Evolution of photosynthetic reaction centers: insights from the structure of the heliobacterial reaction center

The proliferation of phototrophy within early-branching prokaryotes represented a significant step forward in metabolic evolution. All available evidence supports the hypothesis that the photosynthetic reaction center (RC)—the pigment-protein complex in which electromagnetic energy (i.e., photons of visible or near-infrared light) is converted to chemical energy usable by an organism—arose once in Earth’s history. This event took place over 3 billion years ago and the basic architecture of the RC has diversified into the distinct versions that now exist. Using our recent 2.2-Å X-ray crystal structure of the homodimeric photosynthetic RC from heliobacteria, we have performed a robust comparison of all known RC types with available structural data. These comparisons have allowed us to generate hypotheses about structural and functional aspects of the common ancestors of extant RCs and to expand upon existing evolutionary schemes. Since the heliobacterial RC is homodimeric and loosely binds (and reduces) quinones, we support the view that it retains more ancestral features than its homologs from other groups. In the evolutionary scenario we propose, the ancestral RC predating the division between Type I and Type II RCs was homodimeric, loosely bound two mobile quinones, and performed an inefficient disproportionation reaction to reduce quinone to quinol. The changes leading to the diversification into Type I and Type II RCs were separate responses to the need to optimize this reaction: the Type I lineage added a [4Fe–4S] cluster to facilitate double reduction of a quinone, while the Type II lineage heterodimerized and specialized the two cofactor branches, fixing the quinone in the Q_A site. After the Type I/II split, an ancestor to photosystem I fixed its quinone sites and then heterodimerized to bind PsaC as a new subunit, as responses to rising O₂ after the appearance of the oxygen-evolving complex in an ancestor of photosystem II. These pivotal events thus gave rise to the diversity that we observe today.     

Three-dimensional structures of photosynthetic reaction centers

In this article, the three-dimensional structures of photosynthetic reaction centers (RCs) are presented mainly on the basis of the X-ray crystal structures of the RCs from the purple bacteria Rhodopseudomonas (Rp.) viridis and Rhodobacter (Rb.) sphaeroides. In contrast to earlier comparisons and on the basis of the best-defined Rb. sphaeroides structure, a number of the reported differences between the structures cannot be confirmed. However, there are small conformational differences which might provide a basis for the explanation of observed spectral and functional discrepancies between the two species.A particular focus in this review is on the binding site of the secondary quinone (Q_B), where electron transfer is coupled to the uptake of protons from the cytoplasm. For the discussion of the Q_B site, a number of newlydetermined coordinate sets of Rp. viridis RCs modified at the Q_B site have been included. In addition, chains of ordered water molecules are found leading from the cytoplasm to the Q_B site in the best-defined structures of both Rp. viridis and Rb. sphaeroides RCs.Abbreviations B_A accessory bacteriochlorophyll in the active branch - B_B accessory bacteriochlorophyll in the inactive branch - D primary electron donor (special pair) - D_L special pair bacteriochorophyll bound by the L subunit - D_M special pair bacteriochorophyll bound by the M subunit - Q_A primary electron acceptor quinone - Q_B secondary electron acceptor quinone - RC reaction center - Rb. Rhodobacter - Rp. Rhodopseudomonas - _A bacteriopheophytin in the active branch - _B bacteriopheophytin in the inactive branch     

Structure-function investigations of bacterial photosynthetic reaction centers

During photosynthesis light energy is converted into energy of chemical bonds through a series of electron and proton transfer reactions. Over the first ultrafast steps of photosynthesis that take place in the reaction center (RC) the quantum efficiency of the light energy transduction is nearly 100%. Compared to the plant and cyanobacterial photosystems, bacterial RCs are well studied and have relatively simple structure. Therefore they represent a useful model system both for manipulating of the electron transfer parameters to study detailed mechanisms of its separate steps as well as to investigate the common principles of the photosynthetic RC structure, function, and evolution. This review is focused on the research papers devoted to chemical and genetic modifications of the RCs of purple bacteria in order to study principles and mechanisms of their functioning. Investigations of the last two decades show that the maximal rates of the electron transfer reactions in the RC depend on a number of parameters. Chemical structure of the cofactors, distances between them, their relative orientation, and interactions to each other are of great importance for this process. By means of genetic and spectral methods, it was demonstrated that RC protein is also an essential factor affecting the efficiency of the photochemical charge separation. Finally, some of conservative water molecules found in RC not only contribute to stability of the protein structure, but are directly involved in the functioning of the complex.     

