Qian J, Shi W, Pressler M, Hoganson C, Mills D, Babcock G T, Ferguson-Miller S
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Biochemistry. 1997 Mar 4;36(9):2539-43. doi: 10.1021/bi962721+.
Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.
基于突变分析和X射线结构，已经提出了细胞色素c氧化酶中质子转运的几种途径：至少有一种用于将“泵送”的质子从膜内部转移到外部，还有一条独立的途径用于将“底物”质子从内部运输到双核金属中心，与氧结合生成H2O。根据细胞色素c氧化酶的晶体结构，Asp407（球形红细菌编号）位于氧化酶亚基I和亚基II的界面处，在一个负电区域，该区域被认为是泵送途径中的质子出口位点，也是Mg的可能配体[岩田等人（1995年）《自然》376，660 - 669]。已经纯化并表征了球形红细菌细胞色素氧化酶Asp407位置的三个突变体，即Asp407Ala、Asp407Asn和Asp407Cys。所有突变体都表现出电子转移活性以及活性的pH依赖性，与野生型酶相似，并且没有明显的结构变化，可见光谱、电子顺磁共振（EPR）光谱和共振拉曼光谱证明了这一点。当重新组装到人工囊泡中时，纯化的突变体以正常效率泵送质子，并以与野生型相似的方式响应膜pH和电势梯度。此外，在高锰环境中生长的突变体的EPR光谱和锰定量分析表明，Mn/Mg位点没有显著改变。这些结果表明，Asp407在质子转运或Mn/Mg结合中不发挥关键作用。
