Mechanism of quinol oxidation by the bc₁-complex

The structures used in this animation are a composite from the sets of coordinates kindly made available by Dr. Ed Berry, of the bc₁-complex from chicken heart mitochondria, and by Dr. S. Iwata, of the beef heart mitochondrial complex in P6₅22 and P6₅ crystals.

The coordinates from the solved structures were supplemented with models for the quinone, quinol, and semiquinone substrates needed to show their involvement in the catalytic cycle. The composite file includes coordinates for the different components, in different configurations, involved in turnover of the complex. Click here for a summary of the files, and their construction.

The electron transfer reactions

Explanation

1. Cyt c₁ becomes oxidized, and is reduced by the ISP. Since the pH is above the pK of His-161, the ISP looses a proton (represented by a white sphere) on oxidation (see Note 1).
2. The ISP moves to the docking site on cyt b, and forms a reaction complex with the quinol bound in the domain of the Q_o-site distal from heme b_L. The reaction complex involves a H-bond between quinol and His-161 of ISP, represented by the H as a white sphere (see Note 2). The movement of the ISP is represented by successive occupation of the four structures currently available, starting from that in the Iwata et al. P6₅22 crystals, through the Zhang et al. native chicken complex, and the Iwata et al. P6₅ crystals, to that in the Zhang et al. chicken stigmatellin complex.
3. We have suggested that the pH dependence of the rate in the acidic range reflects a requirement that His-161 must be in the dissociated form for formation of the reaction complex with quinol, through formation of the H-bond (see Note 3, and see links below for additional information). The probability of formation of the reaction complex is determined by the availability of the substrates, - QH₂ and the dissociated form of the ISP_ox. The rate of quinol oxidation in the "substrate-limited" range is determined by the probability of formation of the reaction complex; it increases as [QH₂] increases (see Note 4), and increases with increasing pH (over the range below a pK of 7.5 on ISP-His-161), as the concentration of the dissociated form increases. The binding constants for formation of the ES-complex give rise to the 50-fold tighter binding of quinol than quinone, and the shift in apparent pK from the value of 7.6 measured through titration of the E_m value for ISP, to the pK of ~6.3 measured from the pH for half-maximal rate at saturating quinol oxidation. In this animation, the first proton from quinol is transfered to the ISP with the electron, and is lost from His-161 on oxidation of the ISP, regenerating the dissociated form.
4. We have suggested that Glu-272, the second ligand to the occupant indicated in the stigmatellin structure (2bcc), is also a second ligand to quinol. In the native (1bcc) and myxothiazol or MOA-stilbene structures, Glu-272 is rotated by 120^o out of its liganding position, to bring the acidic side chain in contact with a water channel into cyt b from the P-side aqueous phase. It seems likely that a similar movement provides a pathway for release of the second proton from quinol. This would require that the neutral semiquinone, generated at the site after the first electron (and H⁺-) transfer to ISP, donates a H⁺ to Glu-272^- to leave the semiquinone anion as the donor to heme b_H .
5. In the step with a high activation energy, the reaction complex dissociates to products. With an activation barrier of 40-65 kJ/mol, dissociation to products is improbable. Formation of semiquinone in this process is not detected, suggesting a very small equilibrium constant (+ve DG^o'). We had previously suggested that the positive DG^o' for formation of semiquinone might approach the value for the activation barrier, so that both might reflect the same process (see Note 5). If this were the case, DG^o' = DG^# ~= DE^#, the equilibrium constant for formation of products would be very small, and semiquinone would not normally be within the detection range of current instrumentation. However, studies of mutant strains in which the E_m value for ISP was modified have shown that the change in rate with E_m value was less than expected from a simple interpretation along these lines. A detailed study of the activation barriers  of all aprtial reactions, and their pH dependence, has suggested severe constraints on possible mechanisms.
6. The model involves a single quinone at the Q_o-site, rather than two, as in double-occupancy models (see Note 6). We have suggested that, during dissociation of the reaction complex, the semiquinone moves from the distal domain of the Q_o pocket to a domain (the proximal lobe) close to heme b_L. This movement could also contribute to the activation barrier. By positioning the semiquinone close to heme b_L, the movement would allow a more rapid electron transfer reaction. It would also remove the semiquinone from the ISP docking interface, and from potential oxidation by either ISP_ox or O₂. Although the probability of forming semiquinone is low, the increased rate of superoxide formation observed at the Q_o-site in the presence of antimycin, or under static head conditions (see Note 7), suggests that it may nevertheless be kinetically significant.
7. On dissociation of the reaction complex to products, the mobile extrinsic domain (head) of reduced ISP is liberated from the complex, and rotates to dock with cyt c₁, close to the heme. The reaction complex with cyt c₁ involves a H-bond contact between His-161 of ISP and a heme c₁ propionate -C=O (see Note 8). In this animation, the position of the ISP in the P6₅22 crystals from Iwata's structure is used to show this contact. Electrons are transfered from ISP to heme c₁, and (see above) from semiquinone to heme b_L. Because both reactions occur after the rate limiting step, they appear experimentally to be concerted. In this animation, the two electron transfer reactions are shown separately, for clarity.
8. Electron transfer across the membrane through the b-heme chain reduces quinone at the Q_i-site to semiquinone (see Note 9).
9. Quinone leaves the Q_o-site, and is replaced by quinol, so that the second turn-over of the Q_o-site can occur (see Note 10).
10. Steps 2-7 are repeated, and the second electron in the b-heme chain reduces the semiquinone at the Q_i-site to quinol, which vacates the site, to restore the starting condition.