Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

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.     

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii

The photosynthetic membrane in purple bacteria contains several pigment–protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1–RC (light-harvesting complex 1 – reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1–RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC–LH1–PufX cores, as it has been proposed in Rb. sphaeroides.     

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.     

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.     

Protein-Induced Membrane Curvature Investigated through Molecular Dynamics Flexible Fitting

In the photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides, light is absorbed by membrane-bound light-harvesting (LH) proteins LH1 and LH2. LH1 directly surrounds the reaction center (RC) and, together with PufX, forms a dimeric (RC-LH1-PufX)₂ protein complex. In LH2-deficient Rba. sphaeroides mutants, RC-LH1-PufX dimers aggregate into tubular vesicles with a radius of ∼250-550 Å, making RC-LH1-PufX one of the few integral membrane proteins known to actively induce membrane curvature. Recently, a three-dimensional electron microscopy density map showed that the Rba. sphaeroides RC-LH1-PufX dimer exhibits a prominent bend at its dimerizing interface. To investigate the curvature properties of this highly bent protein, we employed molecular dynamics simulations to fit an all-atom structural model of the RC-LH1-PufX dimer within the electron microscopy density map. The simulations reveal how the dimer produces a membrane with high local curvature, even though the location of PufX cannot yet be determined uniquely. The resulting membrane curvature agrees well with the size of RC-LH1-PufX tubular vesicles, and demonstrates how the local curvature properties of the RC-LH1-PufX dimer propagate to form the observed long-range organization of the Rba. sphaeroides tubular vesicles.     

Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc ₁ complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc ₁ complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.     

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.     

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.     

FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical ‘chromatophore’ vesicles. These bacterial ‘organelles’ are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc₁ complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c₂. Proximity between cytochrome bc₁ and RC-LH1-PufX minimises quinone/quinol/cytochrome c₂ diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc₁ with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc₁ complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc₁ observed by spectroscopy, and explain how quinols/quinones and cytochrome c₂ shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.     

Carotenoid to bacteriochlorophyll energy transfer in the RC–LH1–PufX complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> containing the extended conjugation keto-carotenoid diketospirilloxanthin

RC–LH1–PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC–LH1–PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S₂–Q_x pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC–LH1–PufX we observe an additional, minor energy transfer pathway associated with the S₁ state of diketospirilloxanthin. By comparing the spectral properties of the S₁ state of diketospirilloxanthin in solution, in LH2, and in RC–LH1–PufX, we propose that the carotenoid-binding site in RC–LH1–PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S₁/ICT-mediated energy transfer channel.     

Conjugation-length dependence of the T1 lifetimes of carotenoids free in solution and incorporated into the LH2, LH1, RC, and RC-LH1 complexes: possible mechanisms of triplet-energy dissipation

In addition to the roles of antioxidant and spacer, carotenoids (Cars) in purple photosynthetic bacteria pursue two physiological functions, i.e., light harvesting and photoprotection. To reveal the mechanisms of the photoprotective function, i.e., quenching triplet bacteriochlorophyll to prevent the sensitized generation of singlet oxygen, the triplet absorption spectra were recorded for Cars, where the number of conjugated double bonds (n) is in the region of 9-13, to determine the dependence on n of the triplet lifetime. The Cars examined include those in (a) solution; (b) the reconstituted LH1 complexes; (c) the native LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, and Rps. acidophila 10050; (d) the RCs from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, and Rsp. rubrum S1; and (e) the RC-LH1 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, Rps. acidophila 10050, and Rsp. rubrum S1. The results lead us to propose the following mechanisms: (i) A substantial shift of the linear dependence to shorter lifetimes on going from solution to the LH2 complex was ascribed to the twisting of the Car conjugated chain. (ii) A substantial decrease in the slope of the linear dependence on going from the reconstituted LH1 to the LH1 component of the RC-LH1 complex was ascribed to the minor-component Car forming a leak channel of triplet energy. (iii) The loss of conjugation-length dependence on going from the isolated RC to the RC component of the RC-LH1 complex was ascribed to the presence of a triplet-energy reservoir consisting of bacteriochlorophylls in the RC component.     

