Effect of Ca²⁺ on the redox potential of heme a in cytochrome c oxidase.

Subunit I of cytochrome c oxidase (CcO) from mitochondria and many bacteria contains a cation binding site (CBS) located at the outer positively charged aqueous phase not far from heme a. Binding of Ca²⁺ with the CBS in bovine CcO inhibits activity of the enzyme 2-3 -fold [Vygodina, T., Kirichenko, A. & Konstantinov A.A. (2013) Direct Regulation of Cytochrome c Oxidase by Calcium Ions, PLoS One.8 e74436]. Here we show that binding of Ca²⁺ at CBS of bovine CcO shifts E_m of heme a to the positive by 15-20 mV. Na⁺ ions that bind to the same site and compete with Ca²⁺ do not affect E_m of heme a and also prevent and reverse the effect of Ca²⁺. No effect of Ca²⁺ or EGTA is observed on E_m of heme a with the wild type bacterial oxidases from R.sphaeroides or P.denitrificans that contain tightly-bound calcium at the site. In the D477A mutant CcO from P. denitrificans that binds Ca²⁺ reversibly like the mitochondrial CcO, calcium shifts redox titration curve of heme a to the positive by ∼35-50 mV that is in good agreement with the results of electrostatic calculations; however, as shown earlier, it does not inhibit CcO activity of the mutant enzyme. Therefore the data do not support the proposal that the inhibitory effect of Ca²⁺ on CcO activity may be explained by the Ca²⁺-induced shift of E_m of heme a. Rather, Ca²⁺ retards electron transfer by inhibition of charge dislocation in the exit part of the proton channel H in mammalian CcO, that is absent in the bacterial oxidases.

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