Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides. I. Quinone domains and excitation transfer in chromatophores and reaction center.antenna complexes

The purpose of this study was to gain information on the functional consequences of the supramolecular organization of the photosynthetic apparatus in the bacterium Rhodobacter sphaeroides. Isolated complexes of the reaction center (RC) with its core antenna ring (light-harvesting complex 1 (LH1)) were studied in their dimeric (native) form or as monomers with respect to excitation transfer and distribution of the quinone pool. Similar issues were examined in chromatophore membranes. The relationship between the fluorescence yield and the amount of closed centers is indicative of a very efficient excitation transfer between the two monomers in isolated dimeric complexes. A similar dependence was observed in chromatophores, suggesting that excitation transfer in vivo from a closed RC.LH1 unit is also essentially directed to its partner in the dimer. The isolated complexes were found to retain 25-30% of the endogenous quinone acceptor pool, and the distribution of this pool among the complexes suggests a cooperative character for the association of quinones with the protein complexes. In chromatophores, the decrease in the amount of photoreducible quinones when inhibiting a fraction of the centers implies a confinement of the quinone pool over small domains, including one to six reaction centers. We suggest that the crowding of membrane proteins may not be the sole reason for quinone confinement and that a quinone-rich region is formed around the RC.LH1 complexes.     

Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides: II. A study of PufX- membranes

In the bacterium R. sphaeroides, the polypeptide PufX is indispensable for photosynthetic growth. Its deletion is known to have important consequences on the organization of the photosynthetic apparatus. In the wild-type strain, complexes between the reaction center (RC) and the antenna (light-harvesting complex 1 (LH1)) are associated in dimers, and LH1 does not fully encircle the RC. In the absence of PufX, the complexes become monomeric, and the LH1 ring closes around the RC. We analyzed the functional consequences of PufX deletion. Some effects can be ascribed to the monomerization of the RC.LH1 complexes: the number of RCs that share a common antenna for excitation transfer or a common quinone pool become smaller. We examined the kinetic effects of the closed LH1 ring on quinone turnover: diffusion across LH1 entails a delay of approximately 1 ms, and the barrier appears to be located directly against the quinone-binding (secondary quinone acceptor (Q(B))) pocket. The diffusion of ubiquinol from the RC to the cytochrome bc1 complex is approximately 2-fold slower in the mutant, suggesting an increased distance between the two complexes. The properties of the Q(B) pocket (binding of inhibitors, stabilization of Q(B-), and rate of Q(B)-H2 formation) appear to be modified in the mutant. Another specificity of PufX- is the accumulation of closed centers in the Q(A-) (where Q(A) is the primary quinone acceptor) state as the secondary acceptor pool becomes reduced, which is probably the origin of photosynthetic incompetence. We suggest that this is related to the Q(B) pocket alterations. The malfunction of the reaction center is probably due to a faulty association with LH1 that is prevented in the PufX-containing structure.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.     

Possible pathway for ubiquinone shuttling in Rhodospirillum rubrum revealed by molecular dynamics simulation

In the last decade, the structures of many components of the photosynthetic apparatus of purple bacteria, as well as the mutual organization of these components within the purple membrane, were resolved. One key question that emerged concerned the assembly of the core complex consisting of the reaction center (RC) and the light-harvesting 1 (LH1) complex. In some species, like Rhodobacter sphaeroides, the ring-shaped LH1 complex was found to be open, whereas other species, like Rhodospirillum rubrum, have a closed ring surrounding the reaction center. This poses the question of how the ubiquinone molecule that transports electrons and protons from the RC to the cytochrome bc(1) complex overcomes the apparent barrier of the LH1 ring. In this study, we investigated how, in the case of a closed LH1 ring, the ubiquinone molecule diffuses through the LH1 ring. For this purpose, the LH1 structure of R. rubrum was modeled and the potential of mean force along the diffusion pathway through the LH1 was determined by steered molecular-dynamics simulations. The potential was reconstructed using the fluctuation theorem in combination with the stiff spring approximation. An upper limit for the mean first-passage time for diffusion of ubiquinone through the LH1 ring, based on a worst-case scenario potential, was calculated as approximately 8 x 10(-3) s, which is still in agreement with known turnover rates of RC and RC-LH1 complexes in the range of approximately 1000 Hz.     

Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)). Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation. Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex. The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.     

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q(a) has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 degrees C, about half of Q(a)(-) is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q(o) of the cytochrome bc(1) complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q(a)(-) oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc(1) complex, which allows a fast transfer of quinone formed at the level of cyt bc(1) complex to the RCs. In agreement with this model, the fast phase of Q(a)(-) reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc(1). The PufX-deleted mutant displays only the slowest phase of Q(a)(-) oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc(1) and RCs.     

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.     

Three-dimensional reconstruction of a membrane-bending complex: the RC-LH1-PufX core dimer of Rhodobacter sphaeroides

A three-dimensional model of the dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) complex from Rhodobacter sphaeroides, calculated from electron microscope single particle analysis of negatively stained complexes, shows that the two halves of the dimer molecule incline toward each other on the periplasmic side, creating a remarkable V-shaped structure. The distribution of negative stain is consistent with loose packing of the LH1 ring near the 14th LH1 alpha/beta pair, which could facilitate the migration of quinone and quinol molecules across the LH1 boundary. The three-dimensional model encloses a space near the reaction center Q(B) site and the 14th LH1 alpha/beta pair, which is approximately 20 angstroms in diameter, sufficient to sequester a quinone pool. Helical arrays of dimers were used to construct a three-dimensional membrane model, which matches the packing lattice deduced from electron microscope analysis of the tubular dimer-only membranes found in mutants of Rba. sphaeroides lacking the LH2 complex. The intrinsic curvature of the dimer explains the shape and approximately 70-nm diameter of these membrane tubules, and at least partially accounts for the spherical membrane invaginations found in wild-type Rba. sphaeroides. A model of dimer aggregation and membrane curvature in these spherical membrane invaginations is presented.     

Oxidoreductase activity of chromatophores and purified cytochrome bc 1 complex from Rhodobacter sphaeroides: a possible role of cardiolipin

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60?±?2 to 93?±?3?mol cyt c s(-1) (mol total cyt c)(-1). Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc ( 1 ) complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc ( 1 ) complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc ( 1 ) complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophore and in purified cyt bc ( 1 ) complex.     

The cytochrome bc1 complex of Rhodobacter sphaeroides can restore cytochrome c2-independent photosynthetic growth to a Rhodobacter capsulatus mutant lacking cytochrome bc1.

Plasmids encoding the structural genes for the Rhodobacter capsulatus and Rhodobacter sphaeroides cytochrome (cyt) bc1 complexes were introduced into strains of R. capsulatus lacking the cyt bc1 complex, with and without cyt c2. The R. capsulatus merodiploids contained higher than wild-type levels of cyt bc1 complex, as evidenced by immunological and spectroscopic analyses. On the other hand, the R. sphaeroides-R. capsulatus hybrid merodiploids produced only barely detectable amounts of R. sphaeroides cyt bc1 complex in R. capsulatus. Nonetheless, when they contained cyt c2, they were capable of photosynthetic growth, as judged by the sensitivity of this growth to specific inhibitors of the photochemical reaction center and the cyt bc1 complex, such as atrazine, myxothiazol, and stigmatellin. Interestingly, in the absence of cyt c2, although the R. sphaeroides cyt bc1 complex was able to support the photosynthetic growth of a cyt bc1-less mutant of R. capsulatus in rich medium, it was unable to do so when C4 dicarboxylic acids, such as malate and succinate, were used as the sole carbon source. Even this conditional ability of R. sphaeroides cyt bc1 complex to replace that of R. capsulatus for photosynthetic growth suggests that in the latter species the cyt c2-independent rereduction of the reaction center is not due to a structural property unique to the R. capsulatus cyt bc1 complex. Similarly, the inability of R. sphaeroides to exhibit a similar pathway is not due to some inherent property of its cyt bc1 complex.     

