(This publication is substantially the same as Crofts, A.R., Barquera,
B., Bechmann, G., Guergova, M, Salcedo-Hernandez, R., Hacker,
B., Hong, S. and Gennis, R.B. (1995) Structure and function in
the bc1-complex of Rb. sphaeroides. Photosynthesis:
from light to biosphere. (Mathis, P., ed.), Vol. II, pp. 493-500.
Kluwer Academic Publ., Dordrecht. Some corrections to typographical
errors have been made in this document, and some figures have
The photosynthetic chain of Rb. sphaeroides contains two major enzymes, the reaction center and the bc1-complex, which transfer electrons between catalytic sites through redox reactions of ubiquinone-10 (Q) in the lipid phase and cytochrome (cyt) c2 in the periplasmic phase. The turn-over of the chain is well described by a modified Q-cycle (1-3). With the exception of the bc1-complex, structures of the proteins involved in light-harvesting and photosynthetic turn-over are available from X-ray crystallography (4-6), or from the solved structures of closely related proteins (7, 8). A secondary structure for cyt b has been proposed based on profile analysis of aligned sequences (8-11); hydropathy plots suggest that cyt b is organized in eight transmembrane spans (A-H). Spans in the N-terminal stretch (a helix) and between helices A and B (ab helix), C and D (cd helix) and possibly between E and F (ef helix) have been tentatively identified as amphipathic surface helices. We have suggested a tertiary folding pattern based on the location of inhibitor resistance lesions, site-directed mutagenesis to identify heme ligands, molecular engineering of conserved residues and of residues predicted to impinge on the quinone binding sites (12). A crystallographic structure for cyt f of the related b6f-complex from chloroplasts is available (13), but the very weak sequence homology makes this a dubious template for prediction of a structure for cyt c1. A number of laboratories have reported crystals of the mitochondrial complex or its subunit, with diffraction to atomic resolution, and it seems likely that a structure will be available in the near future. The three main subunits are highly conserved, so the mitochondrial complex will likely "contain" the bacterial structure. Our mutant strains are summarized in Table I.
Table I Mutation Em Pho. Growth Kinetic effects Qo-site (Wild type has Vmax ~500 e-/cyt bH/s (t½ ~1 ms), Km ~1.66 mM) F144S None ++++ Slowed QH2 oxidation, myxothiazol resistant. T160A,V None ++++ Polar --> neutral, little effect on function. N279Y None ++++ Mimics myxr strain in yeast; Vmax 174, Km 3.2 mM I292F None - V. slow QH2 oxidation; Vmax 4, Km 2.9 mM P294S None ++++ QH2 oxid. slowed, otherwise normal; Km 3.45 mM E295D Minor ++++ QH2 oxidation slowed 2-fold; Km 2.63 mM E295G Minor +++ QH2 oxidation slowed 9-fold; Km 4 mM E295Q bL -30 ++ QH2 oxidation slowed 50-fold; no reliable Km W296F bL -20 +++ QH2 oxidation weakly inhibited; Km 3.3 mM W296L bL -20 +++ QH2 oxidation slowed 2-fold; Km 4 mM Y297F None ++++ QH2 oxidation weakly inhibited; Km 1.05 mM Y297S Minor ++ QH2 oxidation slowed 25-fold; Km 3.4 mM L305D ND ++ Slow QH2 oxidation (Vmax 8.4); Km 1.55 mM K329G None +++ Slow QH2 oxidation (t½ ~10 ms); Km 1.54 mM G332S None +++ Slow QH2 oxidation (t½ ~5 ms); Km 2.7 mM G332I None - Qo-site blocked, but complex assembly normal G332D None +++ Slow QH2 oxidation (Vmax 15.4); Km 3.2 mM V333M None ++++ Stig. resistant, otherwise normal; Km 2.5 mM Qi-site (Wild-type has Em values: bH 43 5, bL -90 mV. Cyt b150 is 20-30% total cyt bH) G48V None - Qi-site blocked, but AA-induce oxid. present A52V None ++++ Normal electron transfer, weak AA resistance R114A Large - Spectra odd, complex assmbl. but dead, no AA-induced oxid. R114K bH 12 ++++ Kinetics and AA titre normal, AA-induced oxid. low (0.1)* W129F None +++ bc1-complex low but assembled, AA-induced oxid. 0.75* W129A bH 16 +++ Kinetics fairly normal, AA-induced oxid. low (0.33)* V209A None ++++ Kinetics normal, funiculosin sensitive at Qo-site V209T bH 10 ++++ Kinetics normal, no funiculosin sensitivity, b150 low I213L None ++++ E-transfer from cyt bH slightly slowed when pool oxidized V209A/I213L None++++Kinetics, redox centers normal,funiculosin sens. at Qo-site H217A Minor - The bc-complex assembled, but blocked in cyt b oxidation. S218A None ++++ Normal kinetics, AA-induced oxid. 0.85 T219A bL -31 +++ Slowed bH-oxidation, AA-induced oxid. much greater (1.9)* G220V None ++++ Near normal kinetics of Qi-site, AA-induced oxid. low (0.2)* N221I bH 20 +++ Slowed bH-oxidation, AA-induced oxid. much greater (2.3)* N222A bH 20 ++++ Near normal kinetics of Qi-site, AA-induced oxid. low (0.4)* N223Q bH 22 ++++ Normal kinetics, AA-titre, AA-induced oxid. N223V bH 10 +++ Kinetics slightly slowed. AA-induced oxid. low (<0.1)* K251M None ++++ Antr, e-transfer from cyt bH slowed, esp. with oxidized pool K251I None ++++ Antr, e-transfer from cyt bH slowed, esp. with oxidized pool D252A None - Normal complexed assembled, but oxidation of bH blocked D252N None +++ Inhbited Qi-site, but weak turn-over detectable. K251D/D252K (see Table below) Antr, e-transfer from cyt bH slowed *AA-inucded oxid. values are normalized with respect to wild-type. (P294L, L305A, K329E,A, G48D, R114Q, H217Q, F248L No bc1-complex assembled. G48A, N221S, N223A, N223D, P224A Normal kinetics, AA-titre, AA-induced oxidtn.)
Redox titration of the bc1-complex from Rhodobacter sphaeroides shows three cyt b components, titrating with Em,7 values of -90 (bL), 50 (bH) and 150 (b150) mV (2, 14). The isolated complex contains two b-type hemes, associated with the cyt b subunit (15), and the sequence and molecular engineering studies suggests only two conserved heme binding sites (9-11, 16). When titrations are performed in the presence of antimycin A (AA), the cyt b150 component is lost, and when AA is added to preparations poised so that cyt b150 is reduced but cyt bH is oxidized (at Eh,7 ~ 100 mV), AA induces an oxidation of the cyt b150 form (17-19). The higher potential component (cyt b150) was seen in the isolated complex only if the preparation was supplemented with quinone (15, 19), but isolation the bacterial complex using a (His)6 tagged cyt b and a Ni-affinity column gives a complex showing the cyt b150 effects (20). The two hemes of cyt b are assumed to represent cyt bL and cyt bH; as discussed below, cyt b150 is thought to be a form of cyt bH.
The cyt b150 phenomena reflect the mechanism of reduction of Q at the Qi-site of the complex. In the modified Q-cycle, this is thought to involve two sequential one-electron transfers from cyt bH to Q bound at the site, with formation of an intermediate, relatively stable, semiquinone (1-3). Recent attempts to account for cyt b150 have invoked changes in the mid-point potential of cyt bH such that the cyt has different Em values when Q, Q-, or QH2 are bound at the site (18, 19). We suggest here an alternative model based on schemes previously proposed to account for AA-induced oxidation of cyt b (17), and the reduction of cyt bH observed following excitation of chromatophores in the presence of myxothiazol (21), in which the Em values for all components have single values, and the reduction of cyt bH to give cyt b150 occurs by the reactions at the Qi-site which have already been demonstrated. No ad hoc assumptions are needed, and the equilibrium constants are provided by direct measurement from existing data. We propose that formation of cyt b150 occurs through the reverse reaction at the Qi-site which is demonstrated by cyt bH reduction following flash illumination in the presence of myxothiazol (21):
We have previously proposed a mechanism for AA induced oxidation of cyt b150 (or the elimination of cyt b150 from the redox titrations in the presence of AA) based on this reaction (17), which is similar to the well established mechanism by which reduction of QA by QB- is induced on addition of DCMU to photosystem II following formation of the semiquinone at the QB-site (cf. 22). In the reaction below, the semiquinone at the Qi-site accepts an electron from ferrocyt bH-, and leaves as the quinol, so that ferricyt bH is stabilized by AA binding.
