Chromatophore heterogeneity explains effects previously attributed to supercomplexes.

by
A.R. Crofts, Sangjin Hong, and Mariana Guergova-Kuras
Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois

This is an expanded and updated version of a paper published previously as
Crofts, A. R., Guergova-Kuras, M. and Hong, S. (1998) Chromatophore heterogeneity explains effects previously attributed to supercomplexes. Photosynth. Res. 55, 357-362

It is generally considered that metabolic reactions are well described by homogeneous kinetic models in which the reaction phase is statistically uniform. In membranes, especially in photosynthetic systems where the protein complement is high, it has recently been recognized that effects of local inhomogeneity might contribute additional factors which perturb the kinetic behavior, and require more extensive treatment [1]. We show in this paper that statistical inhomogeneity in vesicular systems can also contribute to quite marked discrepancies from the behavior expected from standard kinetic and thermodynamic models, for reactions involving free diffusion in the aqueous phase.

Several recent papers from the Joliots in collaboration with Vermeglio and Lavergne have suggested that the enzymes of photosynthetic electron transfer systems might be organized as supercomplexes [1-5]. The work was based on measurements by kinetic spectrophotometry of the equilibrium poise of reactants after excitation by flashes or continuous light. On continuous illumination of cells of Rb. sphaeroides, the observed values for equilibrium constant (Kobs) for electron transfer in the high potential chain were much lower than those expected from measured redox mid-point potentials [3]. To explain their results, the authors suggested that electron transfer occurred predominantly within supercomplexes, and developed an elegant hypothesis to account for the observed behavior. Two sorts of supercomplex were propose, one with reaction center and cyt c2 in a ratio 2:1, the other with reaction centers, bc1-complex and cyt c2 in a ratio of 2:1:1. Within the supercomplexes, electron transfer reactions were proposed to occur with rates in the µs to ms range, and rapidly attained the poise expected from known equilibrium constants. However, diffusion of cyt c2 from the complex was restricted so that interaction between one supercomplex and another, or with reaction center or bc1-complex not associated into supercomplexes, could occur only on a seconds time scale. In this condition, it was suggested that continuous illumination introduced a statistical heterogeneity into the supercomplex population, leading to a fraction of centers in which the reaction center was more oxidized than expected (because the donor pool was exhausted), accounting for the observed departure from the expected equilibrium poise.

Evidence for separate complexes, connected by diffusion of QH2 and cyt c2

Previous experiments from this lab with chromatophores had suggested a different picture of the organization of the electron transfer chain. We have demonstrated that:
  1. There is a diffusional pathway for QH2 between reaction center and the bc1-complex (giving a lag before reduction of cyt bH when the pool is initially oxidized) [6, 7].
  2. Oxidation of QH2 involves a second-order reaction [6-12].
  3. Both QH2 and cytochrome c2 can "visit" at least 8-10 electron transfer chains, probably representing all bc1-complexes in a chromatophore [13, 14]. In these experiments, stigmatellin or myxothiazol or antimycin were used progressively to titrate out the bc1-complex, and the capacity of the residual active complexes to transfer electrons to the oxidized cyt c2 produced on photoactivation with a saturating flash was tested. A full reduction of cyt (c2 + c1) was observed when more than 80% of bc1-complexes were inactive.
  4. The kinetics slowed in proportion to the fraction of inhibited centers, showing that the rate was determined by the same rate constant in each case, and not effected by the greater diffusional pathway. This implies that there was no impediment to diffusion of cyt c2 in the chromatophore internal volume [13].
More recently, we have looked at kinetics of turn-over of the chain in chromatophores in which the bc1-complex is at a different concentration relative to the reaction center than in "standard" chromatophores. These include chromatophores from strains expressed from a plasmid, strains grown under different conditions, and chromatophores prepared so as to retain or lose a greater fraction of cytochrome c2. Examples from mutant strains are shown below, - a strain with a his-tagged cytochrome b, which overexpressed the bc1-complex to give a stoichiometry >1 bc:RC, a strain that overproduces cyt c2, and a strain in which cyt c2 is replaced by iso-cyt c2, but at a stoichiometry of about 0.3 c2:bc1 complex (26). In all strains, turn-over of the system appears to be relatively homogeneous, and rapid, with kinetic constants similar to wild-type. This shows that, under circumstances in which most chains cannot be in a supercomplex, the diffusion of cyt c2 between RC and bc1 complex is rapid enough to give the wild-type kinetics. If diffusion is rapid under these circumstances, why should it be constrained under conditions in which a supercomplex is postulated?

