Use of a photoactivated ruthenium dimer complex to measure electron transfer between the Rieske iron-sulfur protein and cytochrome c1 in the cytochrome bc1 complex

Electron transfer between the Rieske iron-sulfur protein (Fe2S2) and cytochrome c(1) was studied using the ruthenium dimer, Ru2D, to either photoreduce or photooxidize cytochrome c(1) within 1 mu s. Ru2D 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), Ru2D, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 mu s, followed by electron transfer from cytochrome c(1) to Fe2S2 with a rate constant of 90 000 s(-1). Flash photolysis of reduced beef bc(1), Ru2D, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 mu s, followed by electron transfer from Fe2S2 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 Fe2S2 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.