Identification of the Structural Subunits Required for Formation of the Metal Centers in Subunit I of Cytochrome c Oxidase of Rhodobacter sphaeroides

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• 作者：Melyssa R. Bratton ; Laree Hiser ; William E. Antholine ; Curt Hoganson ; and Jonathan P. Hosler
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：October 24, 2000
• 年：2000
• 卷：39
• 期：42
• 页码：12989 - 12995
• 全文大小：79K
• 年卷期：v.39,no.42(October 24, 2000)
• ISSN：1520-4995

文摘

Genetic manipulation of the aa₃-type cytochrome c oxidase of Rhodobacter sphaeroides wasused to determine the minimal structural subunit associations required for the assembly of the heme Aand copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the genefor subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I_a) that containeda single heme A. No copper was present in this protein, indicating that the heme a₃-Cu_B active site wasnot assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase formswere synthesized that were copurified by histidine affinity chromatography and separated by anion-exchangechromatography. One form was a highly active subunit I-II oxidase containing a full complement ofstructurally normal metal centers. This shows that association of subunit II with subunit I is required forstable formation of the active site in subunit I. In contrast, subunit III is not required for the formation ofany of the metal centers or for the production of an oxidase with wild-type activity. The second productof the cells lacking subunit III was a large amount of a free form of subunit I that appeared identicalto subunit I_a. Since significant amounts of subunit I_a were also isolated from wild-type cells, it is likelythat subunit I_a will be present in any preparation of the aa₃-type oxidase isolated via an affinity tag onsubunit I.