Zinc Ions as Cytochrome c Oxidase Inhibitors: Two Sites of Action

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S. S. Kuznetsova, N. V. Azarkina, T. V. Vygodina, S. A. Siletsky, and A. A. Konstantinov^*

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobjevy Gory, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: konst@genebee.msu.su

^* To whom correspondence should be addressed.

Received September 6, 2004

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Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome-reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50-90%, depending on preparation) with an effective K_i of ~10 µM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes, and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme: full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 µM. The rate of zinc inhibitory action in the slow phase is proportional to Zn²⁺ concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F --> O step corresponding to transfer of the 4th electron in the catalytic cycle).

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KEY WORDS: cytochrome c oxidase, zinc ion, Zn²⁺, proteoliposomes

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