Subunit I of cytochrome c oxidase (CcO) from mitochondria and many bacteria contains a cation binding site (CBS) located at the outer positively charged (P-) 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.

来自线粒体和许多细菌的细胞色素C氧化酶（CcO）的亚基I包含一个阳离子结合位点（CBS），位于外部带正电（P-）的水相，距离血红素a不远。Ca ²⁺与牛CcO中CBS的结合抑制酶的活性2-3倍[Vygodina，T.，Kirichenko，A.＆Konstantinov AA（2013）直接调节钙离子对细胞色素c氧化酶的作用，PLoS。8 e74436]。在这里，我们表明，Ca ²⁺在牛CcO的CBS处的结合将血红素a的E _m移至正值15-20 mV。钠⁺与相同位置结合并与Ca ²⁺竞争的^离子不会影响血红素a的E _m，也不会阻止和逆转Ca ²⁺的作用。没有观察到Ca ²⁺或EGTA对血红素a E _m的影响，而该血红素a与来自球形芽孢杆菌或denitrificans的野生型细菌氧化酶在该位点含有紧密结合的钙。在来自反硝化假单胞菌的D477A突变体CcO像线粒体CcO一样可逆地结合Ca ^2+时，钙移动了血红素a的氧化还原滴定曲线约35-50 mV的正值，这与静电计算的结果非常吻合；但是，如前所述，它不抑制突变酶的CcO活性。因此，数据不支持Ca ²⁺对CcO活性的抑制作用可能是由Ca ²⁺引起的血红素E E _m的移动来解释的。而是，Ca ²⁺通过抑制哺乳动物CcO中质子通道H的出口部分中的电荷错位来阻止电子传递，这在细菌氧化酶中是不存在的。