Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex

Abstract

Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton-coupled electron transfer from quinol to iron-sulfur cluster through a bridging hydrogen bond from histidine that stabilizes the reaction complex.

Figure 1

bc₁ complex from Rhodobacter capsulatus. (a) The studied molecular system consists of a lipid bilayer membrane, water molecules, ions, and the six protein subunits forming the homodimeric bc₁ complex. The bc₁ complex features two monomers (A and B), each consisting of one cytochrome c₁ (cyt. c₁), one cytochrome b (cyt. b), and one iron–sulfur protein (ISP) subunit. (b) Each monomer binds four metal centers, heme c₁ in the cyt. c₁ subunit, and hemes b_L and b_H in the cyt. b subunit, while the ISP binds an iron–sulfur (Fe₂S₂) cluster. The quinol (QH₂) and the quinone (Q) substrate molecules interact with the hemes and the Fe₂S₂ cluster to facilitate electron and proton transfers through the complex (A or B) at two distinct binding sites (Q_o and Q_i). The arrows indicate schematically pathways of electrons and protons at the initial phase of the Q-cycle. (c) The QH₂ substrate molecule at the Q_o-site of the bc₁ complex interacts closely with the H156 residue of the ISP and several other residues of the bc₁ complex. The exact binding mode is addressed in the present study.

Figure 2

Quinol binding at the Q_o-site of the bc₁ complex. Shown are binding and coordination of QH₂ at the Q_o-site. Dashed lines represent key hydrogen bonds that coordinate QH₂ to residues H156 and Y147. The labels next to these lines indicate the corresponding bond lengths that are shown in Figure 3 and discussed in the text. QH₂ binding is primarily coordinated through H156, which is in its ϵ-protonated form in Model I (a), and in its deprotonated form in Model II (b). In the case of Model I, QH₂ binding is additionally stabilized through a water molecule.

Figure 3

Analysis of quinol bonding. Time evolution of the key hydrogen bond lengths stabilizing QH₂ binding at the Q_o-sites of the bc₁ complex in monomer A (left plots) and B (right plots). Blue lines show the bond lengths calculated for Model I (see Figure 2a), while red lines show the bond lengths for Model II (see Figure 2b). The insets in each panel illustrate the corresponding hydrogen bonding motifs, with lengths labeled d₁, d₂, and d₃. Bond length d₁ is defined differently in the case of Models I and II.

Figure 4

Quinol binding at the Q_o-site coordinated by a water molecule. Shown is the evolution of the lengths of hydrogen bonds formed between QH₂ and a water molecule trapped within monomer A and monomer B of the bc₁ complex. Green lines represent the length d_w1 of the bond between the H1 atom of QH₂ and the OH₂ atom of the trapped water molecule, while red and blue lines correspond to the lenghts d_w2 and d_w3 of hydrogen bonds formed between the H1 and H2 atoms of the water molecule and the O atoms of C155 and I292, respectively. The hydrogen bonding network along with d_w1, d_w2 and d_w3 are shown in Figure 2a. A water molecule is bound to the QH₂ molecule throughout the entire simulation in the case of Model I (a) and is seen to bind only sporadically in the case of Model II (b).

Figure 5

Quinol interaction with bc₁ complex. Shown is the time evolution of the interaction energy for the QH₂ head group and the rest of the simulated system, including water molecules, lipids, and the bc₁ complex proteins. Blue and red lines show the energies calculated for Models I and II, respectively. The energies calculated for each step of the simulation are shown in shaded colors, while intense color shows a gliding average with energies averaged over a gliding window of 100 ps. Vertical arrows indicate the time instances for monomers A and B, when the QH₂ molecule unbinds from H156 as seen in the Model II simulation.

Figure 6

Quantum chemistry model of QH₂ binding at the Q_o-site of the bc₁ complex. Included in the quantum chemical description are the components shown here in licorice representation, namely the QH₂ head group and all residues within 10 Å from the head group. The coloring of the bc₁ complex secondary structure illustrates the charge state of the protein amino acids: negative (red), polar uncharged (green), and hydrophobic uncharged (yellow). Side chain groups of polar and negatively charged amino acids surrounding the QH₂ head group (Y147, H135, H156, E295, Y302) were included into the computational model to describe environmental effects on the QH₂ binding, while distant charged side chains that point away from the QH₂ head group and E295 were not included in the quantum chemical description. The Fe₂S₂ cluster and all its coordinating amino acids (licorice) have also been included in the computational model.

Figure 7

Quantum chemical optimization of the binding complex Q_o-site for Model I. Shown are residues of the bc₁ complex directly involved in the binding of QH₂ at the Q_o-site and covered in our quantum chemistry analysis of Model I. The quantum chemical Q_o-site for Model I includes residues Y147, I292, E295, and Y302 from the cyt. b subunit, as well as the Fe₂S₂ cluster together with residues C133, C153, C155, H156, and H135 of the ISP subunit. The initial configuration (a) used in the quantum chemical calculations is a conformational average from a bc₁ complex before equilibration. The optimized structure, shown in part b, features rearrangements of residues Y147 and E295 accompanied by spontaneous proton transfer from Y147 to E295. The C_α atoms of the amino acid residues (cyan spheres) were fixed during the quantum chemical optimization process.

Figure 8

Quantum chemical optimization of the Q_o-site binding complex for Model II. Shown are residues of the bc₁ complex directly involved in the binding of the QH₂ molecule at the Q_o-site and covered in our quantum chemical analysis of Model II. The Q_o-site for Model II is constructed similarly to the Q_o-site for Model I introduced in Figure 7. The key difference here is that residue H156 is deprotonated and that there is no water molecule linking QH₂ with residues I292 and C155. As in case of Model I, the initial configuration of the Q_o-site used for quantum chemical optimization (a) is a conformational average from a bc₁ complex before equilibration. The optimized structure, shown in part b, features rearrangements of residues Y147 and E295. The C_α atoms of the amino acids residues (cyan spheres) were fixed during the quantum chemical optimization process.

Figure 9

Fragments of the Q_o-site and spin densities. The Q_o-sites for Models I (a) and II (b) have been subdivided into 11 fragments, whose total charges were analyzed separately and summarized in Table 4. Atoms belonging to a certain fragment are highlighted with the same color. Transparent surfaces around the Fe-atoms show the difference of the total spin density calculated between all α-electrons (spin up) and all β-electrons (spin down) in the system. The surfaces are shown for the contour values 0.01 (blue) and −0.01 (red) and indicate antiferromagnetic coupling of the two iron ions.