Capitanio N, Capitanio G, Minuto M, De Nitto E, Palese L L, Nicholls P, Papa S
Department of Medical Biochemistry and Biology, University of Bari, Piazza G. Cesare, 70124 Bari, Italy.
Biochemistry. 2000 May 30;39(21):6373-9. doi: 10.1021/bi0003137.
A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.
本文介绍了一项关于一氧化碳抑制的牛心线粒体细胞色素c氧化酶中，血红素a和铜（A）处电子转移与质子转移耦合（氧化还原玻尔效应）的研究。对每个被氧化的血红素a、铜（A）释放的H⁺的耦合数（H⁺/血红素a、铜（A）比率）进行详细分析，是基于直接测量添加到一氧化碳抑制的完全还原的细胞色素c氧化酶中的铁氰化物的氧化当量、血红素a、铜（A）的当量、添加的被氧化的细胞色素c以及氧化时释放并在金属中心再还原时被氧化酶全部重新吸收的H⁺之间的平衡。使用了两种还原剂之一，要么是琥珀酸加微量线粒体膜（提供琥珀酸 - c还原酶来源），要么是六氨合钌（II）氯盐。在6.0 - 8.5的pH范围内，实验测得的H⁺/血红素a、铜（A）比率在0.65至0.90之间变化。H⁺/血红素a、铜（A）比率的pH依赖性可以用一个涉及两个氧化还原连接酸碱基团的函数来最佳拟合，其pKₒ - pKᵣ分别为5.4 - 6.9和7.3 - 9.0。对一氧化碳抑制的氧化酶相同样品进行的氧化还原滴定表明，铜（A）和血红素a表现出叠加的E'(m)值，在6.0 - 8.5范围内，两种金属的该值均随pH单位增加约20 mV而降低。通过分析H⁺/血红素a、铜（A）比率的pH依赖性来表征的一个模型，即血红素a和铜（A）的氧化还原与两个酸碱基团的pK位移相关联，该模型为血红素a和铜（A）的E'(m)的pH依赖性提供了令人满意的拟合。所呈现的结果与血红素a和铜（A）共有的氧化还原玻尔效应在细胞色素c氧化酶的质子泵浦活性中起主要作用一致。
