Time-resolved generation of membrane potential by ba 3 cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and O H states

Sergey A. Siletsky, Ilya Belevich, Nikolai P. Belevich, Tewfik Soulimane, Mårten Wikström

Research output: Contribution to journalArticlepeer-review

Abstract

Two electrogenic phases with characteristic times of ~ 14 μs and ~ 290 μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized “relaxed” O state of ba 3 oxidase from T. thermophilus (O → E transition). The rapid phase reflects electron redistribution between Cu A and heme b. The slow phase includes electron redistribution from both Cu A and heme b to heme a 3 , and electrogenic proton transfer coupled to reduction of heme a 3 . The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a 3 is reduced, but there is no proton pumping and no reduction of Cu B . Single-electron reduction of the oxidized “unrelaxed” state (O H → E H transition) is accompanied by electrogenic reduction of the heme b/heme a 3 pair by Cu A in a “fast” phase (~ 22 μs) and transfer of protons in “middle” and “slow” electrogenic phases (~ 0.185 ms and ~ 0.78 ms) coupled to electron redistribution from the heme b/heme a 3 pair to the Cu B site. The “middle” and “slow” electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach Cu B the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~ 0.1 H + /e , probably due to the formed membrane potential in the experiment.

Original languageEnglish
Pages (from-to)915-926
Number of pages12
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1858
Issue number11
DOIs
Publication statusPublished - Nov 2017

Keywords

  • Catalytic cycle intermediates
  • Charge transfer steps
  • Cytochrome c oxidase
  • Membrane potential
  • Thermus thermophilus

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