Chromatophore heterogeneity explains effects previously
attributed to supercomplexes.
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
. 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 . 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
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:
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?
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,
Oxidation of QH2 involves a second-order
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.
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 .
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.
Cogdell and colleagues have recently solved the structure
of LH2 from Rhodopseudomonas acidophila by X-ray crystallography
, and a similar structure has been computed from diffraction data for
the LH2 of Rps. mollischianum by Schulten, Michel and colleagues
(Prof. Klaus Schulten, personal communication). Ghosh has reported an 8.5
Å resolution structure for the LH1 complex of Rhodospirillum rubrum
using electron diffraction and transmission , which shows a similar
motif, and modelling studies have suggested the the building units for
all these structures are essentially the same. However, the LH2 structures
consisted of rings of 9 or 8 alpha-beta pairs, whereas the LH1 complex
had 16 pairs. Ghosh  pointed out that there was just room for one reaction
center in the LH1 ring. More recently, Kuehlbrandt, in collaboration with
Cogdell, has used electron microscopic techniques to obtain low resolution
structures for LH1/reaction center complexes from Rhodopseudomonas acidophila,
which have confirmed Ghosh's suggestion (Dr. W. Kuehlbrandt, personal communication).
It seems highly likely that the light-harvesting complex of Rb. sphaeroides
is organized in a similar way, and Cogdell and colleagues, and
Schulten and colleagues have constructed models of the reaction center
in the palisade of the LH1 cylinder; images from both labs are shown here.
The picture of the reaction center surrounded by
a cage of LH1 molecules is supported by observations of the kinetic behavior
of cells which do not make the PufX protein. The pufX gene is part
of the puf operon, which codes for the subunits of LH1 and the L
and M subunits of the reaction center. In pufX- strains,
the reaction center is unable to react with quinone produced by the bc1-complex,
and the bc1-complex does not "see" the quinol produced by the
reaction center [17-21], suggesting that an impediment to diffusion between
reaction centers and bc1 complex exists in the absence of PufX.
The PufX gene product has a stoichiometry of ~1-2 per reaction center, and
suppressor strains occur through mutations in LH1 which reduce the ratio
of LH1 to reaction center, or disorder the LH1. We had previously suggested
that PufX might interact with the LH1 ring to facilitate diffusion of quinone
across the barrier created by the palisade of helices surrounding the reaction
center. This mechanism was based on the assumption that a complete ring of LH1 surrounded the
reaction center. In the context of a such a structure, the kinetic
effects seen in pufX- strains would have been difficult
to reconcile with a supercomplex model. However, a new perspective has
come from recent work, initialy reported in contributions at the XIth. International
Congress on Photosynthesis (27, 28), but now published (29, 30). This work has shown that in PufX containing
strains, dimers of the reaction center-LH1 complex can be detected by sucrose
gradient separation after mild detergent solubilization, with a substantial
fraction of centers in the dimeric form. Similar treatment of a pufX-
strain showed only monomeric complexes (27). The structure of the dimers
is not known, but the BChl stoichiometry suggests that there are 24 BChl per reaction
centers in wild-type, compared to 29.5 in pufX- (28).
From previous data suggesting that 2 PufX subunits per reaction center
participate, the loss of 4 BChl would suggest replacement of two a,
b-pairs by 2 PufX. This stoichiometry is compatable with our earlier
hypothesis, but could alternatively indicate that the LH1 ring might be
incomplete or shared. Taken together with the experiments showing dimerization
of the LH1/RC complex, the structures images by Jungas and colleagues (29)
(see below) might represent such dimers.
The bc1-complexes are thought to be structurally
(and possibly functionally ) dimeric. In the mitochondrial complex,
this was clearly evident in low resolution electron microscopy structures
, and has been confirmed in the X-ray structures recently reported [24, 31-33]. The complex isolated from
Rb. sphaeroides runs as a dimer on sizing gels (34). Models constructed
using a dimeric 3-subunit bc1 complex abstracted from the mitochondrial bc1 complex (31), and LH1 models from Schulten's
lab (see above), show that a supercomplex
incorporating such a dimer, would be large enough to be obvious in
electron micrographs, but such structures have not been reported in studies
in which particle sizes seen in freeze-fracture electron microscopy were
carefully tabulated. Until recently, although bc1-complex dimers,
and LH1/reaction center complexes are readily isolated, there have been
no reports of isolation of larger structures. The dimerization of reaction
center - LH1 complexes induced by PufX (see above) is of interest in this
context. The dimeric complexes isolated by Francia et al. (30) do not contain any bc1 complex.
However, Vermeglio has suggested that the dimeric
structures seen in tubular structures in LH2 depleted strains of Rb.
sphaeroides might include a bc1 complex, which is found in isolated membranes in the stoichiometry expected fo the supercomplex. There is no room for a dimeric bc1 complex in the arrays reported by Jungas et al. (29), and it is doubtful that a monomeric 3-subnit complex could fit.
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
free diffusion and equilibration in a statistically
homogeneous pool (classical model);
free diffusion and equilibration in each vesicle
of a heterogeneous population of chromatophores (Crofts model) (Fig. 1,
restricted diffusion in a population of supercomplexes
with heterogeneous illumination (adapted from the Lavergne model )(Fig.
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 .
The Lavergne model is highly constrained ,
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.
i) The observed equilibrium constants should be relatively
independent of inhibitors, and (for random illumination) of fixed value
(Fig. 2, but see Figs. 4, 5).
ii) Rereduction of the oxidized reaction center,
(BChl)2+ (P+), after a saturating flash
should be almost complete in all connected centers reduced before the flash,
independent of inhibitor present, since no heterogeneity is introduced
with a saturating, single-turnover flash, and the natural equilibrium constants
are large [4,13] (but see Figs. 6, 7).
The Crofts model predicts that:
Fig. 3. Predicted behavior
using Crofts model.
i) The observed equilibrium constant will be variable
with preparation, and dependent on the component count, and type of inhibitor
(Fig. 3, 4, 5).
ii) The extent of reduction of (BChl)2+
following a saturating flash will be dependent on component count, and
will depend predictably on the nature of the inhibitor (Fig. 6, 7).
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
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
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).
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
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