Use of a Photoactivated Ruthenium Dimer Complex To Measure Electron Transfer between the Rieske Iron-Sulfur Protein and Cytochrome c sub(1) in the Cytochrome bc sub(1) Complex

Electron transfer between the Rieske iron-sulfur protein (Fe sub(2)S sub(2)) and cytochrome c sub(1) was studied using the ruthenium dimer, Ru sub(2)D, to either photoreduce or photooxidize cytochrome c sub(1) within 1 mu s. Ru sub(2)D has a charge of +4, which allows it to bind with high affinity to the cytochrome bc sub(1) complex. Flash photolysis of a solution containing beef cytochrome bc sub(1), Ru sub(2)D, and a sacrificial donor resulted in reduction of cytochrome c sub(1) within 1 mu s, followed by electron transfer from cytochrome c sub(1) to Fe sub(2)S sub(2) with a rate constant of 90 000 s super(-1). Flash photolysis of reduced beef bc sub(1), Ru sub(2)D, and a sacrificial acceptor resulted in oxidation of cytochrome c sub(1) within 1 mu s, followed by electron transfer from Fe sub(2)S sub(2) to cytochrome c sub(1) with a rate constant of 16 000 s super(-1). Oxidant-induced reduction of cytochrome b sub(H) was observed with a rate constant of 250 s super(-1) in the presence of antimycin A. Electron transfer from Fe sub(2)S sub(2) to cytochrome c sub(1) within the Rhodobacter sphaeroides cyt bc sub(1) complex was found to have a rate constant of 60 000 s super(-1) at 25 degree C, while reduction of cytochrome b sub(H) occurred with a rate constant of 1000 s super(-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 sub(1) and cyt b sub(H) reduction to 25 s super(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.