Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid.

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

Light harvesting by lamellar chromatophores in Rhodospirillum photometricum.

Purple photosynthetic bacteria harvest light using pigment-protein complexes which are often arranged in pseudo-organelles called chromatophores. A model of a chromatophore from Rhodospirillum photometricum was constructed based on atomic force microscopy data. Molecular-dynamics simulations and quantum-dynamics calculations were performed to characterize the intercomplex excitation transfer network and explore the interplay between close-packing and light-harvesting efficiency.

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions.

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum.

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.

Large-Scale Molecular Dynamics Simulations of Cellular Compartments.

Molecular dynamics or MD simulation is gradually maturing into a tool for constructing in vivo models of living cells in atomistic details. The feasibility of such models is bolstered by integrating the simulations with data from microscopic, tomographic and spectroscopic experiments on exascale supercomputers, facilitated by the use of deep learning technologies. Over time, MD simulation has evolved from tens of thousands of atoms to over 100 million atoms comprising an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium. In this chapter, we present a step-by-step outline for preparing, executing and analyzing such large-scale MD simulations of biological systems that are essential to life processes. All scripts are provided via GitHub.

Photosynthetic vesicle architecture and constraints on efficient energy harvesting.

Photosynthetic chromatophore vesicles found in some purple bacteria constitute one of the simplest light-harvesting systems in nature. The overall architecture of chromatophore vesicles and the structural integration of vesicle function remain poorly understood despite structural information being available on individual constituent proteins. An all-atom structural model for an entire chromatophore vesicle is presented, which improves upon earlier models by taking into account the stoichiometry of core and antenna complexes determined by the absorption spectrum of intact vesicles in Rhodobacter sphaeroides, as well as the well-established curvature-inducing properties of the dimeric core complex. The absorption spectrum of low-light-adapted vesicles is shown to correspond to a light-harvesting-complex 2 to reaction center ratio of 3:1. A structural model for a vesicle consistent with this stoichiometry is developed and used in the computation of excitonic properties. Considered also is the packing density of antenna and core complexes that is high enough for efficient energy transfer and low enough for quinone diffusion from reaction centers to cytochrome bc(1) complexes.

Study of effect of molecular mobility in chromatophore membranes of the bacterium E. shaposhnikovii on processes of photoinduced electron transport using the NMR-spin-echo method with isotope substitution and dehydration.

The effect of dehydration and (2)H2O/H2O isotope substitution on electron transport reactions and relaxation of proton-containing groups was studied in chromatophore membranes of Ectothiorhodospira shaposhnikovii. During dehydration (including isotope substitution of hydrate water) of preliminarily dehydrated isolated photosynthetic membranes there was a partial correlation between hydration intervals within which activation of electron transport from high-potential cytochrome c to photoactive bacteriochlorophyll dimer P890 of photosynthetic reaction center and variation of spin-lattice and spin-spin proton relaxation time was observed. Partial correlation between hydration intervals can be considered as evidence of correlation between mobility of non-water proton-containing groups with proton relaxation frequency approximately 10(8) sec(-1) with efficiency of electron transfer at the donor side of the chain.

Phospholipid distributions in purple phototrophic bacteria and LH1-RC core complexes.

In contrast to plants, algae and cyanobacteria that contain glycolipids as the major lipid components in their photosynthetic membranes, phospholipids are the dominant lipids in the membranes of anoxygenic purple phototrophic bacteria. Although the phospholipid compositions in whole cells or membranes are known for a limited number of the purple bacteria, little is known about the phospholipids associated with individual photosynthetic complexes. In this study, we investigated the phospholipid distributions in both membranes and the light-harvesting 1-reaction center (LH1-RC) complexes purified from several purple sulfur and nonsulfur bacteria. 31P NMR was used for determining the phospholipid compositions and inductively coupled plasma atomic emission spectroscopy was used for measuring the total phosphorous contents. Combining these two techniques, we could determine the numbers of specific phospholipids in the purified LH1-RC complexes. A total of approximate 20-30 phospholipids per LH1-RC were detected as the tightly bound lipids in all species. The results revealed that while cardiolipin (CL) exists as a minor component in the membranes, it became the most abundant phospholipid in the purified core complexes and the sum of CL and phosphatidylglycerol accounted for more than two thirds of the total phospholipids for most species. Preferential association of these anionic phospholipids with the LH1-RC is discussed in the context of the recent high-resolution structure of this complex from Thermochromatium (Tch.) tepidum. The detergent lauryldimethylamine N-oxide was demonstrated to selectively remove phosphatidylethanolamine from the membrane of Tch. tepidum.

Intrinsic curvature properties of photosynthetic proteins in chromatophores.

