^(-1); the Q_B semiquinone EPR signal decayed with a rate constant of about 5 s^(-1). Heme b and Cu_B 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 band heme o_3 could not be made, due to interference from dioxygen. In the reaction between the Q_B-depleted enzyme and UQ_2H_2 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 Q_B 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.

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Chan, Sunney I.0000-0002-5348-2723
Additional Information:© 1998 American Chemical Society. Received July 14, 1997; Revised Manuscript Received January 14, 1998. Publication Date (Web): March 5, 1998. This work was supported by Grants GM22432 (S.I.C.) and GM29433 (D.E.E.) from the National Institutes of Health. B.E.S. is the recipient of a National Science Foundation Postdoctoral Fellowship. We would like to thank Siegfried Musser, Robert Gennis, and Jeffrey Osborne for insightful comments, Michael Stowell for the synthesis and purification of the UQ2 used in this work, and Kirk Hansen for experimental assistance.
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NSF Postdoctoral FellowshipUNSPECIFIED
Issue or Number:12
Record Number:CaltechAUTHORS:20170919-153418165
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Official Citation:Reaction of Escherichia coli Cytochrome bo3 with Substoichiometric Ubiquinol-2:  A Freeze-Quench Electron Paramagnetic Resonance Investigation. Brian E. Schultz, Dale E. Edmondson, and Sunney I. Chan. Biochemistry 1998 37 (12), 4160-4168. DOI: 10.1021/bi971714y
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:81590
Deposited By: Tony Diaz
Deposited On:19 Sep 2017 22:46
Last Modified:15 Nov 2021 19:44

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