Structural considerations

In addition to these direct experiments, recent information about the structure of the reaction center and its associated light harvesting apparatus, and about the bc1 complex, makes it seem unlikely that the supercomplex is a real structure. If there are no supercomplexes, it is necessary to find an alternative explanation for the kinetic effects which led Joliot et al. to the hypothesis which required them.

In view of the apparent contradiction between results obtained using cells and chromatophores from Rb. sphaeroides, we have performed comparable experiments in both systems.

In chromatophores we could measure a low apparent equilibrium constant on continuous illumination as seen in cells and attributed to supercomplexes, yet the inhibitor titrations show that supercomplexes are absent in chromatophores.

In contrast to the chromatophore results, when inhibitor titration experiments were performed with whole cells, the electron transfer linked to cyt c2 appeared to be restricted to a local domain of 1-2 units, and diffusion of cyt c2 outside this domain was severely restricted, as would be expected from the supercomplex model.

This paradoxical behavior is open to several interpretations. One possibility is that supercomplexes are present in whole cells, but lost on preparation of chromatophores. A second possibility is that the restricted diffusion seen in cells is not due to supercomplexes, but to some structure in the periplasm, for example the murein sacculus, which is a common structural feature of Gram-negative bacteria.

In order to explore the possibilities, we have modeled the kinetic and thermodynamic behavior of the high-potential chain on illumination under three conditions:

Fig. 1. Models

In the supercomplex model, heterogeneity is introduced by statistically random photon hits. In our alternative model, heterogeneity comes from a statistically random distribution of components within the chromatophore population. In the homogeneous system, the components remain poised at equilibrium throughout the time course of illumination. In both "heterogeneity" models, the apparent equilibrium constant was much less than that in the homogeneous model; indeed theoretical considerations show that this is a general effect of heterogeneity [3].

Predictions

The Lavergne model is highly constrained [3], and gives rise to two predictions. Fig. 2. Predicted kinetics of oxidation of P+ and cyt c on illumination of a population of supercoplexes in the presence of myxothiazol or stigmatellin.

The Crofts model predicts that:

Fig. 3. Predicted behavior using Crofts model.

Experiments with chromatophores favor the Crofts model. They show a variable equilibrium constant, perhaps depending on the amount of trapped cyt c2, or type of inhibitor (Fig. 4, 5). In chromatophores with > 90% of reactions centers rapidly reduced in the absence of inhibitor, a substantial fraction (~20%) of (BChl)2+ remained oxidized after a flash in the presence of myxothiazol, and more (~30%) with stigmatellin, the amount varying with the amount of trapped cyt c2 (Fig. 6, 7). These are within the range predicted by the model.

Fig. 4 Kinetic traces showing the oxidation of P and cyt c on continuous illumination in the presence of myxothiazol or stigmatellin.

Fig. 5. "Magic Square" plot for experimental results on continuous illumination in the presence of myxothiazol or stigmatellin.

Figs. 6, 7. Kinetics during flash illumination in the presence of myxothiazol or stigmatellin.

Figs. 8-10 show kinetics in strains with variable stoichiometries of RC:cyt ct:bc-complex.
In Fig. 8, the difference between the two strains is in the relative stoichiometries of the components of the chain. This can be seen from the amplitudes of the traces after four flashes in the presence of antimycin. The traces show stoichiometric ratios for RC:cyt c2:bc1-complex close to the "normal" 2:1:1 for BC17C, but about 2:1:3 for BH6. The BH6 strain showed a substantial over expression of the bc1-complex, but the rapid reduction of cyt bH observed in the presence of antimycin following the first flash showed kinetics similar to those in a strain with normal stoichi-ometries. In the over-expressing strain, the amount of "connected" bc1-complex was ~3-times greater than that expected from the supercomplex hypothesis, and the amount undergoing rapid reduction after the first flash corresponded to 1 cyt bH / RC, or ~ twice that possible in a super-complex. The behavior is well explained by a diffusional mechanism in the context of the modified Q-cycle (Crofts, 1985).

