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

---

Sergey A. Siletsky^1,a* and Robert B. Gennis^2,b

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

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

^* To whom correspondence should be addressed.

Received October 5, 2020; Revised December 12, 2020; Accepted December 12, 2020

---

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.

---

KEY WORDS: cytochrome c oxidase, zinc ions, proteoliposomes, electrogenic, proton pump, cytochrome aa₃, Rhodobacter sphaeroides

DOI: 10.1134/S0006297921010107

---