Mechanisms of excitation trapping in reaction centers of purple bacteria

The contradiction between two groups of experimental data, which fails to be resolved within the framework of the widely accepted model of excitation migration and trapping (at least in case of purple bacteria), is discussed in the introduction to this review. Three directions of studies intended to resolve this conflict are reviewed in the three further sections: II. Exciton models; III. Water-polarization (water-latch) mechanism of excitation trapping; IV. Quantum-mechanical models. The maximum efficiency of these models in resolving the contradiction mentioned above was assessed. The advantages and disadvantages of the mechanisms described in sections II, III, and IV are discussed in the last section of this review. It is concluded that none of these mechanisms taken alone is able to solve this problem. Therefore, the fundamental problem of the primary excitation conversion in reaction centers remains unsolved and requires additional experimental research.     

Excitation energy trapping in anoxygenic photosynthetic bacteria*

Various aspects of excitation energy conversion in anoxygenic photosynthetic bacteria are surveyed. This minireview discusses different models that have been proposed during the past 60 years to describe excitation energy transfer from an antenna molecule to the reaction center. First, a simple one-dimensional model was suggested, but over time the models became more detailed when structural and dynamic information was included. This review focuses mainly on the picture of purple bacteria and green sulfur bacteria developed during the past decades. This revised version was published online in June 2006 with corrections to the Cover Date.     

Coupling of exciton motion in the core antenna and primary charge separation in the reaction center

The relation between exciton motion in the LH1 antenna and primary charge separation in the reaction center of purple bacteria is briefly reviewed. It is argued that in models based on hopping excitons described strictly by Förster theory, transfer-to-trap-limited kinetics is quite unlikely according to the relation between the exciton trapping kinetics and N, the size of the photosynthetic unit in such models. Because the results of several recent experiments have been interpreted in terms of transfer-to-trap limited kinetics, this presents a conflict between these experimental interpretations and strictly Förster-based theoretical models. Two possible resolutions are proposed. One arises from the random phase-redistribution trapping kinetics of partially coherent excitons, a kinetics uniquely independent of both N and the rate constant for primary charge separation in the reaction center. The other comes from multiple-pathways models of the multipicosecond nonexponentiality of the decay of P^*, the electronically excited primary electron donor in the reaction center. In these models, because it depends only on a certain averaged electron-transfer time constant, the exciton lifetime may be relatively insensivive to variations of individual electrontransfer rate constants-thereby undercutting the argument appearing in recent literature that by default the exciton kinetics must be transfer-to-trap limited.     

A new approach to experimental investigation of dynamic self-organization in reaction centers of purple bacteria

The results of a study of molecular self-organization processes in the reaction centers (RC) ofRb. Sphaeroides purple bacteria by the method of pulsed optical excitation is presented. The existence of a bistability domain for the parameters of RC recovery kinetics is shown. A good agreement between the theory and experimental results is obtained.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Comparative analysis of the primary structure of the reaction center-bound cytochrome subunit in purple bacteria

The amino acid sequences of the reaction center-bound cytochrome subunit of six species of purple bacteria were compared. Amino acid residues thought to be important in controlling the redox midpoint potentials of four hemes in Blastochloris (Rhodopseudomonas) viridis were found to be well conserved. As opposed to all other species studied, the amino acid sequence of the cytochrome subunit of B. viridis had several insertions of more than 10 residues at specific regions close to the LM core, suggesting that interaction of the cytochrome subunit with the LM core in most species is different from that in B. viridis. Distribution of charged amino acid residues on the surface of the cytochrome subunit was compared among six species and discussed from the viewpoint of interaction with soluble electron donors.     

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     

Redox interaction of Mn-bicarbonate complexes with reaction centres of purple bacteria

It is found that dark reduction of photooxidized primary electron donor P870+ in reaction centres from purple anoxygenic bacteria (two non-sulphur Fe-oxidizing Rhodovulum iodosum and Rhodovulum robiginosum, Rhodobacter sphaeroides R-26 and sulphur alkaliphilic Thiorhodospira sibirica) is accelerated upon the addition of Mn2+ jointly with bicarbonate (30-75 mM). The effect is not observed if Mn2+ and HCO3(-) have been replaced by Mg2+ and HCO2(-), respectively. The dependence of the effect on bicarbonate concentration suggests that formation of Mn2+-bicarbonate complexes, Mn(HCO3)+ and/or Mn(HCO3)2, is required for re-reduction of P870+ with Mn2+. The results are considered as experimental evidence for a hypothesis on possible participation of Mn-bicarbonate complexes in the evolutionary origin of oxygenic photosynthesis in the Archean era.     

