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J. Biol. Chem., Vol. 280, Issue 26, 24895-24902, July 1, 2005

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The Iron-Sulfur Cluster of the Rieske Iron-Sulfur Protein Functions as a Proton-exiting Gate in the Cytochrome bc₁ Complex^*

Buddha Gurung, Linda Yu, Di Xia, and Chang-An Yu¶

From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078 and the Laboratory of Cell Biology Center, NCI, National Institutes of Health, Bethesda, Maryland 20892

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc₁ complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable (Miki, T., Yu, L., and Yu, C.-A. (1991) Biochemistry 30, 230-238). To study further the role of this [2Fe-2S] cluster in proton translocation of the bc₁ complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc₁ complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc₁ activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc₁ complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc₁ complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc₁ complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc₁ complexes opened up an otherwise closed proton channel within the bc₁ complex. It was speculated that in the normal catalytic cycle of the bc₁ complex, the [2Fe-2S] cluster may function as a proton-exiting gate.

Received for publication, March 25, 2005 , and in revised form, April 28, 2005.

^* This work was supported by National Institutes of Health Grant GM30721, and the Oklahoma Agricultural Experiment Station Projects 1819, 2371, and 2372, Oklahoma State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: 255 NRC, Oklahoma State University, Stillwater, OK 74078. Tel.: 405-744-6612; Fax: 405-744-7799; E-mail: cayuq{at}okstate.edu.

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