POSSIBLE PROTON RELAY PATHWAYS IN CYTOCHROME-C-OXIDASE
POSSIBLE PROTON RELAY PATHWAYS IN CYTOCHROME-C-OXIDASE
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DOI：
10.1073/pnas.92.5.1604
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发表时间：
1995-02-28
影响因子：
11.1
通讯作者：
FERGUSONMILLER, S
中科院分区：
文献类型：
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作者：
FETTER, JR;QIAN, J;FERGUSONMILLER, S
作为真核生物和许多原核生物呼吸链中的最终电子受体，细胞色素 c 氧化酶 (EC 1.9.3.1) 催化氧还原成水并产生质子梯度。为了测试通过氧化酶的质子途径，对球形红杆菌酶的亚基 I 进行了定点诱变。突变体的特征在于肽的三个高度保守的区域，包括可能的质子加载、卸载和转移位点：螺旋 II 和 III 之间的内部环 (Asp132Asn/Ala)、螺旋 IX 和 X 之间的外部环 (His411Ala、Asp412Asn、Thr413Asn、Tyr414Phe) 以及预测的跨膜螺旋 VIII （Thr352Ala、Pro358Ala、Thr359Ala、Lys362Met）。大多数突变体的活性低于野生型，但只有残基 132 处的突变体失去了质子泵浦，同时保留了电子转移活性。尽管电子转移基本上受到抑制，但 D132 突变体似乎没有发生重大结构改变，因为共振拉曼和可见光吸收光谱正常。然而，较少的 CO 结合（野生型的 70-85%）表明双核中心发生了一些微小的变化。此外，重建的 Asp132 突变体的活性受到离子载体或解偶联剂的抑制而不是刺激。纯化的酶没有观察到抑制作用，并且排除了直接的 pH 效应，表明对电梯度或 pH 梯度的响应发生了改变。结果支持保守的 II-III 环在质子泵送过程中的重要作用，并且与螺旋 VIII 和 IX-X 环中残基参与的可能性一致。
As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Asp132Asn/Ala), an exterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity, Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, fewer CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Asp132 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-III loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.