Origin and evolution of photosynthetic reaction centers

The prototype reaction center may have used protoporphyrin-IX associated with small peptides to transfer electrons or protons across the primitive cell membrane. The precursor of all contemporary reaction centers contained chlorophylla molecules as both primary electron donor and initial electron acceptor and an Fe-S center as secondary acceptor (RC-1 type). The biosynthetic pathway for chlorophylla evolved along with the evolution of a better organized reaction center associated with cytochromes and quinones in a primitive cyclic electron transport system. This reaction center probably functioned initially in photoassimilation, but was easily adapted to CO₂ fixation using H₂ and H₂S as reductants. During this phase bacteriochlorophyllg may have evolved from chlorophylla in response to competition for light, and thereby initiated the gram-positive line of eubacteria. A second reaction center (RC-2) evolved from RC-1 between 3.5 and 2.5 Ga ago in response to the competition for reductants for CO₂ fixation. The new organism containing RC-2 in series with RC-1 would have been able to use poor reducing agents such as the abundant aqueous ferrous ion in place of H₂ and H₂S. This new organism is proposed to be the common ancestor of all phototrophic eubacteria except those related to the gram-positive bacteria. All organisms containing bacteriochlorophylla lost either RC-1 or RC-2, while those organisms containing chlorophylla (ancestors of cyanobacteria) added a water-splitting enzyme to RC-2 between 3.0 and 2.5 Ga ago in order to use H₂O in place of hydrated ferrous ion as electron donor for autotrophic photosynthesis.     

Structural basis of bacterial photosynthetic reaction centers

The photosynthetic reaction center (RC) is the first membrane protein whose three-dimensional structure was revealed at the atomic level by X-ray crystallograph more than fifteen years ago. Structural information about RC made a great contribution to the understanding of the reaction mechanism of the complicated membrane protein complex. High-resolution structures of RCs from three photosynthetic bacteria are now available, namely, those from two mesophilic purple non-sulfur bacteria, Blastochloris viridis and Rhodobacter sphaeroides, and that from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. In addition, a variety of structural studies, mainly by X-ray crystallography, are still being performed to give more detailed insight into the reaction mechanism of this membrane protein. This review deals with structural studies of bacterial RC complexes, and a discussion about the electron transfer reaction between RCs and electron donors is the main focus out of several topics addressed by these structural studies. The structural data from three RCs and their electron donors provided reliable models for molecular recognition in the primary step of bacterial photosynthesis.     

Two-dimensional crystals formed from photosynthetic reaction centers

Photosynthetic reaction centers from the bacterium Rhodopseudomonas viridis were prepared after detergent solubilization of photosynthetic membranes. The purified reaction centers, in agreement with reports from other laboratories, contain four distinct polypeptides ranging in molecular weight from 28,000 to 41,000. When the detergent was gradually removed by dialysis under appropriate conditions, large two- dimensional sheets of reaction centers were formed, suitable for analysis by electron microscopy. The crystals were rectangular, and the dimensions of a single unit cell were 121 X 129 A. Each unit cell contained four distinct subunits, each with approximate dimensions of 45 X 60 A. The thickness of the sheet was 60 A. Preliminary studies of the sheets with negative staining indicated that the sheets show a high degree of order: as many as six orders are visible in transforms of the images. Because of the fact that in R. viridis the native membrane from which these reaction centers were purified also displays a crystal-like structure, comparative studies between a membrane and one of its components, each analyzed by Fourier techniques, are now possible.     

