A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Biochimie. 2018 Jun;149:71-78. doi: 10.1016/j.biochi.2018.04.005. Epub 2018 Apr 7.
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 Ca 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 Ca at CBS of bovine CcO shifts E of heme a to the positive by 15-20 mV. Na ions that bind to the same site and compete with Ca do not affect E of heme a and also prevent and reverse the effect of Ca. No effect of Ca or EGTA is observed on E 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 Ca 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 Ca on CcO activity may be explained by the Ca-induced shift of E of heme a. Rather, Ca 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.
细胞色素 c 氧化酶（CcO）亚基 I 来自线粒体和许多细菌，它含有一个位于带正电荷的外部水相的阳离子结合位点（CBS），距离血红素 a 不远。在牛 CcO 中，CBS 与 Ca 的结合将酶的活性抑制 2-3 倍[Vygodina, T., Kirichenko, A. & Konstantinov A.A. (2013) 钙离子对细胞色素 c 氧化酶的直接调节， PLoS One.8 e74436]。在这里，我们表明，CBS 中 Ca 的结合将血红素 a 的 E 向正方向移动 15-20 mV。与 CBS 结合并与 Ca 竞争的 Na 离子不会影响血红素 a 的 E，也可以防止和逆转 Ca 的作用。在野生型 R.sphaeroides 或 P.denitrificans 细菌氧化酶中，CBS 上结合有紧密结合的 Ca，Ca 或 EGTA 对血红素 a 的 E 没有影响。在从 P.denitrificans 中获得的 D477A 突变型 CcO 中，Ca 可以可逆地结合，类似于线粒体 CcO，Ca 将血红素 a 的氧化还原滴定曲线向正方向移动约 35-50 mV，这与静电计算的结果非常吻合；然而，正如前面所示，它不会抑制突变酶的 CcO 活性。因此，这些数据不支持 Ca 对 CcO 活性的抑制作用可能是由于 Ca 诱导血红素 a 的 E 发生变化的观点。相反，Ca 通过抑制质子通道 H 出口部分的电荷离域来减缓电子转移，而这种作用在细菌氧化酶中不存在。