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.     

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.     

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A)

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.     

Modulation of the redox state of quinones by light in Rhodobacter sphaeroides under anaerobic conditions

Illumination of intact cells of Rhodobacter sphaeroides under anaerobic conditions has a dual effect on the redox state of the quinone pool. A large oxidation of the quinone pool is observed during the first seconds following the illumination. This oxidation is suppressed by the addition of an uncoupler in agreement with a light-induced reverse electron transfer at the level of the complex I, present both in the non-invaginated part of the membrane and in the chromatophores. At longer dark times, this illumination increases the reducing power of the cells leading to a significant reduction of the others reaction centers (RCs). From the observation that a significant proportion of RCs could be reduced by the preillumination without affecting the numbers of charge separation for the RCs, we conclude that there is no rapid thermodynamic equilibrium between the quinones present in the non-invaginated part of the membrane and those localized in the chromatophores. Under anaerobic conditions where the chromatophores quinone pool is fully reduced, we deduce, on the basis of flash-induced fluorescence kinetics, that the reduced RCs are exclusively reoxidized by the quinone generated at the Q _o site of the cyt bc ₁ complex. The supramolecular association between a dimeric RC-LHI complex and one cyt bc ₁ complex allows the confinement of a quinone between the RC-LHI directly associated to the cyt bc ₁ complex.     

Structural basis for the PufX-mediated dimerization of bacterial photosynthetic core complexes

In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 A by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of approximately 300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center (RC). The LH1 consists of 15 resolved alpha/beta heterodimers and is interrupted. Within the opening, PufX polypeptide is assigned at a position facing the Q(B) site of the RC. This core complex is different from a dissociated dimer of the core complex of Rba. sphaeroides revealing that PufX in Rba. veldkampii is unable to dimerize. The absence in PufX of Rba. veldkampii of a G(31)XXXG(35) dimerization motif highlights the transmembrane interactions between PufX subunits involved in the dimerization of the core complexes of Rhodobacter species.     

Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability

Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LH1-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or by the core light-harvesting complexes. The results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.     

Excitation transfer connectivity in different purple bacteria: A theoretical and experimental study

Photosynthetic membranes accommodate densely packed light-harvesting complexes which absorb light and convey excitation to the reaction center (RC). The relationship between the fluorescence yield (φ) and the fraction (x) of closed RCs is informative about the probability for an excitation reaching a closed RC to be redirected to another RC. In this work, we have examined in this respect membranes from various bacteria and searched for a correlation with the arrangement of the light-harvesting complexes as known from atomic force or electron microscopies. A first part of the paper is devoted to a theoretical study analyzing the φ(x) relationship in various models: monomeric or dimeric RC-LH1 core complexes, with or without the peripheral LH2 complexes. We show that the simple “homogeneous” kinetic treatment used here agrees well with more detailed master equation calculations. We also discuss the agreement between information derived from the present technique and from singlet annihilation experiments. The experimental results show that the enhancement of the cross section of open RCs due to excitation transfer from closed units varies from 1.5 to 3 depending on species. The ratio of the core to core transfer rate (including the indirect pathway via LH2) to the rate of trapping in open units is in the range of 0.5 to 4. It is about 1 in Rhodobacter sphaeroides and does not increase significantly in mutants lacking LH2—despite the more numerous contacts between the dimeric core complexes expected in this case. The connectivity in this bacterium is due in good part to the fast transfer between the two partners of the dimeric (RC-LH1-PufX)₂ complex. The connectivity is however increased in the carotenoidless and LH2-less strain R26, which we ascribe to an anomalous LH1. A relatively high connectivity was found in Rhodospirillum photometricum, although not as high as predicted in the calculations of Fassioli et al. (2010). This illustrates a more general discrepancy between the measured efficiency of core to core excitation transfer and theoretical estimates. We argue that the limited core to core connectivity found in purple bacteria may reflect a trade-off between light-harvesting efficiency and the hindrance to quinone diffusion that would result from too tightly packed LH complexes.