Partial reactions giving rise to the overall process of eq (1) are:
At any defined pH, the equilibrium constant for formation of [cyt bH- . Q-(2H+)] (the cyt b150 form) according to equation (1) is given by:
where Ka values are association (binding) constants for QH2 and Q, and Em(cyt bH) and Em(QH./QH2) refer to the mid-point potentials of cyt bH, and of the semiquinone/quinol couple bound at the site, respectively.
Examination of equation (3) shows that an increased value of Keq could be expected if Em(cytbH) is increased, if Em(QH./QH2) is decreased, if the binding of quinol is increased, or if the binding of quinone is decreased. However, the implied importance of the latter pair of terms is misleading.
When expressed with respect to the potential of the quinone pool, the ratio of binding constants for Q and QH2 contributes to the value for Em(QH./QH2) in such a way as to cancel the pre-exponential term. This gives:
(where Em(Q/QH.) refers to the bound couple), which can also be derived directly from eq. 1. The relation between these two expressions for Keq reflects the dependence of the value of Em(Q/QH.)(bound) on the stability constant for the bound reactants in the disproportionation reaction, and on the value for Em(Q/QH2)(bound), which itself depends on the relative affinities of Q and QH2. The important factors in defining a value for Keq are the Em value for cyt bH, and the stability of the semiquinone, while the relative affinities of Q and QH2 determine the apparent mid-point of the cyt b150 form.
Support for the above mechanisms comes from analysis of strains with mutations in the Qi-site (23, 24, Fig. 1 and Tables I and II). In these mutants we measured the fraction of cyt bH which titrates in the high potential cyt b150 form, the extent of AA induced oxidation of cyt b150 and the correlation between these factors and the Em value for cyt bH. Several mutants have a lower fraction of cyt in the b150 form, and a reduced extent of cyt b150 oxidation on addition of AA; most dramatic is the low level of the cyt b150 component in strain N223V, but mutants V209T, G220V, N222A, R114K and W129A all follow this trend. In all of these strains, the Em,7 for cyt bH was lower than in wild-type. Conversely, strain T219A, and the double mutant K251D/D252K, both of which have a higher Em,7 for cyt bH, both showed a higher amplitude of AA induced oxidation, and a higher ratio of cyt b150. Note that in mutants in which the Em of cyt bH was changed the Em values for cyt b150 were relatively little changed, in line with the above mechanism, suggesting that these mutations did not much change the relative affinities of Q and QH2.
Strain Em,7 cyt bH Em,7 cyt b150 AA-induced oxid. _________________________________________________________________ Wild-type 40-50 150-160 0.14 - 0.20 K251D/D252K 71 166 0.48 T219A 55 151 0.28 S218A 42 150 0.13 A52V 32 139 0.07 N222A 20 142 0.06 G220V 20 148 0.04 W129A 16 149 0.05 R114K 12 147 0.01 N223V 10 150 <0.01
Computer simulations show that the redox titration curves for cyt b150 and for AA induced oxidation (showing [cyt bH- . Q-(2H+)]), can be well modeled in wild-type strains using the assumption that reaction (1) is the mechanism for formation of cyt b150, by using the following values: Em(cyt bH) = 45 5 mV; Em(QH./QH2) = 205 5 mV; Ka(QH2)/Ka(Q) = 100; Em(Qpool) = 90 mV; pKbH 7.8; pKQH. 12.5. With these parameters, Em(cyt b150) ~146 mV, and the component accounts for ~ 20-30% of the total cyt bH at pH 7.0. We simulated the titration curves to be expected when Em values for cyt bH were changed (Fig. 1 C). Substitution of the measured Em values for cyt bH from the mutant strains, keeping other values constant, then provided a good fit for the amount of cyt b150 found in these strains (Fig. 2). In addition, the stable level of reduction seen in kinetic experiments in the absence of inhibitors, at Eh values where the pool was oxidized before flash, could also be accounted for by the modeling parameters in terms of the Keq for reduction of Qi by cyt bH.
