Electric field dependence of recombination kinetics in reaction centers of photosynthetic bacteria

Time-resolved charge recombination has been measured by reflectance/absorption spectroscopic analysis of Langmuir-Blodgett films of reaction centers of the photosynthetic bacterium, Rhodopseudomonas sphaeroides over a wide range of applied electric field strengths. The field dependence of the recombination kinetics has been deduced from the time-course of the reduction of the flash-oxidized bacteriochlorophyll dimer [(BChl)⁺₂] recorded at different applied field strengths. Measurements were performed under two different electric field biasing conditions: a constant bias and a high-frequency bipolar square-wave bias. The additional data obtained from bipolar biasing enabled the use of a new deconvolution method to obtain the field dependence of the rate constants from the experimental curves. The deconvolution shows that the rates for charge recombination from the flash-generated state back to the ground state (BChl)₂Q_A approximate exponential functions of the applied electric field. Correlation of the recombination kinetics data with photoinduced electrical response measurements on films with asymmetric up and down populations of reaction centers reveals that fields opposing charge separation result in faster rates of recombination. Although other possibilities are considered, the main source of the effect is believed to be a result of field-induced changes in the free energy gap between and (BChl)₂Q_A. The results presented here are compared to those obtained in experiments with solubilized reaction centers in which the free energy gap between and (BChl)₂Q_A has been changed by quinone replacement.     

Photochemical hole-burning in photosynthetic reaction centers

The change in absorbance (hole spectrum) of the primary electron donor (P870) in bacterial photosynthetic reaction centers has been studied at 1.5–2.1 K following narrow-band excitation at several wavelengths within the P870 absorption band. The hole width is very large, suggesting a homogeneous linewidth on the order of 200–300 cm⁻¹. Possible interpretations of this highly unusual result, including ultra-fast excited-state decay, are discussed.     

叶绿素衍生物的合成及其对紫细菌原初光化学特性 的影响研究

对10个叶绿素衍生物的合成方法进行了研究,在一定的温度(43.5℃)和丙酮的协同作用下,植物脱镁叶绿素a可转换紫细菌RS601光合反应中心的细菌脱镁叶绿素,光化学活性为对照的71.4%,光化学活性的下降与替代色素的变化无对应关系,替代后降低了Bphe^-/Bphe和QA^-/QA电对之间的电子传递速率。     

Magnetic field effects on the recombination of radical ions in reaction centers of photosynthetic bacteria

The amount of triplet products formed upon recombination of the bacteriochlorophyll dimer cation and the bacteriopheophytin anion in modified bacterial reaction centres can be manipulated by external magnetic fields. Making use of this effect, two methods provide information about structural and dynamic properties of the reaction centre. The two methods differ in that one (MARY) uses low static magnetic fields (0 ? B₀ < 1 kG) whereas the other (RYDMR) uses microwaves and high static fields (B₀ > 1 kG). As is shown here the two methods are equivalent from a theoretical point of view, the interpretation of experimental spectra being equally involved in both cases.     

Conformational control of cofactors in nature--functional tetrapyrrole conformations in the photosynthetic reaction centers of purple bacteria

Chemically identical tetrapyrrole cofactors such as hemes and chlorophylls participate in functionally diverse biological roles. An analysis of the available protein structural data for the bacteriochlorophylls in the photosynthetic reaction center gives statistically reliable evidence of the hypothesis that the protein induced cofactor conformation is a modulator of the bio-molecular function of each reaction center. The results serve as a general model to illustrate conformational control of tetrapyrrole cofactors in other proteins.     

Inter- and intraspecific variation in excited-state triplet energy transfer rates in reaction centers of photosynthetic bacteria

In protein-cofactor reaction center (RC) complexes of purple photosynthetic bacteria, the major role of the bound carotenoid (C) is to quench the triplet state formed on the primary electron donor (P) before its sensitization of the excited singlet state of molecular oxygen from its ground triplet state. This triplet energy is transferred from P to C via the bacteriochlorophyll monomer B(B). Using time-resolved electron paramagnetic resonance (TREPR), we have examined the temperature dependence of the rates of this triplet energy transfer reaction in the RC of three wild-type species of purple nonsulfur bacteria. Species-specific differences in the rate of transfer were observed. Wild-type Rhodobacter capsulatus RCs were less efficient at the triplet transfer reaction than Rhodobacter sphaeroides RCs, but were more efficient than Rhodospirillum rubrum RCs. In addition, RCs from three mutant strains of R. capsulatus carrying substitutions of amino acids near P and B(B) were examined. Two of the mutant RCs showed decreased triplet transfer rates compared with wild-type RCs, whereas one of the mutant RCs demonstrated a slight increase in triplet transfer rate at low temperatures. The results show that site-specific changes within the RC of R. capsulatus can mimic interspecies differences in the rates of triplet energy transfer. This application of TREPR was instrumental in defining critical energetic and coupling factors that dictate the efficiency of this photoprotective process.     

