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Time-Resolved Electrometric Study of the F→O Transition in Cytochrome c Oxidase. The Effect of Zn2+ Ions on the Positive Side of the Membrane

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Abstract

The effect of Zn2+ on the P-side of proteoliposomes containing membrane-incorporated Rhodobacter sphaeroides cytochrome c oxidase was investigated by the time-resolved electrometrics following a single electron injection into the enzyme prepared in the F state. The wild-type enzyme was examined along with the two mutants, N139D and D132N. All obtained data indicate that the primary effect of Zn2+ added from the P-side of the membrane is slowing of the pumped proton release from the proton loading site (PLS) to the bulk aqueous phase on the P-side of the membrane. The results strongly suggest the presence of two pathways by which the pumped proton can exit the protein from the PLS and of two separate binding sites for Zn2+. A model is presented to explain the influence of Zn2+ on the kinetics of membrane-potential generation by the wild-type COX, as well as by the N139D and D132N mutants.

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Abbreviations

COX:

cytochrome c oxidase

F, O, and P:

states of cytochrome c oxidase, respectively

P- and N-phases:

positively and negatively charged aqueous phases, respectively separated by the coupling membrane

Rubpy:

tris-bipyridyl complex of ruthenium(II)

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Acknowledgements

The authors are grateful to Ilya Oleynikov for technical assistance in the production of Fig. 10. The authors express their gratitude to anonymous Reviewers, especially to the Reviewer 2 and Reviewer 3.

Funding

This work was supported by the Russian Science Foundation (research project no. 19-14-00063).

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Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    Sergey A. Siletsky

  2. Department of Biochemistry, University of Illinois at Urbana-Champaign, 61801, Urbana, IL, USA

    Robert B. Gennis

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Correspondence to Sergey A. Siletsky.

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Siletsky, S.A., Gennis, R.B. Time-Resolved Electrometric Study of the F→O Transition in Cytochrome c Oxidase. The Effect of Zn2+ Ions on the Positive Side of the Membrane. Biochemistry Moscow 86, 105–122 (2021). https://doi.org/10.1134/S0006297921010107

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