Several mutants with a change in Em for cyt bH do not follow the pattern expected from the hypothesis. We would suggest that in these strains, the other terms in eqs. 3 or 4 which govern the stability of the Q.- or binding of Q or QH2 will be of importance
Two highly conserved charged residues, lysine 251 and aspartate 252 of subunit b in the bc1 complex of Rhodobacter sphaeroides, have previously been implicated in the Qi-site on the basis of AA resistant mutations. In order to test the role of the charge contribution from this pair, the sequence has been inverted by site-directed mutagenesis to give strain K251D/D252K. The inversion of these charged residues has the following effects on the bc1-complex:
1) AA has approximately 2000 times lower affinity than in the wild type (Fig. 1 D).
2) The Em of cyt bH is increased by 30-40 mV, the highest increase so far introduced by mutagenesis (Fig. 1 A, B).
3) The mutant grows photosynthetically at rates close to those of wild-type. The steady-state DBH:cyt c oxidoreductase activity of the bc1 complex is about 3 times slower than in the wild type. The electrogenic processes of the complex, measured from the slow phase of the carotenoid change in the absence of inhibitor, are also slowed about three-fold. The maximal rate of reduction of cyt bH following flash excitation in the presence of AA at 140 M is somewhat slowed, but direct comparison is difficult because the change in Em precludes measurement at the same relative concentrations.
The kinetics show the charging of the membrane after a flash due to turn-over of the complete chain (no inhibitor) or when partial reactions of the bc-complex are inhibited by antimycin or myxothiazol.
4) The reduction of cyt bH by quinol in the presence of myxothiazol at pH 9.0 by a reversal of the quinone reducing site, is 6 times slower, but has a higher amplitude.
5) The formation of cyt b150 at 2 x the level in wild-type, the stable reduction of cyt bH following a flash with the pool oxidized, and the increased cyt bH reduction through reversal of the Qi-site are accounted for by the increased Em of cyt bH (hypothesis above, and Figs. above).
These results suggest that the slowed kinetics reflect changes in rate constant(s) at the Qi-site. Although there is some decrease in steady-state activity in this mutant, K251 and D252 do not seem to be specifically required for catalysis because the turnover is not dramatically altered. Since the increased concentration of cyt b150 is well explained by the change in Em for cyt bH, it does not seem likely that these residues are involved in the stabilization of the semiquinone at the Qi-site. In addition, the change in Em value for cyt b150 is as predicted, suggesting that the affinities of Q and QH2 are unaltered. Loss of charge at D252 has a strong inhibitory effect, but the charge on K251 does not seem to be important (Table I). This pattern of effects is similar to those seen when residues involved in the protonation pathway of the QB-site are changed, suggesting a similar role. Nevertheless, K251 is obviously important for AA binding and may interact directly with the inhibitor. The 30-40 mV increase of the midpoint potential of the cyt bH may reflect a differential coulombic effect due to a longer distance from the heme charge of cyt bH to the positive charge of K251 than to the negative charge of D252 in the wild-type, and a reversal of these distances in the mutant.
