Photoinitiated electron transfer within the Paracoccus denitrificans cytochrome bc1 complex: mobility of the iron-sulfur protein is modulated by the occupant of the Q(o) site

Abstract

Domain rotation of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c(1) redox centers plays a key role in the mechanism of the cyt bc(1) complex. Electron transfer within the cyt bc(1) complex of Paracoccus denitrificans was studied using a ruthenium dimer to rapidly photo-oxidize cyt c(1) within 1 μs and initiate the reaction. In the absence of any added quinol or inhibitor of the bc(1) complex at pH 8.0, electron transfer from reduced ISP to cyt c(1) was biphasic with rate constants of k(1f) = 6300 ± 3000 s(-1)and k(1s) = 640 ± 300 s(-1) and amplitudes of 10 ± 3% and 16 ± 4% of the total amount of cyt c(1) photooxidized. Upon addition of any of the P(m) type inhibitors MOA-stilbene, myxothiazol, or azoxystrobin to cyt bc(1) in the absence of quinol, the total amplitude increased 2-fold, consistent with a decrease in redox potential of the ISP. In addition, the relative amplitude of the fast phase increased significantly, consistent with a change in the dynamics of the ISP domain rotation. In contrast, addition of the P(f) type inhibitors JG-144 and famoxadone decreased the rate constant k(1f) by 5-10-fold and increased the amplitude over 2-fold. Addition of quinol substrate in the absence of inhibitors led to a 2-fold increase in the amplitude of the k(1f) phase. The effect of QH(2) on the kinetics of electron transfer from reduced ISP to cyt c(1) was thus similar to that of the P(m) inhibitors and very different from that of the P(f) inhibitors. The current results indicate that the species occupying the Q(o) site has a significant conformational influence on the dynamics of the ISP domain rotation.

Figure 1

Effects of pH and MOAS on the kinetics of electron transfer between [2Fe2S] and cyt c₁. Approximately 5 μM cyt bc₁ was rapidly oxidized by 20 μM Ru₂D following a laser flash at the indicated pH. Solutions were buffered at pH 6 (A), pH, 7 (B), pH 8 (C), and pH 9 (D) by 20 mM MES, 5 mM phosphate, 20 mM TRIS-HCl, and 20 mM borate, respectively. 5 mM [Co(NH₃)₅Cl]²⁺ was present to act as a sacrificial oxidant of excited state Ru₂D. Cyt c₁ and [2Fe2S] were reduced by 1 mM ascorbate and 4 μM TMPD. All solutions were purged with an N₂ flush and kept at 10 °C. The two transients in each figure correspond to before (lower transient) and after (upper transient) addition of 25 μM MOAS. The upper transient was offset relative to the lower. Transients were fit using either a single, or sum of two exponential function represented by the single white line through each transient. The zero-point in time was set at the very beginning of the transient reduction of cyt c₁, and the arrow shows the time of the laser flash.

Figure 1

Effects of pH and MOAS on the kinetics of electron transfer between [2Fe2S] and cyt c₁. Approximately 5 μM cyt bc₁ was rapidly oxidized by 20 μM Ru₂D following a laser flash at the indicated pH. Solutions were buffered at pH 6 (A), pH, 7 (B), pH 8 (C), and pH 9 (D) by 20 mM MES, 5 mM phosphate, 20 mM TRIS-HCl, and 20 mM borate, respectively. 5 mM [Co(NH₃)₅Cl]²⁺ was present to act as a sacrificial oxidant of excited state Ru₂D. Cyt c₁ and [2Fe2S] were reduced by 1 mM ascorbate and 4 μM TMPD. All solutions were purged with an N₂ flush and kept at 10 °C. The two transients in each figure correspond to before (lower transient) and after (upper transient) addition of 25 μM MOAS. The upper transient was offset relative to the lower. Transients were fit using either a single, or sum of two exponential function represented by the single white line through each transient. The zero-point in time was set at the very beginning of the transient reduction of cyt c₁, and the arrow shows the time of the laser flash.

