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Flash-induced membrane potential generation by cytochrome c oxidase

D Zaslavsky1, A D Kaulen, I A Smirnova

  • 1A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation.

FEBS Letters
|December 28, 1993
PubMed
Summary

Flash-induced electron transfer in cytochrome c oxidase reveals three membrane potential phases. The rapid phase involves electron transfer from CuA to heme a, while slower phases are linked to Compound F reduction and proton pumping.

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Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome C oxidase from bovine heart.

Biochemistry. Biokhimiia·2014

Area of Science:

  • Biochemistry
  • Bioenergetics
  • Membrane Biophysics

Background:

  • Cytochrome c oxidase (CcO) is a key enzyme in cellular respiration, catalyzing the reduction of oxygen to water.
  • Understanding the precise mechanisms of electron transfer and proton pumping in CcO is crucial for elucidating energy transduction in biological systems.
  • Previous studies have investigated CcO function, but the detailed kinetics and stoichiometry of proton translocation during specific catalytic steps remain areas of active research.

Purpose of the Study:

  • To investigate the kinetics and mechanism of membrane potential generation during the flash-induced reduction of CcO Compound F.
  • To elucidate the roles of different electron transfer pathways (CuA to heme a) and redox states (Compound F to Ox state) in proton translocation.
  • To determine the stoichiometry of proton pumping associated with the reduction of Compound F.

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Main Methods:

  • Flash-induced spectroscopy was used to monitor electron transfer events in proteoliposomes reconstituted with CcO.
  • Membrane potential changes were measured using time-resolved electrochromic shifts.
  • Kinetic analysis of membrane potential phases (tau values and contributions) was performed to dissect different steps of the reaction.

Main Results:

  • Three distinct phases of membrane potential generation were observed: 45 microseconds (20%), 1 ms (20%), and 5 ms (60%).
  • The rapid phase (45 µs) was insensitive to ligands and attributed to vectorial electron transfer from CuA to heme a.
  • The slow phases (1 ms and 5 ms) were dependent on peroxide, inhibited by cyanide, and associated with the reduction of Compound F to the Ox state by heme a, involving proton uptake and transmembrane pumping.

Conclusions:

  • The reduction of Compound F to the Ox state is linked to both chemical proton uptake and electrogenic proton pumping across the membrane.
  • The observed stoichiometry suggests approximately 1.5 protons are pumped per electron transferred during the F to Ox transition, in addition to a chemical proton.
  • The biphasic kinetics of the slow phases may indicate the existence of two forms of Compound F, with differential coupling to proton pumping.