Abstract
The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) 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 bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) 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 bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) 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 bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.
Highlights
The Iron-Sulfur Cluster of the Rieske Iron-Sulfur Protein Functions as a Proton-exiting Gate in the Cytochrome bc1 Complex*
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
The three-dimensional structural information for the mitochondrial bc1 complex establishes the location of the redox centers, the number of transmembrane helices, quinone binding at the Qi site, and inhibitor binding at both the Qo and Qi sites [2,3,4, 8, 9]
Summary
Vol 280, No 26, Issue of July 1, pp. 24895–24902, 2005 Printed in U.S.A. The Iron-Sulfur Cluster of the Rieske Iron-Sulfur Protein Functions as a Proton-exiting Gate in the Cytochrome bc Complex*. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc 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. 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 bc complexes opened up an otherwise closed proton channel within the bc complex. The three-dimensional structural information for the mitochondrial bc complex establishes the location of the redox centers, the number of transmembrane helices, quinone binding at the Qi site, and inhibitor binding at both the Qo and Qi sites [2,3,4, 8, 9] It suggests mobility of the extrinsic head domain of ISP during bc catalysis. Restoration of proton-pumping activity was explored by adding excess amounts of ISP head domain containing the [2Fe-2S] cluster to proton-leaking PL vesicles embedding mitochondrial bc complex and thermolysin-digested bacterial bc complex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