In Fig. 9, the kinetics of turn-over of cytochrome c1 and isocyt c2 are shown in strain CYCA65R7 (pVWF1R) (Witthun et al., 1996). In this strain, the gene for cyt c2 was deleted, but expression of the gene for isocyt c2 allowed photosynthetic growth. The isocyt c2 showed some degree of over expression compared to the very low levels normally observed. The stoichiometric ratios of RC:isocyt c2:bc1-complex were 2:<0.3:1, as assayed from kinetic traces using this strain in experiments similar to those in A above. It is clear from the results that the photosynthetic chain in this strain is not organized as a supercomplex. Both in chromatophores, and in cells (not shown), the small amplitude of the fast phase indicates that most of the isocyt c2 does not bind to the reaction center. Such a binding can be observed at higher concentrations in vitro, and gives rise to a rapid (< 2 ms) phase of oxidation (Witthun et al., 1996). The stoichiometry is also incompatible with that expected for a supercomplex; the amount of bc1-complex undergoing redox change is >3-fold greater than the estimated concentration of isocyt c2, but the components of the complex nevertheless undergo rapid electron transfer with a stoichiometry and kinetics similar to the wild type. This clearly indicates that rapid diffusion of isocyt c2 must occur to connect bc1-complexes to RCs. In order to give the kinetics observed, each isocyt c2 molecule must visit ~3 RCs and bc1-complexes during the rise kinetics (t1/2 ~300 ms), and ~6 of each during the reduction phase of the RC (t1/2 ~1.5 ms in the absence of inhibitor).

Fig. 10 shows the kinetics of oxidation of cyt c in strains in which either the bc1 complex or cyt c2 is overproduced. Note that the stoichiometry of the overproduced component is several fold in excess of that in the wild-type. For the cytochrome c2 overproducer, the stoichiometry is ~2.5 per reaction center, and the oxidation is complete in ~50 ms. This shows that reaction centers must have a ready access to cytochrome c2 in stoichiometry about 5 x that expected from the supercomplex model. The traces at higher time resolution show that cyt c oxidation occurs with greater amplitude than in wild-type. With the rate constant used in these experiments, the kinetics are much more rapid than in wild-type, suggesting that most of the phase in excess of the "normal" complement of 1 cyt c2 / RC is due to oxidation of additional cyt c2. The increased rate could be due to increased binding, or to a second-order dependence on concnetration. In either case, the results are not compatible with a supercomplex with a fixed stoichiometry of cyt c2, and a restricted dissociation from the complex.

Additional sources of heterogeneity

In distributing components at random, the program assumed that both reaction center and bc1-complex were monomeric (left in Fig. 3), or that both were dimeric (right in Fig. 2). In both cases, chrompatophores were assumed to be of uniforn size, and "illumination" was assumed to by uniform. The left model represents the least heterogeneity possible with random distribution. Any additional heterogeneity tends to decrease the Kobs; thus smaller values would be obtained in both situations if "illumination" was random, if the size of chromatophores was non-uniform, and if the number of components per chromatophore was smaller. The results of inhibitor titrations suggested that the reaction domain seen by cyt c2 included about 8-10 bc-complexes (rather than the 15 used in the simulation)(11), and that the bc-complex may be dimeric. We have recently developed a new isolation proceedure for the bc-complex with a (His)6-tagged cyt b. This complex appears to be structurally dimeric. We would therefore expect the value for Kobs to be towards the low end of the range shown in Fig.3. The simulation shown in Fig. 11 has a mean distribution ratio of 16:8:8 for RC:cyt c2:bc-complex per chromatophore, and the bc-complex is dimeric.

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