Photoinduced Electron Transfer between the Rieske Iron-Sulfur Protein and Cytochrome c1 in theRhodobacter sphaeroides Cytochromebc1 Complex

Abstract

Electron transfer from the Rieske iron-sulfur protein to cytochrome c 1 (cyt c 1 ) in the Rhodobacter sphaeroides cytochrome bc 1 complex was studied using a ruthenium dimer complex, Ru 2 D. Laser flash photolysis of a solution containing reduced cyt bc 1 , Ru 2 D, and a sacrificial electron acceptor results in oxidation of cyt c 1 within 1 s, followed by electron transfer from the iron-sulfur center (2Fe-2S) to cyt c 1 with a rate constant of 80,000 s ؊1 . Experiments were carried out to evaluate whether the reaction was rate-limited by true electron transfer, proton gating, or conformational gating. The temperature dependence of the reaction yielded an enthalpy of activation of ؉17.6 kJ/mol, which is consistent with either rate-limiting conformational gating or electron transfer. The rate constant was nearly independent of pH over the range pH 7 to 9.5 where the redox potential of 2Fe-2S decreases significantly due to deprotonation of His-161. The rate constant was also not greatly affected by the Rieske iron-sulfur protein mutations Y156W, S154A, or S154A/Y156F, which decrease the redox potential of 2Fe-2S by 62, 109, and 159 mV, respectively. It is concluded that the electron transfer reaction from 2Fe-2S to cyt c 1 is controlled by conformational gating.The cytochrome bc 1 complex (ubiquinol:cytochrome c oxidoreductase) is an integral membrane protein in the energyconserving electron transport chains of mitochondria and many respiratory and photosynthetic prokaryotes (1). The complex contains the Rieske iron-sulfur protein, cyt 1 c 1 , and two b-type hemes (b L and b H ) in the cyt b subunit (1, 2). In the Q-cycle mechanism, the complex translocates four protons to the positive side of the membrane per two electrons transferred from ubiquinol to cyt c (2). In a key bifurcated reaction at the Q o site, the first electron is transferred from ubiquinol to the Rieske iron-sulfur center (2Fe-2S) and then to cyt c 1 and cyt c (1-3). The second electron is transferred from semiquinone in the Q o site to cyt b L and then to cyt b H and ubiquinone in the Q i site. Extensive x-ray crystallographic studies of cyt bc 1 have revealed that the Rieske iron-sulfur protein occurs in several different conformations depending on the crystal form and the presence of Q o site inhibitors ( Fig. 1) (4 -6). In native I4 1 22 bovine crystals an anomalous signal for 2Fe-2S is found close to cyt b L , but its intensity is small, suggesting that the Rieske iron-sulfur protein is conformationally mobile (4, 7). Addition of the Q o inhibitors UHDBT or stigmatellin significantly increased the intensity of 2Fe-2S, indicating that the Rieske iron-sulfur protein was immobilized with 2Fe-2S near the surface of cyt b L (7). In both chicken and yeast cyt bc 1 crystals, grown in the presence of stigmatellin, the Rieske iron-sulfur protein is in a conformation with 2Fe-2S proximal to the cyt b L heme, called the b state (5, 8). However, in native chicken or beef P6 5 22 crystals, the Rieske iron-sulfur protein is in a conformation with 2Fe-2S cl...

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). The rate constant of the reduction of heme b H by ubiquinol was found to be 270 s Ϫ1 with a photoreleasable caged ubiquinol substrate with mitochondrial cytochrome bc 1 (36).…”

Modifications of Protein Environment of the [2Fe-2S] Cluster of the bc1 Complex

“…However, critical as this parameter is, any kinetic mechanism that recognizes the first electron transfer as the determining step would predict that changes in E m on mutation would be expressed in changes in overall rate constant, so this does not represent a feature unique to the mechanism proposed. The reaction in which ISPH is oxidized has a much higher rate constant, low activation barrier, and is relatively independent of pH (13,14,28), and it would not be rate determining under the experimental conditions used here.…”

“…These two residues thus contribute substantially to the electronegativity responsible for the high potential (E m ϳ300 mV in mitochondria and ␣-proteobacteria (30)) and low pK a values (30 -32). Strains mutated at these and several other residue positions were produced in the bc 1 complex from R. sphaeroides (24,28), Paracoccus denitrificans (25), and in yeast (26). From the variation in rate with driving force, each of these laboratories suggested that the first electron transfer must control the overall rate of QH 2 oxidation.…”

Effect of Famoxadone on Photoinduced Electron Transfer between the Iron-Sulfur Center and Cytochrome c 1 in the Cytochrome bc 1 Complex

“…The development of the ruthenium photoreduction method provides an opportunity to measure electron transfer between 2Fe-2S and cyt c 1 in both the forward and reverse directions and thus provides kinetic information on two different initial redox states of cyt bc 1 (10). Moreover, it is becoming clear that the measured rates of electron transfer are probably ratelimited by conformational changes in the ISP (36). The binuclear complex Ru 2 D contains the 2,2Ј:4Ј,4Љ:2Љ,2ٞ-quaterpyridine ligand, which bridges the two ruthenium atoms ( Fig.…”

“…k 1 is attributed to electron transfer from 2Fe-2S to cyt c 1 , whereas k 2 is due to subsequent electron transfer from Q-H 2 to 2Fe-2S and cyt c 1 followed by electron transfer from the semiquinone to cyt b L and cyt b H (Scheme 2) (10). Previous studies of the effects of temperature, pH, and redox potential demonstrated that k 1 is not rate-limited by true electron transfer in the c 1 state but rather is gated by conformational changes from the b state and the mobile state to the active c 1 state (36). According to this analysis, the population of the c 1 state is small but the rate constant for electron transfer from 2Fe-2S to cyt c 1 in the c 1 state is much larger than the observed value of k 1 , 80,000 s…”