Use of a photoactivated ruthenium dimer complex to measure electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the cytochrome bc(1) complex.
Electron transfer between the Rieske iron-sulfur protein (Fe(2)S(2)) and cytochrome c(1) was studied using the ruthenium dimer, Ru(2)D, to either photoreduce or photooxidize cytochrome c(1) within 1 micros. Ru(2)D has a charge of +4, which allows it to bind with high affinity to the cytochrome bc(1) complex. Flash photolysis of a solution containing beef cytochrome bc(1), Ru(2)D, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 micros, followed by electron transfer from cytochrome c(1) to Fe(2)S(2) with a rate constant of 90,000 s(-1). Flash photolysis of reduced beef bc(1), Ru(2)D, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 micros, followed by electron transfer from Fe(2)S(2) to cytochrome c(1) with a rate constant of 16,000 s(-1). Oxidant-induced reduction of cytochrome b(H) was observed with a rate constant of 250 s(-1) in the presence of antimycin A. Electron transfer from Fe(2)S(2) to cytochrome c(1) within the Rhodobacter sphaeroides cyt bc(1) complex was found to have a rate constant of 60,000 s(-1) at 25 degrees C, while reduction of cytochrome b(H) occurred with a rate constant of 1000 s(-1). Double mutation of Ala-46 and Ala-48 in the neck region of the Rieske protein to prolines resulted in a decrease in the rate constants for both cyt c(1) and cyt b(H) reduction to 25 s(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.
Sadoski
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RC
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Engstrom
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G
Tian
H
H
Zhang
L
L
Yu
C A
CA
Yu
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L
Durham
B
B
Millett
F
F
D019943:Amino Acid Substitution / Q000235:genetics; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002417:Cattle; D003575:Cytochromes c1 / Q000378:metabolism*; D019281:Dimerization; D004579:Electron Transport / Q000187:drug effects; D014450:Electron Transport Complex III / Q000737:chemistry / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009942:Organometallic Compounds / Q000378:metabolism*; D009994:Osmolar Concentration; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010782:Photolysis; D018583:Pliability; D011090:Polyenes / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides; D012428:Ruthenium / Q000378:metabolism*; D055672:Static Electricity
D000814:Aniline Compounds; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D009942:Organometallic Compounds; D016877:Oxidants; D011090:Polyenes; C037989:Rieske iron-sulfur protein; C409198:bis(bis(2,2'-bipyridine)ruthenium(II))(2,2'-4',4''-2'',2'''-quaterpyridine); D000968:Antimycin A; D012428:Ruthenium; D003575:Cytochromes c1; C041573:stigmatellin; D014450:Electron Transport Complex III; C023650:aniline