Experimental evidence of dynamic self-organization in the electron transfer system (example of reaction centers of purple bacteria)

The results of an experimental study of nonlinear dynamic processes in the electron transfer system, the reaction centers (RCs) of purple bacteria are presented. A difference was observed in the absorption spectra of RCs exposed to a rising intensity of acting light compared to a descending intensity of acting light. We observed the hysteresis of the RC optical transmission coefficient at =865 nm, with a quasistationary increase and subsequent decrease of the optical excitation level. The kinetics of charge recombination in an RC containing two quinone acceptors revealed a dependence on the prehistory of the RC illumination. The results were interpreted in terms of the existence of a light-induced memory effect in the electron-conformational system and the appearance of bifurcation in the system at critical values of the photoinduced electron flux through the macromolecule.     

Recombination reduction of photooxidized cytochrome c in reaction centers of Rhodopseudomonas viridis at low temperature

The temperature dependence of dark reduction of photooxidized cytochrome c was studied in isolated preparations of Rhodopseudomonas viridis reaction centers. Within the range from room temperature to 260 K this process was found to be mediated by thermal diffusion of exogenous donor molecules, whereas at lower temperatures photooxidized cytochrome is reduced as a result of indirect recombination with photoreduced primary quinone acceptor. Kinetic simulation allowed certain thermodynamic characteristics of this reaction to be calculated. To the first approximation, these characteristics correlate with the estimates obtained from the results of direct redox titration.     

Effects of oxygen on the dark recombination between photoreduced secondary quinone and oxidized bacteriochlorophyll in Rhodobacter sphaeroides reaction centers

The influence of duration of exposure to actinic light (from 1 sec to 10 min) and temperature (from 3 to 35^°C) on the temporary stabilization of the photomobilized electron in the secondary quinone acceptor (Q_B) locus of Rhodobacter sphaeroides reaction centers (RC) was studied under aerobic or anaerobic conditions. Optical spectrophotometry and ESR methods were used. The stabilization time increased significantly upon increasing the exposure duration under aerobic conditions. The stabilization time decreased under anaerobic conditions, its dependence on light exposure duration being significantly less pronounced. Generation of superoxide radical in photoactivated aerobic samples was revealed by the ESR method. Possible interpretation of the effects is suggested in terms of interaction between the semiquinone Q_B with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.     

Role of HiPIP as electron donor to the RC-bound cytochrome in photosynthetic purple bacteria

High-Potential Iron-Sulfur Proteins (HiPIP) are small electron carriers, present only in species of photosynthetic purple bacteria having a RC-bound cytochrome. Their participation in the photo-induced cyclic electron transfer was recently established for Rubrivivax gelatinosus, Rhodocyclus tenuis and Rhodoferax fermentans (Schoepp et al. 1995; Hochkoeppler et al. 1996a, Menin et al. 1997b). To better understand the physiological role of HiPIP, we extended our study to other selected photosynthetic bacteria. The nature of the electron carrier in the photosynthetic pathway was investigated by recording light-induced absorption changes in intact cells. In addition, EPR measurements were made in whole cells and in membrane fragments in solution or dried immobilized, then illuminated at room temperature. Our results show that HiPIP plays an important role in the reduction of the photo-oxidized RC-bound cytochrome in the following species: Ectothiorhodospira vacuolata, Chromatium vinosum, Chromatium purpuratum and Rhodopila globiformis. In Rhodopseudomonas marina, the HiPIP is not photo-oxidizible in whole cells and in dried membranes, suggesting that this electron carrier is not involved in the photosynthetic pathway. In Ectothiorhodospira halophila, the photo-oxidized RC-bound cytochrome is reduced by a high midpoint potential cytochrome c, in agreement with midpoint potential values of the two iso-HiPIPs (+ 50 mV and + 120 mV) which are too low to be consistent with their participation in the photosynthetic cyclic electron transfer.     