Dielectric and photoelectric properties of photosynthetic reaction centers

A brief review of studies of dielectric and photoelectric properties of photosynthetic reaction centers of purple bacteria as well as photosystem I and photosystem II of cyanobacteria and higher plants is given. A simple kinetic model of the primary processes of electron transfer in photosynthesis is used to discuss possible mechanisms of correlation between rate constant of charge transfer reaction, free energy of electron transition, and effective dielectric constant in the locus of corresponding carriers.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 315–322.Original Russian Text Copyright © 2005 by Chamorovsky, Chamorovsky, Semenov.This revised version was published online in April 2005 with corrections to the post codes.     

Electron donors and acceptors in photosynthetic reaction centers

Summary A review is given of primary and associated electron transport reactions in various division of photosynthetic bacteria and in the two photosystems of plant photosynthesis. Two types of electron acceptor chains are distinguished: type Q, found in purple bacteria, Chloroflexus and system II of oxygenic photosynthesis and type F, found in green sulfur bacteria, Heliobacterium and photosystem I. Secondary donor reactions are discussed in relation to plant photosystem II.Dedicated to the memory of Warren L. Butler     

Recombination dynamics in bacterial photosynthetic reaction centers.

The time dependence of magnetic field effects on light absorption by triplet-state and radical ions in quinone-depleted reaction centers of Rhodopseudomonas sphaeroides strain R-26 has been investigated. Measurements on the time scale of the hyperfine interaction in the radical pair [(BChl)2+. ...BPh-.)] provided kinetic data characterizing the recombination process. The results have been interpreted in terms of a recently proposed model that assumes an intermediate electron acceptor (close site) between the bacteriochlorophyll "special pair" (BChl)2 and the bacteriopheophytin BPh (distant site). Recombination is assumed to proceed through this intermediate acceptor. The experiments led to effective recombination rates for the singlet and triplet channel: k(Seff) = 3.9 . 107 s-1 and k(Teff) = 7.4 . 10(8) s-1. These correspond to recombination rates ks = 1 . 10(1) s-1 and kT = 7.1 . 10(11) s-1 in the close configuration. The upper bound of the effective spin dephasing rate k2eff approximately equal to 1 . 10(9) s-1 is identical with the rate of the electron hopping between the distant site of zero spin exchange interaction and the close site of large interaction. Interpretation of data for the case of direct recombination yields the recombination rates, spin dephasing rate, and exchange interaction in a straightforward way.     

Protein fluorescence of photosynthetic reaction centers from Rhodopseudomonas sphaeroides

Luminescence emitted by tryptophan residues of reaction center (RC) preparations was studied. The RG preparations were isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides by treatment with lauryl dimethyl amine oxide (LDAO). After excitation at lambda 280 nm the quantum yield of luminescence is 0,02. It is shown that 60% of tryptophanyls are located inside the protein globule in the surrounding of relaxating polar groups and the rest approximately 40% on the outer surface of the globule--predominantly in the positively charged region of the LDAO-RC protein--in the surrounding of protein-bound water molecules. There is a correlation between the pH dependencies of the position of the peak of luminescence from tryptophanyls and effectivity of electron transfer from the primary (quinone) to secondary acceptor. The two parameters are invariant at pH from 7 to 9 and vary at pH less than 7 and pH greater than 9. The phenomena responsible for the observed correlation are discussed on the basis of pH-dependent changes in the RC protein which govern electron transport activity at the reaction center.     

Characterization of photosynthetic reaction centers with specific isotope labels

In this minireview the information at the atomic level that has been obtained by studying photosynthetic reaction centers with site-directed isotope labelling is discussed. The required isotopically labelled RCs are prepared using isotopically labelled amino acids and cofactors that have been prepared via organic total synthetic chemistry. In some cases the results of isotopically labelled RCs that are prepared via other methods are also discussed.     

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.     

Electrostatic interaction between redox cofactors in photosynthetic reaction centers

Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921-3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them.     

