The photosynthetic reaction centre from the purple bacteriumRhodopseudomonas viridis

We first describe the history and methods of membrane protein crystallization, and show how the structure of the photosynthetic reaction centre from the purple bacteriumRhodopseudomonas viridis was solved. The structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure.Published inLes Prix Nobel—The Nobel Prizes 1988 (Nobel Foundation, Stockholm, 1989) and republished here with the permission of the Nobel Foundation the copyright holders.     

Research on photosynthetic reaction centers from 1932 to 1987

The history of research on photosynthetic reaction centers is outlined, starting with the implication of their existence through the discovery of the photosynthetic unit, as reported by R. Emerson and W. Arnold in 1932, and culminating in the crystallization and X-ray analysis of the anoxygenic bacterial reaction centers, reported by J. Deisenhofer, H. Michel, and coworkers, over the period 1982–1987. Reaction centers of purple photosynthetic bacteria have received the most attention because they have been well purified and characterized. Structures of cyanobacterial reaction centers of Photosystems I and II are now available from the laboratories of H. Witt and W. Saenger. This revised version was published online in June 2006 with corrections to the Cover Date.     

Investigation of ubiquinol formation in isolated photosynthetic reaction centers by rapid-scan Fourier transform IR spectroscopy

Light-induced formation of ubiquinol-10 in Rhodobacter sphaeroides reaction centers was followed by rapid-scan Fourier transform IR difference spectroscopy, a technique that allows the course of the reaction to be monitored, providing simultaneously information on the redox states of cofactors and on protein response. The spectrum recorded between 4 and 29 ms after the second flash showed bands at 1,470 and 1,707 cm(-1), possibly due to a QH(-) intermediate state. Spectra recorded at longer delay times showed a different shape, with bands at 1,388 (+) and 1,433 (+) cm(-1) characteristic of ubiquinol. These spectra reflect the location of the ubiquinol molecule outside the Q(B) binding site. This was confirmed by Fourier transform IR difference spectra recorded during and after continuous illumination in the presence of an excess of exogenous ubiquinone molecules, which revealed the process of ubiquinol formation, of ubiquinone/ubiquinol exchange at the Q(B) site and between detergent micelles, and of Q(B)(-) and QH(2) reoxidation by external redox mediators. Kinetics analysis of the IR bands allowed us to estimate the ubiquinone/ubiquinol exchange rate between detergent micelles to approximately 1 s. The reoxidation rate of Q(B)(-) by external donors was found to be much lower than that of QH(2), most probably reflecting a stabilizing/protecting effect of the protein for the semiquinone form. A transient band at 1,707 cm(-1) observed in the first scan (4-29 ms) after both the first and the second flash possibly reflects transient protonation of the side chain of a carboxylic amino acid involved in proton transfer from the cytoplasm towards the Q(B) site.     

Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate

Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I _exp, obeys a simple exponential law with the rate constant , in which α is a parameter relating the light intensity, measured in mW/cm², to a corresponding theoretical rate in units of reciprocal seconds, and k _rec is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the α parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer–Lambert–Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation. An erratum to this article can be found at     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

The Photosynthetic Reaction Centre from the Purple Bacterium Rhodopseudomonas viridis

We first describe the history and methods of membrane protein crystallization, and show how the structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis was solved. The structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure.Published in Les Prix Nobel–The Nobel Prizes 1988 (Nobel Foundation, Stockholm, 1989) and republished here with the permission of the Nobel Foundation the copyright holders.     

Proton transfer in the photosynthetic reaction center of Blastochloris viridis

Photosynthetic reaction centers of Blastochloris viridis require two quanta of light to catalyse a two-step reduction of their secondary ubiquinone Q(B) to ubiquinol. We employed capacitive potentiometry to follow the voltage changes that were caused by the accompanying transmembrane proton displacements. At pH 7.5 and 20 degrees C, the Q(B)-related voltage generation after the first flash was contributed by a fast, temperature-independent component with a time constant of approximately 30 micros and a slower component of approximately 200 micros with activation energy (E(a)) of 50 kJ/mol. The kinetics after the second flash featured temperature-independent components of 5 micros and 200 micros followed by a component of 600 micros with E(a) approximately 60 kJ/mol.     