We have used site directed mutagenesis and biophysical assay to probe the structure-function interface at the Qo-site. A number of inhibitor resistance mutations have mapped to the span between helices E and F, and in addition, the span contains an almost totally conserved - PEWY- region, P294, E295, W296, Y297. Mutational replacement in this span shows that none of the residues is essential to function, but replacement of any lowers activity. Since resistance mutations occur on either side of the -PEWY- span, it seems likely that this region is near to the Qo-site (25). Many residue changes in this region show marked effects on the site, as reflected in modified kinetics of quinol oxidation, and binding of inhibitors (see Table I). Several of these have not previously been identified as contributing to the site. In general, although the maximal rates vary >200-fold, the mutants with modified kinetics all show the increase in rate of QH2 oxidation as the pool becomes reduced over the range Eh < 180 mV, with an approach to saturation at potentials where the pool is half-reduced, which in wild-type has been interpreted as showing a site at which QH2 binds in preference to Q by a factor 10-100 (3). This behavior in the mutant strains shows that the relative affinities of Q and QH2, and Km for QH2, are not greatly affected. The main effects of mutation appear to be on kcat.
Although there is a consensus that QH2 oxidation follows the pattern expected from the modified Q-cycle, details of the mechanism at the structural level, and interpretation of mutational effects, are more controversial. In addition to ligands for quinone, the Qo-site has contributions from the Fe.S subunit, and interactions with cyt c1 (26, and Daldal, F., personal communication), which might effect function if perturbed. Following earlier work showing an effect of the redox state of the quinone pool on the EPR spectrum of the 2Fe.2S center of the complex (27), Ding et al. (28), using wild-type and mutants strains from Rb. capsulatus, have investigated the effects of extraction of ubiquinone, and concluded that different spectral changes can be detected at different local concentrations. They suggested that these reflect two different populations of bound quinone at the Qo-site, called Qo,s and Qo,w for strongly and weakly binding species. They have extended these observations to mutant strains, and kinetic studies of turn-over and inhibitor binding, concluding that the mechanism might reflect a double occupancy of the Qo-site, with both sites showing equal binding for Q and QH2. More recently, they have discussed possible mechanisms and atomic details of ligation (29, 30). In general, the mechanisms recognize the need for a rapidly exchanging site required by the kinetic data (1-3); it would seem likely that Qo,w provides this function, and that Qo,s acts as a prosthetic group regulating the delivery of electrons to the metallic acceptors (2Fe.2S and cyt bL) of the complex. In collaboration with Dutton's lab, we have looked at similar effects in some of the strains from Rb. sphaeroides summarized in the Table above.
The double-occupancy mechanism requires that the catalytic site must contain two distinct sets of ligands to provide for the differential binding of the two species. If the protein provided these, it would be expected that ligands specific for one or other species would be detected by mutation, but no such differential effects have been seen among the many mutants generated by our two groups (25). Furthermore, there is no clear correlation between effects of mutation on the spectral changes attributed to Qo,w, and interpreted as showing substantial changes in affinity, and the small changes in Km for QH2 oxidation. Myxothiazol, stigmatellin and UHDBT eliminate the spectral changes associated with both strong and weak binding, with the same titration (<1mol/mol cyt c1 for stigmatellin) as that giving rise to inhibition of electron flow (28). These inhibitors compete for a common site (30, 31); however, stigmatellin and UHDBT, but not myxothiazol, directly effect the redox potential and spectra of the 2Fe.2S center, indicating differences in the liganding, and these differences are reflected in a differential resistance to the inhibitors in different mutant strains (25, 30). These effects are difficult to explain in the double-occupancy model. The failure to detect differential effects of mutation on Q-binding could mean that the ligands for Qo,w are provided by Qo,s; in this context, the titration effects could be explained by suggesting that the inhibitors displace Qo,s (29), and the relative lack of effect of mutation on Km(QH2) by the fact that binding does not involve protein ligands. Still unexplained is the discrepancy between the tighter binding of quinone indicated by kinetic experiments, and the equal binding of Q and QH2 from the EPR data. If the structure shows that the Qo-site has only a single quinone, alternative explanations of the binding effects on the EPR spectrum of 2Fe.2S will have to be found.
Acknowledgments. We thank Les Dutton and Fevzi Daldal for extensive helpful discussion and access to unpublished material, and NIH for support though a grant RO1 GM35438.