Notes on the mechanism of the bc₁ complex.

Note 1. Dependence on pH of the redox potential of the ISP

    Ugulava and Crofts (1998, see also Crofts et al. (1998a) have recently investigated the redox potential of the ISP in chromatophores and the isolated bc₁ complex from Rb. sphaeroides using CD-spectroscopy. The redox potential at pH 7.0 for the Rieske center in the isolated bc₁ complex, and in chromatophore membranes from the R-26 strain of Rb. sphaeroides, was 300 ± 5 mV. The E_m varied with pH in the range above pH 7, and the pH dependence was fit either by a single pK at ~7.5, or by two pK values, pK₁ ~7.6 and pK₂ ~9.8. Similar titrations and pK values were found for the ISP in the isolated bc₁ complex and membranes from R-26 strain of Rb. sphaeroides. These values are similar to those found in the isolated beef ISP (Link, 1994, and see above).

Note 2. The complex between stigmatellin and the reduced ISP as a model for the reaction complex

    Lancaster and Michel (1996, 1997) have suggested from structures of the Rps. viridis photochemical reaction center co-crystallized with stigmatellin, and from the H-bonding pattern observed, that stigmatellin bound at the Q_B-site mimics an intermediate reaction complex involving either quinol anion or neutral semiquinone. Link (1997) has suggested that the complex between stigmatellin and the ISP might similarly mimic the reaction complex between semiquinone and the reduced ISP in the bc₁ complex, and has proposed that the redox properties of the complex (shifted E_m for ISP) might gate the system to prevent reduction of cyt c₁ by the semiquinone. Crofts and Wang (1989) (see below) had earlier suggested a model in which quinol oxidation proceeded from an enzyme-substrate (ES-) complex complex formed by binding quinol from the membrane to a catalytic site on cyt b which included the 2Fe2S center. The movement of the ISP (Zhang et al., 1998) has highlighted the importance of formation of an ES-complex including both the ISP (at its docking interface on cyt b), and quinol, both of which are effectively diffusible substrates, and Crofts et al. (1998a) have suggested that the stigmatellin complex might mimic a complex between quinol and the oxidized ISP. Because the transfer of an electron in such a complex might not give rise to EPR detectable products, the complex could include internal equilibration with a state like that suggested by Link (1997), which, as noted by Link and by Jünemann et al. (1998), would not be readily detected by EPR.

QH₂ + Fe₂S₂^ox  + b_L <==>   [QH₂-Fe₂S₂^ox b_L --- QH^•-HFe₂S₂^red b_L]  <==> QH^• b_L + HFe₂S₂^red  ........  (1)

We have more recently suggested that the formation of the enzyme-substrate complex at the Q_o-site involves a second ligand from Glu-272 of the -PEWY- loop. In the stigmatellin containing structure 2bcc, and in updated coordinates for the structure published as 3bcc, this residue is shown as providing a H-bond to the -OH of stigmatellin (Crofts et al., 1999a, b, c, d).

Note 3. Dependence of electron transfer rate on pH.

    Crofts and Wang (1989) showed that, when the concentration of reactants was adjusted to be the same, the activation barrier for quinol oxidation was the same at pH 7 and 8.9. Crofts et al. (1998a, Hong et al. (1999), and see Ugulava and Crofts, 1998) have measured the partial reactions of quinol oxidation following flash activation of chromatophores from Rb. sphaeroides, and shown, in agreement with the work on the mitochondrial complex, that the rate of oxidation of bound quinol was strongly pH dependent in the acidic range. However, the activation energy was independent of pH over the range 5.5 - 8.9. They have interpreted their data as showing that formation of the reaction complex between oxidized ISP and quinol bound at the Q_o-site is dependent on prior dissociation of a group with a pK in the range ~7.6, and have suggested that this is the same as the group with pK ~7.6 on the oxidized form seen in the pH dependence of redox tirtrations of the ISP. Following Iwata et al. (1996), they have suggested that His-161 (in beef numbering) is the group involved, and that this residue must be in the dissociated form before it can form the H-bond with QH₂ leading to formation of the reaction complex. This has the effect of making the ISP a H-carrier (as shown in equation (1) above) rather than an electron carrier, as generally assumed.

