Subunit III of cytochrome c oxidase is not involved in proton translocation: a site‐directed mutagenesis study.
Subunit III of cytochrome c oxidase is not involved in proton translocation: a site‐directed mutagenesis study.
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细胞色素 c 氧化酶的亚基 III 不参与质子易位：一项定点诱变研究。
作者：
T. Haltia;Matti Saraste;Mårten Wikström
亚基III（COIII）是aa3型细胞色素c氧化酶的三个核心亚基之一。COIII不含任何氧化还原中心，可以从纯化的酶中除去，但在酶的生物合成过程中具有功能。二环己基碳二亚胺（DCCD）修饰COIII中保守的谷氨酸残基，并消除酶的质子转运活性。在这项研究中，COIII的不变羧酸E98（DCCD结合谷氨酸）和D259通过定点突变来改变，以研究它们在质子泵中的作用。光谱和活性测量表明，在诱变的COIII的存在下，形成了一种结构正常的酶，这是积极的电子转移。细菌原生质球实验表明，突变氧化酶是完全胜任质子易位。在没有COIII基因的情况下，只有一部分氧化酶被组装成具有低但显著活性的酶。这种残余活性也与质子移位有关。我们的结论是，与许多早期的建议，COIII不是质子泵的基本要素。
Subunit III (COIII) is one of the three core subunits of the aa3‐type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD‐binding glutamic acid) and D259 of COIII were changed by site‐directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.