Figure 2

Electron transfer in P. den cyt bc₁ in the presence of P_m and P_f type inhibitors Approximately 5 μM cyt bc₁ was rapidly oxidized by 20 μM Ru₂D following laser flash ₂₊ in a 300 μL anaerobic solution of 20 mM TRIS pH 8.0 at 10°C. 5 mM [Co(NH₃)₅Cl] was present to act as a sacrificial oxidant of excited state Ru₂D. Cyt c₁ and [2Fe2S] were reduced by 1 mM ascorbate and 4 μM TMPD. P_m inhibitors myxothiazol (A) and azoxystrobin (B) were added to 25 μM. Effects of P_f inhibitors stigmatellin (C, 25 μM), JG-144 (D, 40 μM), and famoxadone (E, 25 μM) are also shown. Note the difference in time scale of the P_f inhibitors compared to the P_m inhibitors. Each transient was fit by the sum of two exponentials represented by the white line, with the kinetic parameters given in Table 2.

Figure 2

Electron transfer in P. den cyt bc₁ in the presence of P_m and P_f type inhibitors Approximately 5 μM cyt bc₁ was rapidly oxidized by 20 μM Ru₂D following laser flash ₂₊ in a 300 μL anaerobic solution of 20 mM TRIS pH 8.0 at 10°C. 5 mM [Co(NH₃)₅Cl] was present to act as a sacrificial oxidant of excited state Ru₂D. Cyt c₁ and [2Fe2S] were reduced by 1 mM ascorbate and 4 μM TMPD. P_m inhibitors myxothiazol (A) and azoxystrobin (B) were added to 25 μM. Effects of P_f inhibitors stigmatellin (C, 25 μM), JG-144 (D, 40 μM), and famoxadone (E, 25 μM) are also shown. Note the difference in time scale of the P_f inhibitors compared to the P_m inhibitors. Each transient was fit by the sum of two exponentials represented by the white line, with the kinetic parameters given in Table 2.

Figure 2

Electron transfer in P. den cyt bc₁ in the presence of P_m and P_f type inhibitors Approximately 5 μM cyt bc₁ was rapidly oxidized by 20 μM Ru₂D following laser flash ₂₊ in a 300 μL anaerobic solution of 20 mM TRIS pH 8.0 at 10°C. 5 mM [Co(NH₃)₅Cl] was present to act as a sacrificial oxidant of excited state Ru₂D. Cyt c₁ and [2Fe2S] were reduced by 1 mM ascorbate and 4 μM TMPD. P_m inhibitors myxothiazol (A) and azoxystrobin (B) were added to 25 μM. Effects of P_f inhibitors stigmatellin (C, 25 μM), JG-144 (D, 40 μM), and famoxadone (E, 25 μM) are also shown. Note the difference in time scale of the P_f inhibitors compared to the P_m inhibitors. Each transient was fit by the sum of two exponentials represented by the white line, with the kinetic parameters given in Table 2.

Figure 3

Normalized difference spectra at 540 nm of 7.7 μM P. den cyt bc₁ after addition of 1.5 mM succinate and 50 nM SCR (grey), and after subsequent addition of 100 μM decylubiquinone (black). Theoretical fits to the respective difference spectra based on the published difference values of Shinkarev et al. (68) revealed the grey line prior to the addition of substrate to consist of 0.8 μM reduced cyt c₁ and 0.8 μM reduced heme b_H. The black line after addition of substrate revealed that 7.7 μM cyt c₁ and 6.7 μM heme b_H were reduced.

Figure 4

Electron transfer in P. den cyt bc₁ in the presesence of quinol substrate at 552nm (A) and 560nm (B). Approximately 5 μM cyt bc₁ was rapidly oxidized by 20 μM Ru₂D following laser flash in a 300 μL anaerobic solution of 20 mM TRIS pH 8.0 at 10°C. 5 mM [Co(NH₃)₅Cl]²⁺ was present to act as a sacrificial oxidant of excited state Ru₂D. Cyt c₁ and [2Fe2S] were reduced by 1 mM ascorbate and 4 μM TMPD. 100 μM decylubiquinone was added and reduced by the addition of 1 mM succinate and 50 nM SCR. The white line through each transient is an exponential fit. The 552 nm transient was fit by a sum of two exponentials using the values given in table 2. The 560 nm transient was fit by a single exponential function using the values given in Table 2.

Scheme 1

Scheme 2