Note 4. Quinol is oxidized in a second-order reaction, which shows saturation

Crofts et al. (1999a) and Hong et al. (1999) have noted that a similar argument pertains to the binding of the ISP_ox in the ES-complex. The association constant pulls the H⁺ dissociation reaction over so as to give an apparent pK of ~6.3 for the dependence of electron transfer rate on pH, as compared to the pK of ~7.6 measured from the dependence of E_{m, ISP} on pH.

Note 5. The activation barrier in quinol oxidation

    Jünemann et al. (1998) have re-examined the de Vries et al. (1982) experiment, and shown that the semiquinone observed under their conditions was not sensitive to classical inhibitors of the Q_o-site (stigmatellin, myxothiazol or MOA-stilbene). They concluded, in agreement with the results of Andrews and Crofts, that no semiquinone is detectable at the Q_o-site. They discussed their results in the context of a simple kinetic scheme (effectively a sub-set of the Crofts-Wang scheme, but without the explicit values for rate constants provided by the latter authors), and suggested that "The chemical basis for obligatory bifurcation of electron flow was not addresses in the original Q-cycle proposal. However, ... in principle, the basis could lie simply in the fact that the first reaction step of oxidation of quinol (QH₂) to semiquinone (Q^.-) is thermodynamically very unfavorable":
                                           k₁                   k₂
                        QH₂FeSb_L <==> Q^.-FeS^-b_L  --> QFeS^-b_L^-
                                           k_-1

    Jünemann et al. (1998) noted that their results limited plausible reaction mechanisms to those, such as the Crofts-Wang scheme, in which semiquinone does not accumulate, but noted that formation of a reaction complex such as that suggested by Link (1997), in which semiquinone and reduced ISP were both present, could not be excluded if the two unpaired electrons were antiferromagnetically coupled. This had previously been suggested by Link (1997).

    Hong and Crofts (see Crofts et al., 1998a, Hong et al., 1999) have re-examined the activation barriers in the partial reactions of quinol oxidation in Rb. sphaeroides, and concluded that there is no pH dependence over the range 5.5-9.0 for quinol oxidation, as assayed by the rate of reduction of cyt b_H (or cyt b_L at high pH) following flash activation in the presence of antimycin. The rate of quinol oxidation increased strongly with pH in the acidic range, but leveled off above pH 7.5. The rate and activation energy of the reactions of the high potential chain (oxidation of cyt c₁ and ISP) were both relatively independent of pH. At all pH values, the process with the high activation energy was that assayed by reduction of cyt b (see above). The results confirm and extend the work of Crofts and Wang (1989), and are consistent with the idea that the activation barrier is in processes of quinol oxidation after formation of the ES-complex. The results are compatible with the minimal hypothesis of Crofts and Wang (1989), and the limitations suggested by Jünemann et al. (1998) . The results provide no evidence for an activation barrier due to dissociation of QH₂ (Brandt and Okun, 1997). They are compatible with the suggestion that the effect of pH on the rate over the acidic range is associated with a group with pK ~7.6, required in the dissociated state, and tentatively identified as His-161 of the ISP, as discussed above (Crofts et al., 1998a, 1999a, Hong etal., 1999). The mobility of the ISP requires an extension of the simple model to allow binding of both mobile substrates to form the reaction complex:

Note 6. Occupancy of the bifurcated Q_o-site: single or double-occupancy of the site by quinones during turnover.

    The differential binding of inhibitors, the bifurcated volume of the Q_o-site, and the involvement of the ISP in inhibitor binding, provide confirmation at the structural level of features that had been predicted on the basis of 16 years of biochemical, biophysical and mutagenesis studies. Following the first explicit recogniton by Meinhardt and Crofts (1982), many groups have demonstrated:

1. the differential binding of UHDBT, UHNQ, or stigmatellin (as one class (I)), and myxothiazol or MOA-type inhibitors (as another class (II));
2. displacement of one class by the other (interpreted as overlapping binding sites);
3. differential interaction of these two classes with either ISP or heme b_L;
4. contributions of both cyt b and ISP to the catalytic site.