Oxidation of cytochrome c 2 by photosynthetic reaction centers of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in vivo. Effect of viscosity on the rate of reaction

In Rhodospirillum rubrum and Rhodopseudomonas sphaeroides it is shown that the oxidation of cytochrome c ₂ involves a diffusion limited process. From analysis of the results it follows that the electron transfer probability must be very low. This is corroborated by in vitro studies using the isolated components.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural analysis of photosynthetic membranes by cryo-electron tomography of intact Rhodopseudomonas viridis cells

During the photosynthetic process, highly organized membranal assemblies convert light into biochemical energy with high efficiency. We have used whole-mount cryo-electron tomography to study the intracellular architecture of the photosynthetic membranes of the anaerobic purple photosynthetic bacterium Rhodopseudomonas viridis, as well as the organization of the photosynthetic units within the membranes. Three-dimensional reconstruction demonstrates a continuity of the plasma membrane with the photosynthetic membranes that form tunnel-like structures with an average diameter of 31 nm ± 8 nm at the connection sites. The spacing between the photosynthetic membranes at their cytoplasmic faces was found to be 11 nm, thus enforcing a highly close packaging of the photosynthetic membranes. Analysis of successive tomographic slices allowed for derivation of the spacing between adjacent photosynthetic core complexes from a single-layered photosynthetic membrane, in situ. This analysis suggests that most, if not all, photosynthetic membranes in R. viridis are characterized by a similar two-dimensional hexagonal lattice organization.     

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800–B850 gap from 750 cm^\s-1 at 0.1 MPa to 900 cm^-1 at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800–850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800–B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C₉-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (-0.4 cm⁰¹/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm⁰¹/MPa). The shift rate for B850 at 4.2 K is-0.28 cm^–1/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.Abbreviations B800 BChl antenna band absorbing (at room temperature) at 800 nm (B850, B875, B1015 are defined similarly) - CD circular dichroism - FC factor Franck-Condon factor - FMO comple Fenna-Matthews-Olson complex - L-S theory Laird-Skinner theory - LH1 core light-harvesting complex of the BChl antenna complexes - LH2 peripheral light-harvesting complex of the BChl antenna complexes - NPHB non-photochemical hole burning - P960 absorption band of special pair of Rhodopseudomonas viridis absorbing at 960 nm (room temperature). P870 of Rhodobacter sphaeroides is defined similarly - QM/MM results quantum mechanical/molecular mechanical results - RC reaction center - ZPH zero phonon hole     

A comparison of stigmatellin conformations, free and bound to the photosynthetic reaction center and the cytochrome bc1 complex

We describe in detail the conformations of the inhibitor stigmatellin in its free form and bound to the ubiquinone-reducing (Q(B)) site of the reaction center and to the ubiquinol-oxidizing (Q(o)) site of the cytochrome bc(1) complex. We present here the first structures of a stereochemically correct stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the stigmatellin side-chain relative to the chromone head group, and on the interaction of the stigmatellin side-chain with these membrane protein complexes. The different conformations of stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the chi1 dihedral angle is an essential factor for allowing stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.     

Comparative Analyses of Three-Dimensional Models of Bacterial Reaction Centers

Despite the fact that the three-dimensional structure of an integral membrane protein was first determined 20 years ago, structures have been solved for very few membrane proteins. The reaction center is an exception with many mutant and modified structures available from 3 different bacterial species. In order to relate these structures to the function of the reaction center, an accurate assessment of the reliability of the structural models is required. Here we describe the quality of the structures of the bacterial reaction center based upon different criteria, such as evaluation of the geometry of the models and comparison of different models. Overall, the structures are found to be most accurate in the membrane-embedded regions with the periplasmic and cytoplasmic exposed regions having more disorder and differences among the structural models. In general, the cofactors and the surrounding protein regions are among the most accurately determined regions of the protein, except for the secondary quinone and its binding pocket that shows a large variation among structures. The limited accuracy of the secondary quinone is due to its partial occupancy as a consequence of its functional role and to the presence of surface features, including lipids and detergent molecules.     

Quantification of non-Q B -reducing centers in leaves using a far-red pre-illumination

An alternative approach to quantification of the contribution of non-Q_B-reducing centers to Chl a fluorescence induction curve is proposed. The experimental protocol consists of a far-red pre-illumination followed by a strong red pulse to determine the fluorescence rise kinetics. The far-red pre-illumination induces an increase in the initial fluorescence level (F_{25 μs}) that saturates at low light intensities indicating that no light intensity-dependent accumulation of Q_A⁻ occurs. Far-red light-dose response curves for the F_{25 μs}-increase give no indication of superimposed period-4 oscillations. F_{25 μs}-dark-adaptation kinetics following a far-red pre-pulse, reveal two components: a faster one with a half-time of a few seconds and a slower component with a half-time of around 100 s. The faster phase is due to the non-Q_B-reducing centers that re-open by recombination between Q_A⁻ and the S-states on the donor side. The slower phase is due to the recombination between Q_B⁻ and the donor side in active PS II reaction centers. The pre-illumination-induced increase of the F_{25 μs}-level represents about 4–5% of the variable fluorescence for pea leaves (∼2.5% equilibrium effect and 1.8–3.0% non-Q_B-reducing centers). For the other plant species tested these values were very similar. The implications of these values will be discussed.     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Acquisition of photosynthetic capacity by a reaction centre that lacks the QA ubiquinone; possible insights into the evolution of reaction centres?

