Aspartate-407 in Rhodobacter sphaeroides Cytochrome c Oxidase Is Not Required for Proton Pumping or Manganese Binding

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• 作者：Jie Qian ; Wenjun Shi ; Michelle Pressler ; Curt Hoganson ; Denise Mills ; Gerald T. Babcock ; and Shelagh Ferguson-Miller
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：March 4, 1997
• 年：1997
• 卷：36
• 期：9
• 页码：2539 - 2543
• 全文大小：254K
• 年卷期：v.36,no.9(March 4, 1997)
• ISSN：1520-4995

文摘

Several pathways for proton transport in cytochrome coxidase have been proposed on thebasis of mutational analysis and X-ray structure: at least one formoving "pumped" protons from theinterior to exterior of the membrane and a separate route fortransporting "substrate" protons from theinterior to the binuclear metal center to combine with oxygen to makeH₂O. According to the crystalstructures of cytochrome c oxidase, Asp407(Rhodobacter sphaeroides numbering) is at the interfaceofsubunit I and subunit II of the oxidase, in a negative patch proposedto be the proton exit site in a pumpingpathway, as well as a possible ligand to Mg [Iwata et al. (1995)Nature 376, 660-669]. Three mutantsat the Asp407 position of R. sphaeroides cytochrome oxidase,Asp407Ala, Asp407Asn, and Asp407Cys,have been purified and characterized. All showed electron transferactivity, and pH dependence of activity,similar to that of the wild type enzyme and no major structuralchanges, as evidenced by visible, EPR,and resonance Raman spectroscopy. When reconstituted intoartificial vesicles, the purified mutants pumpedprotons with normal efficiency and responded to the membrane pH andelectrical gradients in a mannersimilar to that of wild type. Furthermore, the EPR spectra and Mnquantitation analysis of mutants grownin high Mn indicated no significant alteration in the Mn/Mg site.These results suggest that Asp407 doesnot play a critical role in proton translocation or in Mn/Mgbinding.