    None of the structures show the quinone(s) bound at the Q_o-site, which had been expected from the binding data of Ding, Dutton and colleagues (Ding et al., 1992, 1995, 1995a) (see below). Based on the electron density, a quinone was modeled at the Q_i-site by Zhang et al. (1998), and evidence for occupancy by a quinone at the Q_i-site was presented by Xia et al. (1997). Since both preparations contained sub-stoichiometric amounts of quinone, the results show that, at least in the crystals, the Q_i-site bound quinone much more tightly than the Q_o-site.

    Brandt et al. (1988) showed that the binding of MOA-type inhibitors to the bc₁ complex, assayed by the red-shift induced in the spectrum of heme b_L, was decreased by increasing concentrations of the ubihydroquinone-10 analogue nonylUQ. From Eadie-Hofstee plots of the effect of inhibitors on steady-state turnover, or Scatchard plots of the effect of nonylUQ on inhibitor binding, they concluded that the decreased binding reflected a non-competitive effect, and that nonylUQ and the inhibitors bound at separate sites. The interpretation of their kinetic data was based on a classical scheme in which inhibitor equilibrated reversibly with both free enzyme and the enzyme with bound quinol. However, because of the tight binding of the inhibitors used, it is clear that this kinetic treatment was not appropriate. Brandt and von Jagow (1991) extended this work to explore the competition between inhibitors, using quenching of fluorescence of inhibitors to assay binding. They showed differential effects of the redox state of ISP on the binding of two classes of inhibitors at the Q_o-site, with reduction of ISP favoring binding of class I inhibitors. This was expected from the change in redox potential induced by binding, which had previously been interpreted as showing that class I inhibitors bound more tightly to the reduced form of the ISP (Bowyer et al., 1980; von Jagow and Ohnishi, 1985).  Brandt and colleagues (Brandt and von Jagow, 1991; Brandt et al., 1991) confirmed previous observations of displacement from the binding site of inhibitors of one class by those of the other class (Meinhardt and Crofts, 1982). In agreement with Meinhardt and Crofts (1982), they suggested distinct but overlapping binding sites for the two classes, and proposed a 'catalytic switch' mechanism to explain both the bifurcation of electron flow and the inhibition at the Q_o center. In their discussion of this work, the authors highlighted the problems associated with the bifurcated reaction, and the question of why the second electron does not follow the first into the high potential chain. The 'catalytic switch' mechanism was an ingenious model, which proposed conformational changes for different redox states to prevent the 'futile' electron transfer. The movement of the ISP now demonstrated in the structures (Zhang et al., 1998) provides some support for these ideas, but not for specific features of the mechanism. More recently, Brandt and colleagues (Brandt 1996, 1996a; Brand and Okun, 1997), based on their earlier observations of a non-competitive binding, have proposed a proton-gated charge transfer mechanism for the turnover of the Q_o-site, involving occupancy of the Q_o-site by two quinone species, and a rate controlled by deprotonation of quinol.

From Brandt (1996).

    Ding, Dutton and collaborators (Ding et al., 1992, 1995, 1995a; Saribas et al., 1997) were the first to propose that the Q_o-site functions with an occupancy of two quinone species. The experimental basis for this hypothesis was the effect of extraction of quinone on the EPR spectra associated with interaction between the reduced ISP and the occupant of the Q_o-site in chromatophores from Rb. capsulatus. With a Q pool containing >4 Q/bc₁ complex, the well characterized band at g_x=1.800 was observed, and was attributed to the presence of a weakly bound quinone at the Q_o-site (Q_ow). When the Q-pool was reduced, or Q completely extracted, or myxothiazol bound at the Q_o-site, the g_x line-shape changed from a sharp peak at 1.800 to a shallow peak at 1.765. An intermediate line-shape at 1.783 was seen when ~2 Q / bc₁ complex were present, and was attributed to a strongly binding quinone, Q_os. From the differential effects of extraction, Ding et al. (1992, 1995, 1995a) concluded that Q_os was bound >10-fold more tightly than Q_ow or the quinone at the Q_i-site. In view of the affinities, it was assumed that the strongly bound species was always present when the weakly bound species was present, to give a double-occupancy.

