Proton uptake of rhodobacter capsulatus reaction center mutants modified in the primary quinone environment

Flash-induced absorbance spectroscopy was used to analyze the proton uptake and electron transfer properties of photosynthetic reaction centers (RC) of Rhodobacter capsulatus that have been genetically modified near the primary quinone electron acceptor (Q(A)). M246Ala and M247Ala, which are symmetry-related to the positions of two acidic groups, L212Glu and L213Asp, in the secondary quinone electron acceptor (QB) protein environment, have been mutated to Glu and Asp, respectively. The pH dependence of the stoichiometry of proton uptake upon formation of the P+Q(A)- (H+/P+Q(A)-) and PQ(A) (H+/Q(A)-) (P is the primary electron donor, a noncovalently linked bacteriochlorophyll dimer) states have been measured in the M246Ala --> Glu and the M247Ala --> Asp mutant RC, in the M246Ala-M247Ala --> Glu-Asp double mutant and in the wild type (WT). Our results show that the introduction of an acidic group (Glu or Asp) in the QA protein region induces notable additional proton uptake over a large pH region (approximately 6-9), which reflects a delocalized response of the protein to the formation of Q(A)-. This may indicate the existence of a widely spread proton reservoir in the cytoplasmic region of the protein. Interestingly, the pH titration curves of the proton release caused by the formation of P+ (H+/P+: difference between H+/P+Q(A)- and H+/PQ(A)- curves) are nearly superimposable in the WT and the M246Ala --> Glu mutant RC, but substantial additional proton release is detected between pH 7 and 9 in the M247Ala --> Asp mutant RC. This effect can be accounted for by an increased proton release by the P+ environment in the M247Ala --> Asp mutant. The M247Ala --> Asp mutation reveals the existence of an energetic and conformational coupling between donor and acceptor sides of the RC at a distance of nearly 30A.     

Attenuated total reflection Fourier transform infrared spectroscopy of redox transitions in photosynthetic reaction centers: comparison of perfusion- and light-induced difference spectra

Chemically induced Fourier transform infrared difference spectra associated with redox transitions of several primary electron donors and acceptors in photosynthetic reaction centers (RCs) have been compared with the light-induced FTIR difference spectra involving the same cofactors. The RCs are deposited on an attenuated total reflection (ATR) prism and form a film that is enclosed in a flow cell. Redox transitions in the film of RCs can be repetitively induced either by perfusion of buffers poised at different redox potentials or by illumination. The perfusion-induced ATR-FTIR difference spectra for the oxidation of the primary electron donor P in the RCs of the purple bacteria Rb. sphaeroides and Rp. viridis and P700 in the photosystem 1 of Synechocystis 6803, as well as the Q(A)/Q(A) transition of the quinone acceptor (Q(A)) in Rb. sphaeroides RCs are reported for the first time. They are compared with the light-induced ATR-FTIR difference spectra P+Q(A)/PQ(A) for the RCs of Rb. sphaeroides and P700+/P700 for photosystem 1. It is shown that the perfusion-induced and light-induced ATR-FTIR difference spectra recorded on the same RC film display identical signal to noise ratios when they are measured under comparable conditions. The ATR-FTIR difference spectra are very similar to the equivalent FTIR difference spectra previously recorded upon photochemical or electrochemical excitation of these RCs in the more conventional transmission mode. The ATR-FTIR technique requires a smaller amount of sample compared with transmission FTIR and allows precise control of the aqueous environment of the RC films.     

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.     

Dipyridamole and its derivatives modify the kinetics of the electron transport in reaction centers from Rhodobacter sphaeroides

A well known vasodilator dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d]pyrim idine, and its derivatives have recently been shown as potential co-activators (modulators) in the phenomenon of multidrug resistance (MDR) in cancer therapy. They inhibit the specific function of a transmembrane P-glycoprotein responsible for the ex-flux of anti-cancer drugs from tumor cells. To clarify molecular mechanisms of the anti-MDR activity of DIP and its two derivatives, RA25 and RA47, we have studied their effects on electron transport in reaction centers (RC) from purple photosynthetic bacteria Rb. sphaeroides, using RC as a model system. Increasing concentrations of DIP and RA47 progressively accelerate the back electron transfer from the primary quinone acceptor QA to the bacteriochlorophyll dimer Bchl2 (Bchl2+ -QA- recombination). In the absence of o-phenantroline, when both quinone acceptors QA and QB are involved in the electron transport, RA47 is more effective than DIP. DIP stabilizes the electron on the secondary quinone acceptor QB, the effect manifested as the retardation of Bchl2+ -QB- recombination. Effects of RA25 are negligible in all cases. The drugs are proposed to change the electron transport affecting the RC structural dynamics and the stabilization of the electron on quinone acceptors through modification of H-bonds in the system.     