Rhodopseudomonas acidophila strain 10050 contains photosynthetic LH2 antenna complexes that are not enriched with phosphatidylglycerol,and the phospholipids have a fatty acyl composition that is unusual for purple non-sulfur bacteria

The phospholipid composition of Rhodopseudomonas acidophila strain 10050 grown aerobically or anaerobically in the light was determined. The major phospholipids present in the aerobic cells were phosphatidylethanolamine (PE; 54%), phosphatidylglycerol (PG; 24%) and cardiolipin (diphosphatidylglycerol, DPG) (14%), together with phosphatidylcholine (PC; 5%). On moving the cells to anaerobic photosynthetic growth in the light PE remained the major phospholipid (37-49%), but there was a major change in the proportion of PC, which increased to 31-33%, and corresponding reductions in the contents of PG to 11-16% and DPG to 4-5%. The fatty acid composition of the phospholipids was unusual, compared with other purple non-sulfur photosynthetic bacteria, in that it contained 16:0 (29%), 17:1 (20%) and 19:1 (9%) plus several mainly unsaturated 2-OH fatty acids (9% total) as major components, when grown aerobically in the dark. In contrast when grown photosynthetically under anaerobic conditions there was <2% 17:1 or 19:1 present, while the amounts of 16:1 and 18:1 increased, and 16:0 decreased. The phospholipid composition of the purified light-harvesting complex 2 (LH2) complex was PE (43%), PC (42%) and DPG (15%). Unexpectedly, there was no PG associated with the purified LH2. These findings contrast with previous studies on several other photosynthetic bacteria, which had shown an increase in PG upon photosynthetic growth [Biochem. J. 181 (1979) 339]. The prior hypothesis that phosphatidylglycerol has some specific role to play in the function of light-harvesting complexes cannot be true for Rps. acidophila. It is suggested that specific integral membrane proteins may strongly influence the phospholipid content of the host membranes into which they are inserted.     

Energy transfer in purple bacterial photosynthetic units from cells grown in various light intensities

Three photosynthetic membranes, called intra-cytoplasmic membranes (ICMs), from wild-type and the ?pucBA_abce mutant of the purple phototrophic bacterium Rps. palustris were investigated using optical spectroscopy. The ICMs contain identical light-harvesting complex 1–reaction centers (LH1–RC) but have various spectral forms of light-harvesting complex 2 (LH2). Spectroscopic studies involving steady-state absorption, fluorescence, and femtosecond time-resolved absorption at room temperature and at 77 K focused on inter-protein excitation energy transfer. The studies investigated how energy transfer is affected by altered spectral features of the LH2 complexes as those develop under growth at different light conditions. The study shows that LH1 → LH2 excitation energy transfer is strongly affected if the LH2 complex alters its spectroscopic signature. The LH1 → LH2 excitation energy transfer rate modeled with the Förster mechanism and kinetic simulations of transient absorption of the ICMs demonstrated that the transfer rate will be 2–3 times larger for ICMs accumulating LH2 complexes with the classical B800–850 spectral signature (grown in high light) compared to the ICMs from the same strain grown in low light. For the ICMs from the ?pucBA_abce mutant, in which the B850 band of the LH2 complex is blue-shifted and almost degenerate with the B800 band, the LH1 → LH2 excitation energy transfer was not observed nor predicted by calculations.     

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides

Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (τ_f = 980 ps) for excitation densities of up to 3·10¹³ photons/(pulse·cm²), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides. Guest editor: Dr. Conrad Mullineaux.     

The architecture of Rhodobacter sphaeroides chromatophores

The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc₁ complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc₁ complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc₁ complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.     

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.     

The effect of different levels of the B800-850 light-harvesting complex on intracytoplasmic membrane development in Rhodobacter sphaeroides

A role for the peripheral (B800-850) light-harvesting complex in vesicularization of the Rhodobacter sphaeroides intracytoplasmic membrane (ICM), suggested from studies in mutant strains lacking one or more of the pigment-protein complexes, was examined further in the wild-type strain NCIB 8253 grown at high (∼1000 W m^–2), moderate (∼300 W m^–2), and low (∼100 W m^–2) light intensities. The resulting ICM vesicles (chromatophores) had B800-850 levels related inversely to irradiance and banded in rate-zone sedimentation at ∼1.10, 1.09, and 1.07 g ml^–1, respectively. Equilibrium centrifugation on iso-osmotic gradients indicated that this distinct sedimentation behavior resulted solely from differences in hydrodynamic radii. These size differences were confirmed by gel-exclusion chromatography and in electron micrographs of thin-sectioned cells. A pulse-chase study of ICM growth following a tenfold reduction in light intensity showed a relatively slow equilibration of membrane proteins during adaptation, and that new protein was incorporated largely into additional ICM formed at the lowered illumination level, giving rise to chromatophores of reduced size and elevated B800-850 content. These results provide further evidence for a model in which the B800-850 complex both drives development of vesicular ICM in Rba. sphaeroides and determines the size of resulting vesicles. Received: 12 October 1995 / Accepted: 21 December 1995