The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex.
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.
Gurung
Buddha
B
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Yu
Linda
L
Xia
Di
D
Yu
Chang-An
CA
D000818:Animals; D001692:Biological Transport; D055592:Biophysical Phenomena; D001703:Biophysics; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004925:Escherichia / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008027:Light; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D013053:Spectrophotometry; D013997:Time Factors
D007506:Iron-Sulfur Proteins; D008024:Ligands; D010743:Phospholipids; D011522:Protons; D006639:Histidine; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III; D010100:Oxygen