In purple bacteria, photosynthesis is carried out on large indentations of the bacterial plasma membrane termed chromatophores. Acting as primitive organelles, chromatophores are densely packed with the membrane proteins necessary for photosynthesis, including light harvesting complexes LH1 and LH2, reaction center (RC), and cytochrome bc(1). The shape of chromatophores is primarily dependent on species, and is typically spherical or flat. How these shapes arise from the protein-protein and protein-membrane interactions is still unknown. Now, using molecular dynamics simulations, we have observed the dynamic curvature of membranes caused by proteins in the chromatophore. A membrane-embedded array of LH2s was found to relax to a curved state, both for LH2 from Rps. acidophila and a homology-modeled LH2 from Rb. sphaeroides. A modeled LH1-RC-PufX dimer was found to develop a bend at the dimerizing interface resulting in a curved shape as well. In contrast, the bc(1) complex, which has not been imaged yet in native chromatophores, did not induce a preferred membrane curvature in simulation. Based on these results, a model for how the different photosynthetic proteins influence chromatophore shape is presented.

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: size dispersion and ATP synthase distribution.

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.

AFM studies of the supramolecular assembly of bacterial photosynthetic core-complexes.

The atomic force microscope (AFM) allows visualization of the assembly and molecular interactions of single proteins. Most recently, AFM images of bacterial membranes have revealed details of the supramolecular architecture of bacterial photosynthetic apparatus in different species. The near-native experimental conditions used in AFM imaging reduce artefacts and make AFM ideal for studying native conformations. High-resolution AFM of native membranes has revealed variation in core-complex architectures amongst species.

The 17-propionate function of (bacterio)chlorophylls: biological implication of their long esterifying chains in photosynthetic systems.

Molecular structures of (bacterio)chlorophylls [= (B)Chls] in photosynthetic apparatus are surveyed, and a diversity of the ester groups of the 17-propionate substituent is particularly focused on in this review. In oxygenic photosynthetic species including green plants and algae, the ester of Chl molecules is limited to a phytyl group. Geranylgeranyl and farnesyl groups in addition to phytyl are observed in (B)Chl molecules inside photosynthetic proteins of anoxygenic bacteria. In main light-harvesting antennas of green bacteria (chlorosomes), a greater variety of ester groups including long straight chains are used in the composite BChl molecules. This diversity is ascribable to the fact that chlorosomal BChls self-aggregate to form a core part of chlorosomes without any specific interaction of oligopeptides. Biological significance of the long chains is discussed in photosynthetic apparatus, especially in chlorosomes.

Determination of Cell Doubling Times from the Return-on-Investment Time of Photosynthetic Vesicles Based on Atomic Detail Structural Models.

Cell doubling times of the purple bacterium Rhodobacter sphaeroides during photosynthetic growth are determined experimentally and computationally as a function of illumination. For this purpose, energy conversion processes in an intracytoplasmic membrane vesicle, the chromatophore, are described based on an atomic detail structural model. The cell doubling time and its illumination dependence are computed in terms of the return-on-investment (ROI) time of the chromatophore, determined computationally from the ATP production rate, and the mass ratio of chromatophores in the cell, determined experimentally from whole cell absorbance spectra. The ROI time is defined as the time it takes to produce enough ATP to pay for the construction of another chromatophore. The ROI time of the low light-growth chromatophore is 4.5-2.6 h for a typical illumination range of 10-100 µmol photons m-2 s-1, respectively, with corresponding cell doubling times of 8.2-3.9 h. When energy expenditure is considered as a currency, the benefit-to-cost ratio computed for the chromatophore as an energy harvesting device is 2-8 times greater than for photovoltaic and fossil fuel-based energy solutions and the corresponding ROI times are approximately 3-4 orders of magnitude shorter for the chromatophore than for synthetic systems.

Transmission electron microscopic study on supramolecular nanostructures of bacteriochlorophyll self-aggregates in chlorosomes of green photosynthetic bacteria.

Supramolecular nanostructures of bacteriochlorophyll (BChl) self-aggregates in major light-harvesting complexes (chlorosomes) of green photosynthetic bacteria were successfully observed by freeze-fracture transmission electron microscope. Rod-shaped nanostructures with approximately 10 nm in diameter could be visualized in three types of green sulfur bacteria (Chlorobium). Diameter of the rod-shaped nanostructures in Chlorobium chlorosomes was independent of the molecular structures of their light-harvesting pigments, namely BChl-c or d. In contrast, chlorosomes of the green filamentous bacterium Chloroflexus aurantiacus had rod-shaped nanostructures with approximately 5 nm in diameter. The present results support that BChl self-aggregates in chlorosomes form rod-shaped nanostructures called rod-elements with approximately 10- and 5-nm diameters for Chlorobium and Chloroflexus, respectively.

Computational Methodologies for Real-Space Structural Refinement of Large Macromolecular Complexes.