    Brandt (1998) has suggested that the bifurcated volume of the Q_o-site observed in the structures provides strong support for double-occupancy models. However, the failure to find the tightly bound quinone expected of the Ding and Dutton model, and the overlapping of the volumes of inhibitors, especially of the tails in the exit channel, which would also be expected on occupancy by two UQ-10 molecules, suggest that alternatives should be considered. Other experimental observations that are difficult to reconcile with a double occupancy model have been discussed by Crofts et al. (1995, 1998, 1999d):

1. Experiments with inhibitors have been interpreted as showing that occupation of the Q_o-site is exclusive, so that a tightly binding inhibitor will displace a weakly binding inhibitor (Meinhardt and Crofts, 1982; Brandt et al., 1988; Brandt and von Jagow, 1991; reviewed by von Jagow and Link, 1986; Brandt and Trumpower, 1994). The structures provide strong support for this observation, since the volumes of inhibitors in different structures overlap when mapped to a commmon structure. Ding et al. (1995) noted that either stigmatellin or myxohiazol, at 1 mol/mol bc₁ complex, eliminated signals ascribed to both Q_ow and Q_os, suggesting that quinone occupants are displaced by these inhibitors, and that the site could accommodate only one occupant.
2. The competition between inhibitor and quinone seen by Ding et al. is in contrast to the conclusions of Brandt et al. (1988) that both nonylUQ and an inhibitor could occupy the Q_o-site simultaneously, by binding to different domains. This discrepancy could possibly be explained by the fact that the inhibitors used by Brandt et al. (1988) (MOA-stilbene, oudemansin, strobilurin) all had short tails, so the conflict for volume would be less, especially with the nonylUQ used as the competitor in most experiments. However, Brandt et al. (1991) and Brandt and von Jagow (1991) showed competitive displacement of MOA-stilbene by UHNQ, which would seem like an appropriate model for nonylUQ. This competitive effect therefore seems to be in contradiction to the earlier finding from this lab of non-competitive binding. The apparent contradiction between the results above might be resolved through a critical re-examination of the experimental basis for the non-competitive effects suggested by Brandt et al. (1988), since it is clear that the kinetic treatment they used was not appropriate for a tightly binding inhibitor.
3. Although there is general agreement that stigmatellin, UHDBT, and myxothiazol occupy overlapping volumes (Xia et al., 1997; Zhang et al., 1998; Iwata et al., 1998), Kim et al. (1998) state that "(t)he Q_o inhibitors occupy different subsites in the Q_o pocket. Except for the combination MOA-stilbene/UHDBT, their binding sites overlap, which explains why binding of these Q_o inhibitors is mutually exclusive...". This non-overlap of MOA-stilbene and UHDBT would support the Brandt et al. (1988) conclusion that two occupants (nonylUQ and MOA-type inhibitors) could both reside in the site, but it seems contradictory to their later inhibitor studies (Brandt et al., 1991; Brandt and von Jagow, 1991) showing displacement of MOA-stilbene by UHNQ. Refined structures from Berry and collaborators (Crofts et al., 1999d) show that a substantial overlap occurs between stigmatellin and MOA-stilbene in the volume where the channel to the lipid phase connects to the bifurcated volume. The ring of MOA-stilbene distal to the pharmacophore sits in the common volume of the site through which the tails emerge, and would likely overlap any tail emerging from the site.

    Recent experiments by Sharp et al. (1999) on inhibition by diphenylamine (DPA) are interesting from this point of view. They showed that, over the 100 µM range, DPA could inhibit quinol oxidation, but not formation of the complex giving rise to the g_x=1.80 or 1.783 bands. As the concentration was raised above that giving inhibition of electron transfer, the g_x=1.80 signal was lost before the 1.783 signal. They interpreted this as showing a triple occupancy of the Q_o-site, with DPA binding in a non-competitive manner in addition to Q_os and Q_ow. An alternative explanation is that DPA, having no tail, can bind in the proximal domain without causing displacement of quinone bound in the distal domain, but would inhibit electron transfer by competing for occupancy of the proximal domain. In a single-occupancy model, this might prevent a movement of semiquinone to the proximal domain during turnover. Consistent with this, DPA at concentrations in the 100 µM range increased the titre for mucidin, as would be expected if both compete for occupancy of the proximal domain (Guergova-Kuras, M and Crofts, A.R., unpublished).
4. From the double-occupancy mechanism of Ding et al. (1992, 1995, 1995a) it might be expected that the catalytic site would contain two distinct sets of ligands to provide for the differential binding of the two species. From the structure, it might be anticipated that the two quinones would occupy the two lobes. Although numerous single-site mutations that differentially affect binding of stigmatellin or myxothiazol (or inhibitors of equivalent class) have been characterized, no similar differential effects on Q_ow and Q_os have been observed. Analysis of the location in the structure of mutations giving rise to resistance to different inhibitors shows, not unexpectedly, that (with some exceptions) residues at which mutations give resistance to myxothiazol or mucidin cluster around the proximal domain where the pharmacophore sits, those giving resistance to stigmatellin are around the distal lobe, and those giving resistance to both cluster around the common volume (Crofts et al., 1998).  If the two quinones sit in the two lobes, it would be expected that their binding would be differentially affected, just as those of the inhibitors in the two lobes are.
5. Analysis of the location of mutations showing differential effects on the ability to form the complex monitored by the g_x=1.800 EPR signal of ISP_red, and rate of elecron transfer, suggest i) that the g_x=1.800 complex requires correct binding of both ISP_red and of quinone in the distal domain, and ii) that occupation of the proximal lobe of the Q_o-site is essential for catalysis, but not for the binding of quinone, and its interaction with ISP_red in the g_x=1.800 complex (Crofts et al., 1998, 1999d). The latter conclusion comes from analysis of mutations around the proximal lobe that prevent electron transfer, but do not affect the g_x=1.800 signal. Mutations in this latter category do not affect the g_x=1.783 signal either. It is difficult to explain these effects in terms of two quinones occupying the two lobes of the site.
6. The results reported by Crofts and Wang (1989) and Jünemann et al. (1998), showing that no semiquinone is detectable at the Q_o-site, would appear to exclude the specific mechanism suggested by Brandt (1996) (see above) for quinol oxidation through a double-occupancy involving symproportionation through a semiquinone anion pair in a quinhydrone-like arrangement.

    It is important to note that the model does not imply (as suggested by Jünemann et al., 1998) that the semiquinone would be formed at detectable concentration. Although the observed rate of reduction of cyt b_L is that of the rate limiting step (Meinhardt and Crofts, 1983), the intrinsic rate constant for the electron transfer reaction to heme b_L is likely rapid compared to the rate limiting step (Crofts and Wang, 1989). The ~6.0 Å distance from the semiquinone to the heme edge expected from occupancy of the proximal domain would allow electron transfer rate constants in the >10¹⁰ s^-1 range (Moser et al., 1996; k_max =  5.01 x 10¹¹ s^-1 when -DG = l), so the time-averaged relative concentration could be < 10^-6 that of a fully occupied site, and still achieve 100 ms kinetics. A kinetically significant concentration of semiquinone for "side" reactions could also be very low, and undetectable (see Hong et al., 1999 for a detailed discussion).

    We have attempted to generate a structural file to show an alternative model involving two quinones, and electron transfer from a semiquinone produced at the distal lobe (as in this model), but transferred to heme b_L via a quinone in the proximal lobe. Unfortunately, the program SCULPT, used to energy minimize the structure to generate a feasible configuration, ejects one of the quinones while expanding the structure to accommodate both. This does not mean that a double-occupancy should be excluded. However, if a double-occupancy is to form the basis of a mechanism, it will be necessary to demonstrate without ambiguity that two quinone species can occupy the site.