A photosynthetically impaired strain of Rhodobacter sphaeroides containing reaction centres with an alanine to tryptophan mutation at residue 260 of the M-polypeptide (AM260W) was incubated under photosynthetic growth conditions. This incubation produced photosynthetically competent strains containing suppressor mutations that changed residue M260 to glycine or cysteine. Spectroscopic analysis demonstrated that the loss of the Q_A ubiquinone seen in the original AM260W mutant was reversed in the suppressor mutants. In the mutant where Trp M260 was replaced by Cys, the rate of reduction of the Q_A ubiquinone by the adjacent (H_A) bacteriopheophytin was reduced by three-fold. The findings of the experiment are discussed in light of the X-ray crystal structures of the wild-type and AM260W reaction centres, and the possible implications for the evolution of reaction centres as bioenergetic complexes are considered.     

Microscopic simulation of quantum dynamics and nuclear tunneling in bacterial reaction centers

The dispersed polaron version of the semiclassical trajectory approach is used to evaluate the quantum mechanical nuclear tunneling effects in the charge recombination reaction, P⁺Q^–PQ, in photosynthetic bacterial reaction centers, The cclculations are based on the crystallographic structure of reaction centers from Rhodopseudomonas viridis. They succeed in capturing the temperature dependence of the rate constant without using adjustable parameters. This provides the first example of a microscopic simulation of quantum mechanical nuclear tunneling in a biological system.Abbreviations P bacteriochlorophyll dimer - Q ubiquinone in Rhodobacter sphaeroides, menaquinone in Rhodopseudomonas viridis - Rps. Rhodopseudomonas - Rb. Rhodobacter - QCFF/PI quantum mechanical extension of the consistent force field to -electron     

Localisation of reaction centre and light harvesting complexes in the photosynthetic unit of Rhodopseudomonas viridis

The photosynthetic unit of Rhodopseudomonas viridis contains a reaction centre (P960) and a light harvesting complex (B1015). Immune electron microscopy combined with image processing has allowed the central core of the photosynthetic unit to be identified as the reaction centre and the surrounding protein ring as the light harvesting complex. This light harvesting complex, subdivided into twelve subunits was shown to contain 24 bacteriochlorophyll b molecules. A model is presented which may account for the far red shift of the Qy absorption of the bacteriochlorophyll b molecules in vivo.     

Structure and function of the photosynthetic reaction center fromRhodobacter sphaeroides

The three-dimensional structure of the photosynthetic reaction center fromRhodobacter sphaeroides is described. The reaction center is a transmembrane protein that converts light into chemical energy. The protein has three subunits: L, M, and H. The mostly helical L and M subunits provide the scaffolding and the finely tuned environment in which the chromophores carry out electron transfer. The details of the protein-chromophore interactions are from studies of a trigonal crystal form that diffracted to 2.65-Å resolution. Functional studies of the multi-subunit complex by site-specific replacement of key amino acid residues are summarized in the context of the molecular structure.This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38 and by Public Health Service Grant GM36598.     

Theoretical studies on the mechanism of primary electron transfer in the photosynthetic reaction center of Rhodobacter sphaeroides

The mechanism of the primary electron transfer (ET) process in the photosynthetic reaction center (PRC) of Rhodobacter sphaeroides has been studied with quantum chemistry method of ab initio density functional theory (DFT) (B3LYP/6-31G) based on the optimized X-ray crystallographic structure. The calculation was carried out on different structural levels. The electronic structure of pigment molecules was first studied, and then the influence of the neighboring protein was taken into account at three approximation levels: (a) the surrounding proteins were treated as a homogeneous medium with a uniform dielectric constant (SCRF); (b) both the influence of axial coordination of His to the special pair P and ABChl as, and the hydrogen bonds between related residues and P and also BPhas were included; and (c) the influence of the electronic structure of the protein subunit chains as a whole was studied. The results suggest that: (1) according to the composition of the HOMO and LUMO of P, there might be a charge-separated state of (BChl_L ⁺BChl_M ⁻) for the excited state of P; (2) to treat the protein surroundings as a homogeneous medium is not sufficient. Different interactions between pigment molecules and related residues play different roles in the ET process; (3) the axial coordination of His to P raises the E _LUMO of P greatly, and it is very important for the ET process to occur in the PRC of wild-type bacterium; the axial coordination of His to ABChl as also raises their E _LUMO significantly; (4) the hydrogen-bonds between amino acid residues and P and also BPh as depress the E _LUMO of the pigment molecules to some extent, which makes the E _LUMO of P lower than those of ABChlas, and the E _LUMO of BPh a _L lower than that of BPh a _M. Consequently, the ET process from P to BPh a _L does not, according to our calculation model, occur via ABChl a _L. The possibility of the ET pathway from P to BPh a _L via ABChl a _L was discussed; (5) the frontier orbitals of protein subunit chains L and M are localized at the random coil area and the α–helix areas, respectively. Results mentioned above support the fact that the ET process proceeds in favourable circumstances along the branch L. This revised version was published online in June 2006 with corrections to the Cover Date.