The rise of the computer as a powerful tool for model building and refinement has revolutionized the field of structure determination for large biomolecular systems. Despite the wide availability of robust experimental methods capable of resolving structural details across a range of spatiotemporal resolutions, computational hybrid methods have the unique ability to integrate the diverse data from multimodal techniques such as X-ray crystallography and electron microscopy into consistent, fully atomistic structures. Here, commonly employed strategies for computational real-space structural refinement are reviewed, and their specific applications are illustrated for several large macromolecular complexes: ribosome, virus capsids, chemosensory array, and photosynthetic chromatophore. The increasingly important role of computational methods in large-scale structural refinement, along with current and future challenges, is discussed.

Spectral identification of the electrochromically active carotenoids of Rhodobacter sphaeroides in chromatophores and reconstituted liposomes.

Reaction centers with both light harvesting complexes I and II (B875 and B800/850; i.e., RCLHILHII complexes) have been isolated from Rhodobacter sphaeroides. These complexes have been incorporated into liposomes made from lipids purified from Escherichia coli. The electrochromic bandshift of carotenoids, present in these reconstituted complexes, shows shifted minima and maxima with respect to a similar spectrum in chromatophores of Rb. sphaeroides in a potassium diffusion potential induced difference spectrum (see also Crielaard, W., Hellingwerf, K.J. and Konings, W.N. (1989) Biochim. Biophys. Acta 973, 205-211). The absorbance spectrum, at room temperature or at 77 K, of both membrane preparations did not, however, reveal differences in the carotenoid region. The long-wavelength carotenoid peak in both preparations is located at 513 nm (77 K). A small difference could be observed between the 77 K excitation spectra of the B850 fluorescence. Reconstituted complexes show a carotenoid peak at 513 nm, whereas in chromatophores this peak is located at 514.5 nm. When fluorescence was recorded at 805 nm, to detect B800 excitation, there was a marked difference between both preparations. In liposomes the long wavelength B800-associated carotenoid peak is located at 512.5 nm, whereas in chromatophores this peak is located at 516 nm. These results explain the shifted minima and maxima in a potassium diffusion induced difference spectrum in proteoliposomes. The prediction of two carotenoid pools in chromatophores (De Grooth, B.G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 247-258) is confirmed, and the field sensitive carotenoids are identified as the pool that is associated with the B800 band (Kramer, H.J.M., Van Grondelle, R., Hunter, C.N., Westerhuis, W.H.J. and Amesz, J. (1984) Biochim. Biophys. Acta 765, 156-165).

Escape probability and trapping mechanism in purple bacteria: revisited.

Despite intensive research for decades, the trapping mechanism in the core complex of purple bacteria is still under discussion. In this article, it is attempted to derive a conceptionally simple model that is consistent with all basic experimental observations and that allows definite conclusions on the trapping mechanism. Some experimental data reported in the literature are conflicting or incomplete. Therefore we repeated two already published experiments like the time-resolved fluorescence decay in LH1-only purple bacteria Rhodospirillum rubrum and Rhodopseudomonas viridis chromatophores with open and closed (Q(A)(-)) reaction centers. Furthermore, we measured fluorescence excitation spectra for both species under the two redox-conditions. These data, all measured at room temperature, were analyzed by a target analysis based on a three-state model (antenna, primary donor, and radical pair). All states were allowed to react reversibly and their decay channels were taken into consideration. This leads to seven rate constants to be determined. It turns out that a unique set of numerical values of these rate constants can be found, when further experimental constraints are met simultaneously, i.e. the ratio of the fluorescence yields in the open and closed (Q(A)(-)) states F(m)/F(o) approximately 2 and the P(+)H(-)-recombination kinetics of 3-6 ns. The model allows to define and to quantify escape probabilities and the transfer equilibrium. We conclude that trapping in LH1-only purple bacteria is largely transfer-to-the-trap-limited. Furthermore, the model predicts properties of the reaction center (RC) in its native LH1-environment. Within the framework of our model, the predicted P(+)H(-)-recombination kinetics are nearly indistinguishable for a hypothetically isolated RC and an antenna-RC complex, which is in contrast to published experimental data for physically isolated RCs. Therefore RC preparations may display modified kinetic properties.

Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg-->Ile substitution at sites 207 and 217 in the L-subunit.

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation.

The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc1 complex (bc1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b(h) (in the absence of Zn2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c(1) reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ <100 microM the reduction of heme b(h) remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b(h) is the main electrogenic reaction in the cytochrome bc1 complex, (2) imply an allosteric link between the site of heme b(h) oxidation and the site of cytochrome c1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc1 complex.

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry?

The basic photosynthetic unit containing the reaction centre and the light-harvesting I complex (RC-LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resolution of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was observed also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational analysis developed here showed that the reaction centres take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit.