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 micros, 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.
Highlights
Electron transfer from the Rieske iron-sulfur protein to cytochrome c1 in the Rhodobacter sphaeroides cytochrome bc1 complex was studied using a ruthenium dimer complex, Ru2D
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 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
Summary
Materials—Ru2D was prepared by a modification of the method of Downard et al [21]. Cytochrome c (horse heart, Type III) was purchased from Sigma Chemical Co. Succinate cytochrome c reductase (SCR) was purified as previously described [23]. The His-tagged bc complex with fully oxidized, or reduced cytochrome c1 was obtained by addition of K3Fe(CN) or sodium ascorbate, respectively, followed by passage through the nickel-nitrilotriacetic acid column to remove excess oxidant or reductant. The redox status of heme c1 and the 2Fe-2S in the partially reduced wild-type and mutant complexes were determined as previously described [9]. Reduction of 2Fe-2S was followed by measuring the negative circular dichroism peak, at 500 nm, of partially reduced complex minus fully oxidized complex in a JASCO J-715 spectropolarimeter (26 –28). Samples typically contained 5 M R. sphaeroides bc complex and 20 M Ru2D in 20 mM sodium borate or Tris-Cl buffer with 0.02% lauryl maltoside. The error for each parameter reported is the asymptotic standard error representing 95% confidence limits given by the algorithm
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