The fifth electron in the fully reduced caa3 from Thermus thermophilus is competent in proton pumping

Sergey A. Siletsky, Ilya Belevich, Tewfik Soulimane, Michael I. Verkhovsky, Mårten Wikström

Research output: Contribution to journalArticlepeer-review

Abstract

The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa3 cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a3 to heme a and further to the cytochrome c/CuA has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a3 to heme a and further to the cytochrome c/CuA pair is resolved as a single transition with τ ∼ 40 μs. In the reaction of the fully reduced cytochrome caa3 with oxygen, the first electrogenic phase (τ ∼ 30 μs) is linked to OO bond cleavage and generation of the P R state. The next electrogenic component (τ ∼ 50 μs) is associated with the PR → F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ∼ 0.25 ms, ∼ 1.4 ms and ∼ 4 ms, are linked to the conversion of the binuclear center through the F → OH → EH transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.

Original languageEnglish
Pages (from-to)1-9
Number of pages9
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1827
Issue number1
DOIs
Publication statusPublished - Jan 2013

Keywords

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

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