Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a₃ (Fe_a3) and copper (Cu_B). Here, the influence of the ligation at the oxidized Fe_a3³⁺–Cu_B²⁺ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (E_m(a)) for the cyanide (the low-spin Fe_a3³⁺) and the formate-ligated CcO (the high-spin Fe_a3³⁺). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of E_m(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fe_a³⁺ between His–Fe_a³⁺–His and His–Fe_a³⁺–OH⁻ occurs only at pH above 9.5 with the estimated pK >11.0.

在牛细胞色素c氧化酶（CcO）的质子泵过程中，细胞色素a被认为是关键的氧化还原中心。最近的研究表明，血红素a及其附近区域的结构都对由细胞色素a ₃（Fe _a3）的铁和铜（Cu _B）组成的遥远催化中心的连接和氧化还原状态敏感。在这里，氧化的Fe _a3 ³⁺ –Cu _B ²⁺中心的连接对血红素a处电子-质子耦合的影响在较宽的pH范围（6.5-11）中进行了检测。通过确定血红素a（E _m（a））对氰化物（低自旋Fe _a3 ³⁺）和甲酸酯连接的CcO（高自旋Fe _a3 ³⁺）。当其他三个CcO氧化还原中心被氧化时，在实验条件下进行测量。对于CN–和甲酸酯连接的CcO，发现E _m（a）有两个略有不同的线性pH依赖性，其斜率分别为−13 mV / pH单位和−23 mV / pH单位。这些线性相关性表明在细胞色素a处仅弱且非特异性的电子-质子耦合两种形式的CcO。氰化物连接的氧化CcO的UV-Vis吸收和电子-顺磁共振波谱研究也证实了在生理pH值下缺乏强电子-质子耦合的情况。结果表明，在铁的配位体交换_一个³⁺的His-铁之间_一个³⁺ -His和His-铁_一个³⁺ -OH ^-仅在pH大于9.5与所估计的pK> 11.0出现。