Electron-transfer dynamics of photosynthetic reaction centers in thermoresponsive soft materials

Poly(ethylene glycol)-grafted, lipid-based, thermoresponsive, soft nanostructures are shown to serve as scaffolding into which reconstituted integral membrane proteins, such as the bacterial photosynthetic reaction centers (RCs) can be stabilized, and their packing arrangement, and hence photophysical properties, can be controlled. The self-assembled nanostructures exist in two distinct states: a liquid-crystalline gel phase at temperatures above 21 degrees C and a non-birefringent, reduced viscosity state at lower temperatures. Characterization of the effect of protein introduction on the mesoscopic structure of the materials by 31P NMR and small-angle X-ray scattering shows that the expanded lamellar structure of the protein-free material is retained. At reduced temperatures, however, the aggregate structure is found to convert from a two-dimensional normal hexagonal structure to a three-dimensional cubic phase upon introduction of the RCs. Structural and functional characteristics of the RCs were determined by ground-state and femtosecond transient absorption spectroscopy. Time-resolved results indicate that the kinetics of primary electron transfer for the RCs in the low-viscosity cold phase of the self-assembled nanostructures are identical to those observed in a detergent-solubilized state in buffered aqueous solutions (approximately 4 ps) over a wide range of protein concentrations and experimental conditions. This is also true for RCs held within the lamellar gel phase at low protein concentrations and at short sample storage times. In contrast are kinetics from samples that are prepared with high RC concentrations and stored for several hours, which display additional kinetic components with extended electron-transfer times (approximately 10-12 ps). This observation is tentatively attributed to energy transfer between RCs that have laterally (in-plane) organized within the lipid bilayers of the lamellar gel phase prior to charge separation. These results not only demonstrate the use of soft nanostructures as a matrix in which to stabilize and organize membrane proteins but also suggest the possibility of using them to control the interactions between proteins and thus to tune their collective optical/electronic properties.     

Lipid binding to the carotenoid binding site in photosynthetic reaction centers

Lipid binding to the carotenoid binding site near the inactive bacteriochlorophyll monomer was probed in the reaction centers of carotenoid-less mutant, R-26 from Rhodobacter sphaeroides. Recently, a marked light-induced change of the local dielectric constant in the vicinity of the inactive bacteriochlorophyll monomer was reported in wild type that was attributed to structural changes that ultimately lengthened the lifetime of the charge-separated state by 3 orders of magnitude (Deshmukh, S. S.; Williams, J. C.; Allen, J. P.; Kalman, L. Biochemistry 2011, 50, 340). Here in the R-26 reaction centers, the combination of light-induced structural changes and lipid binding resulted in a 5 orders of magnitude increase in the lifetime of the charge-separated state involving the oxidized dimer and the reduced primary quinone in proteoliposomes. Only saturated phospholipids with fatty acid chains of 12 and 14 carbon atoms long were bound successfully at 8 °C by cooling the reaction center protein slowly from room temperature. In addition to reporting a dramatic increase of the lifetime of the charge-separated state at physiologically relevant temperatures, this study reveals a novel lipid binding site in photosynthetic reaction center. These results shed light on a new potential application of the reaction center in energy storage as a light-driven biocapacitor since the charges separated by ～30 ? in a low-dielectric medium can be prevented from recombination for hours.     

The role of the accessory bacteriochlorophyll in reaction centers of photosynthetic bacteria: intermediate acceptor in the primary electron transfer?

Time-resolved spectroscopy in conjunction with magnetic-field-dependent recombination dynamics of the primary radical ion pair in reaction centers of Rb, sphaeroides R26, were used to analyze the mechanism of electron transfer from the bacteriochlorophyll dimer in its excited singlet state (¹P^*) to bacteriopheophytin (H). This analysis provides evidence against the participation of the accessory bacteriochlorophyll (B) as a kinetic intermediate and thus favours a single-step electron transfer, which is mediated by superexchange electronic interactions.     