Photochemistry and electron transfer in borohydride-treated photosynthetic reaction centers

The photochemistry and electron-transfer activities of sodium-borohydride-treated reaction centers from the purple photosynthetic bacterium Rhodopseudomonas sphaeroides R26 have been investigated by both milliand picosecond absorption techniques. Separation from the treated reaction center of the reduction product, apparently a reduced form of one of the two molecules of bacteriochlorophyll contributing to the 800 nm ground-state absorption band, is also reported. In the near-infrared region, differences between treated and untreated reaction centers are observed in both milli- and picosecond light-induced difference spectra. However, borohydride-treated reaction centers exhibit photochemistry and electron transfer which are indistinguishable from those observed in untreated reaction centers. These results indicate that normal activity occurs in reaction centers that contain both molecules of bacteriopheophytin, but only three of the usual four molecules of bacteriochlorophyll.     

Localization of light-induced structural changes in bacterial photosynthetic reaction centers

Light-induced structural changes in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated using two approaches. Cu²⁺ was used as a paramagnetic structural probe. The EPR spectrum of Cu²⁺ incorporated into the metal-depleted reaction centers was affected by 1,10-phenanthroline, an electron transfer inhibitor substituting Q_B, which suggests a localization of Cu²⁺ in a vicinity of the Q _B ^– site. However, the spectrum was not influenced by low temperature (77 K) illumination of the sample which suggests that the copper ion position is not exactly the same as that of the iron ion. Freezing the reaction centers under illumination in the presence of potassium ferricyanide and 1,10-phenanthroline caused a change in the shape of the Cu²⁺ EPR spectrum in comparison to that of a sample frozen in darkness. These data indicate a change of the Cu²⁺ ligand symmetry owing to light-induced structural changes which are probably located near the acceptor side of the reaction center. Partial trypsinolysis of reaction centers was also used to locate the structural changes. Trypsin treatment in the dark and under illumination resulted in different peptide patterns as detected by gel electrophoresis and reverse-phase high-performance liquid chromatography. Partial amino-acid sequence analysis of a number of peptides, characteristic of either light- or dark-treated reaction centers, showed that they originated from the acceptor sides of the H and M subunits. The occurrence of light-induced structural differences in the H-subunit is consistent with the suggestion that it may be involved in regulating electron transfer in this part of the reaction center.     

Excited states of photosynthetic reaction centers at low redox potentials

In preparations of photochemical reaction centers from Rhodopseudomonas spheroides R-26, lowering the redox potential so as to reduce the primary electron acceptor prevents the photochemical transfer of an electron from bacteriochlorophyll to the acceptor. Measuring absorbance changes under these conditions, we found that a 20-ns actinic flash converts the reaction center to a new state, P^F, which then decays with a half-time that is between 1 and 10 ns at 295 °K. At 25 °K, the decay half-time is approx. 20 ns. The quantum yield of state P^F appears to be near 1.0, both at 295 and at 15 °K. State P^F could be an intermediate in the photochemical electron-transfer reaction which occurs when the acceptor is in the oxidized form.Following the decay of state P^F, we detected another state, P^R, with a decay half-time of 6 μs at 295 °K and 120 μs at 15 °K. The quantum yield of state P^R is approx. 0.1 at 295 °K, but rises to a value nearer 1.0 at 15 °K. The kinetics and quantum yields are consistent with the view that state P^R forms from P^F. State P^R seems likely to be a side-product, rather than an intermediate in the electron-transfer process.The decay kinetics indicate that state P^F cannot be identical with the lowest excited singlet state of the reaction center. One of the two states, P^F or P^R, probably is the lowest excited triplet state of the reaction center, but it remains unclear which one.     

Structural aspects of vectorial electron transfer in photosynthetic reaction centers

Structural aspects of photosynthetic reaction centers in bacteria and plants are discussed in relation with the ability of these structures to perform a photoinduced electron transfer from one side of the membrane to the other. A comparison is made with recently synthesized artificial models. Functional similarities between the acceptor sides of bacterial and of Photosystem-II centers are utilized to hypothesize on their structure.This review corresponds to a lecture delivered at the 3rd European Bioenergetics Conference, Hannover, September 1984.