Kinetic study of PF and CarT states in the LM subunit purified from the wild-type Rhodobacter sphaeroides reaction centers

The kinetics of absorbance changes related to the charge-separated state, P^F, and to the formation and decay of the carotenoid triplet state (Car^T) were studied in the LM reaction center subunit isolated from a wild-type strain of the purple bacterium Rhodobacter sphaeroides (strain Y). The P^F lifetime is lengthened (20±1.5 ns) in the LM complex as compared to the intact reaction centers (11±1 ns). The yield of the carotenoid triplet formation is higher (0.28±0.01) in the LM complex than in native reaction centers. We interpret our results in terms of perturbations of a first-order reaction connecting the singlet and the triplet state of the radical-pair state. Our results, together with those of a recent work (Agalidis, I., Nuijs, A.M. and Reiss-Husson, F. (1987) Biochim. Biophys. Acta (in press)) are consistent with a high I to Q_A electron transfer rate in this LM subunit, which is metal-depleted.The LM complex is considerably more sensitive than the reaction centers to photooxidative damage in the presence of oxygen. This is not readily accounted for simply by the higher carotenoid triplet yield, and may suggest a greater accessibility of the internal structures in the absence of the H-subunit.The lifetime of the carotenoid triplet decay (6.4±0.3 s) in the LM subunit is unchanged compared to the native reaction centers.Abbreviations BChl bacteriochlorophyll - Bph bacteriopheophytin - Car carotenoid - Chl chlorophyll - cyt cytochrome - L, M and H subunits light, medium and heavy subunits of the reaction center complex - P^R triplet electronic state of the primary electron donor - P; Q_A the first stable electron acceptor, a bound quinone - RC reaction center - LDAO lauryldimethylamine N-oxide - SDS sodium dodecyl sulfate - UQ ubiquinone This paper is published in our new format. All future authors are requested to follow our new instructions (see Photosynthesis Research 10:519–526, 1986)—Editor.     

Why Is Electron Transport in the Reaction Centers of Purple Bacteria Unidirectional?

Although the two electron-transfer branches in the reaction centers (RC) of purple bacteria are virtually symmetric, it is well known that only one of them is functionally active (the A-branch). The mechanisms of functional asymmetry of structurally symmetric branches of the electron transport system are analyzed in this work within the framework of the theory of bimolecular charge-transfer complexes (CTC). CTC theory is shown to provide an explanation of this phenomenon. According to the CTC theory, the dominance of one branch is required to implement the CTC state in special bacteriochlorophyll pairs of RC, in which more than 30% of the excited electron density in the CTC is shifted toward one of the bacteriochlorophyll molecules. This causes a significant increase in the efficiency of further electron transfer to the primary quinone acceptor as compared to a system with two absolutely symmetric electron transfer branches. Specific features of dielectric asymmetry near the RC special pair are discussed. It is emphasized that a strong CTC is able to provide effective trapping of electronic excitation energy from antenna chlorophyll, which is a main function of the RC. Hypothetical stages of CTC formation in other classes of photosynthesizing bacteria during evolution are discussed.     

Oscillations of the energy gap for the initial electron-transfer step in bacterial reaction centers

Oscillations in the electrostatic energy gap [V_elec(t)] for electron transfer from the primary electron donor (P) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers are examined by molecular-dynamics simulations. Autocorrelation functions of V_elec in the reactant state (PB) include prominent oscillations with an energy of 17 cm^–1. This feature is much weaker if the trajectory is propagated in the product state P⁺B^–. The autocorrelation functions also include oscillations in the regions of 5, 80 and 390 cm^–1 in both states, and near 25 and 48 cm^–1 in P⁺B^–. The strong 17-cm^–1 oscillation could involve motions that modulate the distance between P and B, because a similar oscillation occurs in the direct electrostatic interactions between the electron carriers.     

