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

Abstract

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.

Simultaneous reduction of iron–sulfur protein and cytochrome b L during ubiquinol oxidation in cytochrome bc 1 complex

“…A rate constant of 1,200 s Ϫ1 was reported when using a binuclear ruthenium complex to rapidly photooxidize cytochrome c 1 in the purified R. sphaeroides bc 1 complex (47). Using the same technique of rapid photoreduce/photooxidize cytochrome c 1 with ruthenium dimer (Ru 2 D), oxidant-induced reduction of cytochrome b H was observed with a rate constant of 250 s Ϫ1 in the presence of antimycin A with bovine bc 1 complex and 1,000 s Ϫ1 with R. sphaeroides bc 1 complex (48).…”

Electron Transfer from the Rieske Iron-Sulfur Protein (ISP) to Cytochrome f in Vitro

“…From the observed decrease in turnover rate in site-directed mutants of the ISP of the cytochrome bc 1 complex that associated with a decreased ISP E m (44,45), it was inferred that the quinol-ISP electron transfer step is rate-limiting in the bc 1 complex. A slow phase of cytochrome c 1 reduction, k 2 (c1-ISP) ϭ 250 to 180 s Ϫ1 and 1000 s Ϫ1 , respectively, observed in photoactivated electron transfer using isolated mitochondrial and bacterial bc 1 complexes, was attributed to the quinol-ISP electron transfer step (11). The fast phase of the photoactivated cytochrome c 1 reduction, k ET (c1-ISP) ϳ 16,000 -60,000 s Ϫ1 (11), was associated with electron transfer from ISP docked to cytochrome c 1 , implying that electron transfer from docked ISP to cytochrome c 1 or f (see above) is also not rate-limiting.…”

“…5A), and reduction with k 1,slow (ISP-f) Ϸ 150 -250 s Ϫ1 . The missing 50% of the amplitude of cytochrome f oxidation-reduction can be accounted for if an additional microsecond kinetic component, k 1,fast (ISP-f) , similar to that observed in the reduction of cytochrome c 1 (11), is included in describing the reduction of cytochrome f (Fig. 5A).…”

“…b Rate constants obtained from stopped-flow data in the present study. c Rate constants were obtained from measurements in isolated cytochrome bc 1 complexes; 2/3 and 1/3 of the amplitude of the cytochrome c 1 reduction is associated with the large and small rate constants (11). re-reduction of 50% of the cytochrome f prevents observation of the full oxidation of the total chemical content of cytochrome f, in this case k 1 (f-PC) ϭ 2700 s Ϫ1 and k 1,fast (ISP-f) ϭ 3200 s Ϫ1 .…”

“…Only about half of the chemical content of cytochrome f is observed to turn over with this half-time after a short, 5-s flash (8 -10). The other half has been inferred to be reduced too rapidly to be observed (10), with a rate constant, ϳ10 4 s Ϫ1 , presumably similar to the fast phase of reduction of cytochrome c 1 in the bc 1 complex (11).…”