Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc1 complex

Abstract

Long-range movement of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in electron transfer within the cyt bc1 complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc1 were prepared to examine the role of this loop in controlling the capture and release of the ISP from cyt b. Electron transfer in the cyt bc1 complex was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 mus and initiate the reaction. The rate constant for electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c1 was k1 = 60 000 s-1. Famoxadone binding to the Qo site decreases k1 to 5400 s-1, indicating that a conformational change on the surface of cyt b decreases the rate of release of the ISP from cyt b. The mutation I292A on the surface of the ISP-binding crater decreased k1 to 4400 s-1, while the addition of famoxadone further decreased it to 3000 s-1. The mutation L286A at the tip of the ef loop decreased k1 to 33 000 s-1, but famoxadone binding caused no further decrease, suggesting that this mutation blocked the conformational change induced by famoxadone. Studies of all of the mutants provide further evidence that the ef loop plays an important role in regulating the domain movement of the ISP to facilitate productive electron transfer and prevent short-circuit reactions.

Figure 1

Electron transfer within wild-type R. sphaeroides cyt bc₁ following photooxidation of cyt c₁ (9). The 300 μL sample contained 5 μM cyt bc₁, 20 μM Ru₂D, 5 mM [Co(NH₃)₅Cl]²⁺, in 20 mM sodium borate, pH 9.0 with 0.01% dodecylmaltoside. The cyt bc₁ was treated with 10 μM Q_oC₁₀BrH₂, 1 mM succinate, and 50 nM SCR to completely reduce [2Fe2S] and cyt c₁, and reduce cyt b_H by about 30%. The sample was then excited with a 480 nm laser flash to photooxidize cyt c₁ within 1 μs. (Top two traces) The 552 nm transient indicates that cyt c₁ was photooxidized within 1 μs, and then reduced in a biphasic reaction with rate constants of 60,000 s⁻¹ and 2,000 s⁻¹. The rate constant for the reduction of cyt b_H measured at 561 − 569 nm was 2,300 s⁻¹. (Bottom two traces) Addition of 30 μM famoxadone decreased the rate of reduction of cyt c₁ to 5,400 s⁻¹ and eliminated reduction of cyt b_H.

Figure 2

X-ray crystal structure of bovine cyt bc₁ with bound famoxadone (31). The cyt c₁ and cyt b_L hemes are colored red, the [2Fe2S] center is represented by a CPK model, the ISP is blue, and cyt b is grey. Residues 252−268 in the ef loop are colored orange while residues 269−283 in the PEWY sequence and the ef helix are red. Residues 136−152 in the cd1 helix are green and residues 163−171 in the neck-contacting domain are colored yellow. The residues in cyt b that were mutated are shown as sticks and labeled with R. sphaeroides sequence numbering.

Figure 3

Sequence alignment of residues near the Q_o pocket of the cyt b subunit in cyt bc₁ complexes from bovine (BT) and R. sphaeroides (RS). Residues with a high sequence similarity are in boldface. Helices determined crystallographically are indicated by gray rectangles. Residues in direct contact with the ISP are indicated with a black dot. Adopted from reference .

Figure 4

Electron transfer from [2Fe2S] to cyt c₁ in R. sphaeroides cyt bc₁ mutants following photooxidation of cyt c₁. The conditions were the same as described in Figure 1. (A) I292A cyt bc₁. The rate constant k₁ is 4,400 s⁻¹ in the absence of inhibitor, and 3000 s⁻¹ in the presence of 30 μM famoxadone. (B) I292L cyt bc₁. The rate constant k₁ is 10,000 s⁻¹ in the absence of inhibitor, and 4,700 s⁻¹ in the presence of famoxadone. (C) I292M cyt bc₁. The rate constant k₁ is 6,200 s⁻¹ in the absence of inhibitor, and 2,800 s⁻¹ in the presence of famoxadone.

Figure 4

Electron transfer from [2Fe2S] to cyt c₁ in R. sphaeroides cyt bc₁ mutants following photooxidation of cyt c₁. The conditions were the same as described in Figure 1. (A) I292A cyt bc₁. The rate constant k₁ is 4,400 s⁻¹ in the absence of inhibitor, and 3000 s⁻¹ in the presence of 30 μM famoxadone. (B) I292L cyt bc₁. The rate constant k₁ is 10,000 s⁻¹ in the absence of inhibitor, and 4,700 s⁻¹ in the presence of famoxadone. (C) I292M cyt bc₁. The rate constant k₁ is 6,200 s⁻¹ in the absence of inhibitor, and 2,800 s⁻¹ in the presence of famoxadone.

Figure 4

Electron transfer from [2Fe2S] to cyt c₁ in R. sphaeroides cyt bc₁ mutants following photooxidation of cyt c₁. The conditions were the same as described in Figure 1. (A) I292A cyt bc₁. The rate constant k₁ is 4,400 s⁻¹ in the absence of inhibitor, and 3000 s⁻¹ in the presence of 30 μM famoxadone. (B) I292L cyt bc₁. The rate constant k₁ is 10,000 s⁻¹ in the absence of inhibitor, and 4,700 s⁻¹ in the presence of famoxadone. (C) I292M cyt bc₁. The rate constant k₁ is 6,200 s⁻¹ in the absence of inhibitor, and 2,800 s⁻¹ in the presence of famoxadone.

Scheme 1