Electron transfer kinetics in photosynthetic reaction centers embedded in polyvinyl alcohol films

The coupling between electron transfer and protein dynamics has been studied at room temperature in isolated reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in polyvinyl alcohol (PVA) films of different water/RC ratios. The kinetic analysis of charge recombination shows that dehydration of RC-containing PVA films causes reversible, inhomogeneous inhibition of electron transfer from the reduced primary quinone acceptor (Q(A)(-)) to the secondary quinone Q(B). A more extensive dehydration of solid PVA matrices accelerates electron transfer from Q(A)(-) to the primary photooxidized electron donor P(+). These effects indicate that incorporation of RCs into dehydrated PVA films hinders the conformational dynamics gating Q(A)(-) to Q(B) electron transfer at room temperature and slows down protein relaxation which stabilizes the primary charge-separated state P(+)Q(A)(-). A comparison with analogous effects observed in trehalose-coated RCs suggests that protein motions are less severely reduced in PVA films than in trehalose matrices at comparable water/RC ratios.     

Self-assembly and sensor response of photosynthetic reaction centers on screen-printed electrodes

Photosynthetic reaction centers were immobilized onto gold screen-printed electrodes (Au-SPEs) using a self-assembled monolayer (SAM) of mercaptopropionic acid (MPA) which was deliberately defective in order to achieve effective mediator transfer to the electrodes. The pure Photosystem II (PS II) cores from spinach immobilize onto the electrodes very efficiently but fair badly in terms of photocurrent response (measured using duroquinone as the redox mediator). The cruder preparation of PS II known as BBY particles performs significantly better under the same experimental conditions and shows a photocurrent response of 20-35 nA (depending on preparation) per screen-printed electrode surface (12.5mm(2)). The data was corroborated using AFM, showing that in the case of BBY particles a defective biolayer is indeed formed, with grooves spanning the whole thickness of the layer enhancing the possibility of mass transfer to the electrodes and enabling biosensing. In comparison, the PS II core layer showed ultra-dense organization, with additional formation of aggregates on top of the single protein layer, thus blocking mediator access to the electrodes and/or binding sites. The defective monolayer biosensor with BBY particles was successfully applied for the detection of photosynthesis inhibitors, demonstrating that the inhibitor binding site remained accessible to both the inhibitor and the external redox mediator. Biosensing was demonstrated using picric acid and atrazine. The detection limits were 1.15 nM for atrazine and 157 nM for picric acid.     

Transmembrane charge transfer in photosynthetic reaction centers: some similarities and distinctions

This mini review presents a general comparison of structural and functional peculiarities of three types of photosynthetic reaction centers (RCs)--photosystem (PS) II, RC from purple bacteria (bRC) and PS I. The nature and mechanisms of the primary electron transfer reactions, as well as specific features of the charge transfer reactions at the donor and acceptor sides of RCs are considered. Comparison of photosynthetic RCs shows general similarity between the core central parts of all three types, between the acceptor sides of bRC and PS II, and between the donor sides of bRC and PS I. In the latter case, the similarity covers thermodynamic, kinetic and dielectric properties, which determine the resemblance of mechanisms of electrogenic reduction of the photooxidized primary donors. Significant distinctions between the donor and acceptor sides of PS I and PS II are also discussed. The results recently obtained in our laboratory indicate in favor of the following sequence of the primary and secondary electron transfer reactions: in PS II (bRC): Р(680)(Р(870)) → Chl(D1)(В(А)) → Phe(bPhe) → Q(A); and in PS I: Р(700) → А(0А)/A(0B) → Q(A)/Q(B).     

pH-sensitive fluorescent dye as probe for proton uptake in photosynthetic reaction centers

Isolated and purified reaction centers (RC) from Rhodobacter sphaeroides R-26.1 were solubilised in detergent with excess quinone and external electron donors and illuminated in the presence of pyranine. The pH change accompanying the reaction center photocycle was monitored by recording the variation of the pyranine fluorescence intensity. Using Q(B)-depleted reaction centers or blocking the photocycle with terbutryne strongly reduced the pH change. The usefulness and limits of this technique in monitoring the pH changes during the RC photocycle are also discussed.     

Functionality of photosynthetic reaction centers in polyelectrolyte multilayers: toward an herbicide biosensor

The bacterial reaction center (RC), a membrane photosynthetic protein, has been adsorbed onto a glass surface by alternating deposition with the cationic polymer poly(dimethyldiallylammonium chloride) (PDDA) obtaining as an end result an ordinate polyelectrolyte multilayer (PEM) where the protein retains its integrity and photoactivity over a period of several months. Such a system has been characterized from the functional point of view by checking the protein photoactivity at different hydration conditions, from extensive drought to full hydration. The kinetic analysis of charge recombination indicates that incorporation of RCs into dehydrated PEM hinders the conformational dynamics gating QA- to QB electron-transfer leaving unchanged the protein relaxation that stabilizes the primary charge separated state P+QA-. The herbicide-induced inhibition of the QB activity was studied in some detail. By dipping the PEM in herbicide solutions for short times, kinetics of herbicide binding and release have been determined; binding isotherms have been studied using PEM immersed in herbicide solution. QB functionality of RC has been restored by rinsing the PEM with water, thus allowing the reuse of the same sample. This last point has been exploited to design a simple optical biosensor for herbicides. A suitable kinetic model has been proposed to describe the interplay between forward and back electron-transfer processes upon continuous illumination, and the use of the PDDA-RC multilayers in herbicide bioassays was successfully tested.     

Isolation and analysis of tetraheme-bound-cytochrome from photosynthetic reaction centers of Rhodopseudomonas viridis

A tetraheme cytochrome (BCytc) was isolated from the photosynthetic reaction centers (RC) of Rhodopseudomonas viridis while maintaining the redox activity. BCytc was removed from the H-subunit-detached RC by polyacrylamide electrophoresis using an alkyl ether sulfate mixed with sodium dodecyl sulfate. Redox titration of BCytc showed a simple one-step redox titration curve and a lowered midpoint potential than that of one in RC. Direct electron transfer between BCytc and electrode surfaces, such as indium tin oxide, was successfully performed, indicating a potential for molecular electronic material.     

Protein dynamics control of electron transfer in photosynthetic reaction centers from Rps. sulfoviridis

In the cycle of photosynthetic reaction centers, the initially oxidized special pair of bacteriochlorophyll molecules is subsequently reduced by an electron transferred over a chain of four hemes of the complex. Here, we examine the kinetics of electron transfer between the proximal heme c-559 of the chain and the oxidized special pair in the reaction center from Rps. sulfoviridis in the range of temperatures from 294 to 40 K. The experimental data were obtained for three redox states of the reaction center, in which one, two, or three nearest hemes of the chain are reduced prior to special pair oxidation. The experimental kinetic data are analyzed in terms of a Sumi-Marcus-type model developed in our previous paper,1 in which similar measurements were reported on the reaction centers from Rps. viridis. The model allows us to establish a connection between the observed nonexponential electron-transfer kinetics and the local structural relaxation dynamics of the reaction center protein on the microsecond time scale. The activation energy for relaxation dynamics of the protein medium has been found to be around 0.1 eV for all three redox states, which is in contrast to a value around 0.4-0.6 eV in Rps. viridis.1 The possible nature of the difference between the reaction centers from Rps. viridis and Rps. sulfoviridis, which are believed to be very similar, is discussed. The role of the protein glass transition at low temperatures and that of internal water molecules in the process are analyzed.     

Stabilization effect of single-walled carbon nanotubes on the functioning of photosynthetic reaction centers

The interaction between single-walled carbon nanotubes and photosynthetic reaction centers purified from purple bacterium Rhodobacter sphaeroides R-26 has been investigated. Atomic force microscopy studies provide evidence that reaction center protein can be attached effectively to the nanotubes. The typical diameter of the nanotube is 1-4 nm and 15 +/- 2 nm without and with the reaction centers, respectively. Light-induced absorption change measurements indicate the stabilization of the P+(Q(A)Q(B))- charge pair, which is formed after single saturating light excitation after the attachment to nanotubes. The separation of light-induced charges is followed by slow reorganization of the protein structure. The stabilization effect of light-initiated charges by the carbon nanotubes opens a possible direction of several applications, the most promising being in energy conversion and storage devices.