紫细菌光合色素指纹图谱的建立与色素分析

【目的】探索快速高效的色素样品制备方法;为建立紫细菌全色素TLC和HPLC标准指纹图谱和数据库提供研究方法和思路;为色素代谢与调控等研究提供快速的色素分析方法。【方法】选择沼泽红假单胞菌(Rhodopesudomonas palustris CQV97)为材料,采用改良丙酮甲醇提取法、TLC重复展层法、图像灰度分析法、吸收光谱法、HPLC和MS法进行色素分析。【结果】甲醇或丙酮可选择性地提取样品的细菌叶绿素和类胡萝卜素,通过对丙酮甲醇法的改良,使色素提取总量提高了13.5%。建立了CQV97菌株全色素的TLC和HPLC指纹图谱,二者均含有11种色素组分。图像灰度分析法估算了TLC指纹图谱各色素组分的表观相对含量。以TLC图谱的各色素组分为外标物,明确了HPLC图谱中各色素组分的保留时间(Rt)与TLC图谱中各色素组分的迁移率(Rf)之间的对应关系。结合色素代谢途径、光谱分析和MS,定性分析了指纹图谱中11种色素组分。TLC或HPLC分析结果表明,理论样品与对照样品色素组分和含量一致,而实际样品与对照样品色素组分一致,但含量不同,重复测定3次,样品中色素相对含量的RSD均小于5%。【结论】改良的丙酮甲醇法可以快速高效地提取光合细菌的色素。采用TLC重复展层法和HPLC法建立的全色素指纹图谱色素组成一致,重复稳定性好,各具特色。TLC和HPLC两种指纹图谱分析方法均能进行光合色素的快速分析,适合于紫细菌色素合成途径中主要积累色素的组分和含量变化规律研究。     

Stabilization of the electron in the quinone acceptor part of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> reaction centers

The evolution of the light-induced absorption difference spectrum (380–500 nm) of the reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides has been examined over 200 μs. The observed changes are interpreted as the effects of proton movement along the H-bond between the primary quinone acceptor and its protein surroundings. A theoretical analysis of the spectral evolution, considering the proton tunneling kinetics, corroborates this interpretation. The electronic state of the primary quinone is stabilized within tens of microseconds; the process is retarded upon deuteration of the reaction center as well as in 90% glycerol, and accelerated upon nondestructive heating to 40°C.     

Free energy dependence of the direct charge recombination from the primary and secondary quinones in reaction centers from Rhodobacter sphaeroides

The direct charge recombination rates from the primary quinone, k _AD (D⁺Q _A ^– DQ_A) and the secondary quinone, k _BD (D⁺Q _B ^– DQ_B), in reaction centers from Rhodobacter sphaeroides were measured as a function of the free energy differences for the processes, G _AD ⁰ and G _BD ⁰ , respectively. Measurements were performed at 21 °C on a series of mutant reaction centers that have a wide range of dimer midpoint potentials and consequently a large variation in G _AD ⁰ and G _BD ⁰ . As –G _AD ⁰ varied from 0.43 to 0.78 eV, k _AD varied from 4.6 to 28.6 s^–1. The corresponding values for the wild type are 0.52 eV and 8.9 s^–1. Observation of the direct charge recombination rate k _BD was achieved by substitution of the primary quinone with naphthoquinones in samples in which ubiquinone was present at the secondary quinone site, resulting specifically in an increase in the free energy of the D⁺Q _A ^– state relative to the D⁺Q_AQ _B ^– state. As –G _BD ⁰ varied from 0.37 to 0.67 eV, k _BD varied from 0.03 to 1.4 s^–1. The corresponding values for the wild type are 0.46 eV and 0.2 s^–1. A fit of the two sets of data to the Marcus theory for electron transfer yielded significantly different reorganization energies of 0.82 and 1.3 eV for k _AD and k _BD, respectively. In contrast, the fitted values for the coupling matrix element, or equivalently the maximum possible rate, were comparable (25 s^–1) for the two charge recombination processes. These results are in accord with Q_B having more interactions with dipoles, from both the surrounding protein and bound water molecules, than Q_A and with the primary determinant of the maximal rate being the quinone-donor distance.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a