Kim et al. (1998) have also recently discussed a single-occupancy model, similar to that previously discussed by Zhang et al. (1998) and Crofts et al. (1998, 1999a, d), and shown here:

   "..... we assume two transient quinone binding sites in the inhibitor binding pocket. One of these putative sites, designated P1, is closer to ISP where UHDBT binds; the other site, designated P2, is closer to b_L heme where MOA-stilbene binds. Analogous to inhibitor binding, binding of quinol to P1 will cause the fixation of ISP, and binding of semiquinone to P2 will release the ISP from this fixed state. With these assumptions, the ET events at the Q_o site can be described as follows: A cycle starts with ubiquinol bound to P1, and the oxidized ISP in the fixed state. The first electron is transferred from ubiquinol to the FeS; the two protons are released. The ubisemiquinone moves to P2, causing a conformational transition in which the ISP is in the loose state. The reduced ISP in the loose state approaches cytochrome c₁ and may form a transient complex with it; this allows rapid ET from the FeS to the c₁ heme. The second electron is transferred from ubisemiquinone in P2 to heme b_L, and subsequently to b_H and to ubiquinone or ubisemiquinone in the Q_i site. The ubiquinone in P2 dissociates from the bc₁ complex to be replaced by another ubiquinol molecule, which will occupy P1 and bring the ISP back to the fixed conformational state."

    Kim et al. (1998) have also considered another model, similar to that previously suggested by Link (1997), in which the semiquinone remains in the distal (P1) lobe, and electron transfer occurs simultaneously to heme b_L and the ISP, directly from the complex with the ISP. This is the model they appear to prefer.

    "One problem with (the above) model is that especially Q_o-I inhibitors are chemically dissimilar to quinones, so that it is not clear whether ubisemiquinone binds in P2, and, if it does, whether it can cause the same structural changes as MOA-stilbene. Another problem is the lack of a detectable ubisemiquinone radical at the Q_o site. The reported detection of a transient, antimycin A-insensitive, ubisemiquinone radical at the Q_o site (..) was recently questioned (P. R. Rich, personal communication), as it was not sensitive to the Q_o site inhibitors such as myxothiazol, MOA-stilbene, or stigmatellin. An alternative hypothesis for the ET event at the Q_o site could avoid this difficulty by assuming that the two electrons of ubiquinol in the complex are transferred simultaneously, one to FeS, and the second to heme b_L and further to heme b_H; thus, no ubisemiquinone would be generated. In this scenario, movement of the quinone between Q_o subsites would probably not occur, and the conformational change of the cytochrome b protein, which switches the reduced ISP from the fixed to the loose state, would have a different cause. An attractive candidate for the switching event would be the ET from heme b_L to heme b_H; this mechanism would make sure that the second electron of ubiquinol would reach its destination before or at the same time as the first electron reaches heme c₁. Thus, because the oxidation of the reduced FeS would depend on the transfer of an electron from heme b_L to heme b_H, bifurcation would be obligatory for the oxidation of ubiquinol in the bc₁ complex."

     Since the complex is presumed to form with quinol at the distal lobe (the P1 site), this would require electron transfer through an edge-to-edge distance of ~11 Å, with a maximal rate constant, k_max = 2.51 x 10⁸. Given the high activation energy, and therefore the high reorganization energy, and the low probability of the transition state, this is too slow for electron transfer to be the process by which the transition state progresses to products. It is clear from a detailed consideration of the distances, reorganization energy, and free-energy for the reaction that such a simultaneous electron transfer could not occur from the distal domain (Hong et al., 1999).
 

Note 7. Role of Q_o-site in production of super oxide anion.

Note 9. Reaction complex between ISP and cyt c₁.

Note 9. Mechanism of the Q_i-site.

Note 10. Modified Q-cycle mechanism.

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