Mechanism of quinol oxidation by the bc1-complex

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The initial view is of the three subunits from a "functional" monomer. Coordinates from the Zhang et al. native complex from chicken heart mitochondrial complex have been supplemented by additional coordinates to provide the working model (see below). Click here to view the complete chicken complex using the coordinate data set submitted to the Brookhaven Protein Data Bank. Click here for a tutorial on the structure/function interface, based on the three catalytic subunits of a "functional" monomer.

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


The model is set up to operate under "conditions" in which both cyt c1 and the ISP are initially, reduced, and the Q-pool is about half reduced. The pH is 8.0. A quinol occupies the Qo-site, and a quinone occupies the Qi-site. The animation shows the turnover of the complex, following delivery of an oxidizing equivalent to cyt c1. This would be appropriate for activation of the Rb. sphaeroides photosynthetic chain by a ~2 ms saturating flash (Click here to see a brief summary of the modified Q-cycle as it operates in Rb. sphaeroides).
  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 Qo-site distal from heme bL. 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 ISPox. 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 Em 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 120o 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 bH .
  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 DGo'). We had previously suggested that the positive DGo' 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, DGo' = 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 Em value for ISP was modified have shown that the change in rate with Em 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 Qo-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 Qo pocket to a domain (the proximal lobe) close to heme bL. This movement could also contribute to the activation barrier. By positioning the semiquinone close to heme bL, 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 ISPox or O2. Although the probability of forming semiquinone is low, the increased rate of superoxide formation observed at the Qo-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 bL. 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 Qi-site to semiquinone (see Note 9).
  9. Quinone leaves the Qo-site, and is replaced by quinol, so that the second turn-over of the Qo-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 Qi-site to quinol, which vacates the site, to restore the starting condition.
Note: The stoichiometry of three electrons delivered to the high potential chain, shown in the animation, would not be seen experimentally after flash activation of the Rb. sphaeroides photosynthetic chain. The stoichiometry of RC:cyt c2:bc1 is 2:1:1, so the number of oxidizing equivalents provided by the reaction centers is only 2/bc1 complex, and the ISP would remain reduced after the second turnover of the Qo-site.
©Copyright 1996, Antony Crofts, University of Illinois at Urbana-Champaign,