Temperature dependence of charge recombination in Heliobacillus mobilis

Transient absorption difference spectroscopy was used to study the temperature dependence of the P798+ decay kinetics in heliobacteria. For membrane samples, two components were obtained from the fitting of kinetic traces in the temperature range of 4-29 degrees C. A 3-9 ms component representing the cytochrome (cyt) c oxidation has an activation energy of 33.0 +/- 2.8 kJ/mol. A 12-22 ms component representing either P798+FX- or P798+FA/B- recombination has an activation energy of 15.3 +/- 2.4 kJ/mol. In isolated reaction centers (RC), only one 14 ms component due to P798+FX- recombination was obtained in this temperature range. The Arrhenius plot shows that the recombination rate of this P798+FX- state is temperature independent in the near room temperature range. For RC in the temperature range of 60-298 K, a 12-15 ms decay was obtained at temperatures greater than 240 K. Biphasic decay traces (12-15 ms and 2-4 ms components) were obtained at temperatures between 170 K and 230 K. Only one 2-4 ms component was found at temperatures lower than 160 K. The gradual switchover from the 12-15 ms to the 2-4 ms component upon cooling may indicate the shift of the P798+FX- recombination state to a state that is prior to P798+FX-, although other interpretations can not be excluded. The absorption difference spectrum (delta A @ 160 K - delta A @ 240 K) in the blue region shows a positive amplitude below 405 nm and a negative amplitude above 405 nm implying that the 2-4 ms decay component may be due to the recombination of P798+A1-, where A1 is a quinone-type acceptor.     

Conformational control of the Q(A) to Q(B) electron transfer in bacterial reaction centers: evidence for a frozen conformational landscape below -25 degrees C

The competition between the P(+)Q(A)(-) --> PQ(A) charge recombination (P, bacteriochlorophyll pair acting as primary photochemical electron donor) and the electron transfer to the secondary quinone acceptor Q(A)(-)Q(B) --> Q(A)Q(B)(-) (Q(A) and Q(B), primary and secondary electron accepting quinones) was investigated in chromatophores of Rb. capsulatus, varying the temperature down to -65 degrees C. The analysis of the flash-induced pattern for the formation of P(+)Q(A)Q(B)(-) shows that the diminished yield, when lowering the temperature, is not due to a homogeneous slowing of the rate constant k(AB) of the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer but to a distribution of conformations that modulate the electron transfer rate over more than 3 orders of magnitude. This distribution appears "frozen", as no dynamic redistribution was observed over time ranges > 10 s (below -25 degrees C). The kinetic pattern was analyzed to estimate the shape of the distribution of k(AB), showing a bell-shaped band on the high rate side and a fraction of "blocked" reaction centers (RCs) with very slow k(AB). When the temperature is lowered, the high rate band moves to slower rate regions and the fraction of blocked RCs increases at the expense of the high rate band. The RCs that recombine from the P(+)Q(A)Q(B)(-) state appear temporarily converted to a state with rapid k(AB), indicating that the stabilized state described by Kleinfeld et al. (Biochemistry 1984, 23, 5780-5786) is still accessible at -60 degrees C.     

Thermodynamics of light-induced and thermal degradation of bacteriochlorins in reaction center protein of photosynthetic bacteria

The rate constants of thermal (irreversible) damage of bacteriochlorin pigments (bacteriochlorophyll monomer [B], bacteriochlorophyll dimer [P] and bacteriopheophytine [H]) in reaction center [RC] protein from the photosynthetic bacterium Rhodobacter sphaeroides were studied in the dark and during intense (400 mW x cm(-2)) laser light excitation (wavelengths 488 and 515 nm) under deoxygenated conditions. While the kinetics of degradation of P and B were monoexponential, the decay kinetics of H were overlapped by an initial lag phase at elevated (>40 degrees C) temperature. This is explained by removal of the central metal ion from the bacteriochlorophylls as part of their degradation processes. At all temperatures, the rates of damage were very similar for all bacteriochlorin pigments and were larger in the light than in the dark. The logarithm of the rate constant of pigment degradation and loss of photochemistry as a function of reciprocal (absolute) temperature (Arrhenius/Eyring plot) showed single phase in the light and double phases in the dark. Below 20 degrees C, the rate of pigment degradation in the RC decreased so dramatically in the dark that it became limited by the natural degradation process of bacteriochlorophyll measured in solution. The function of loss of photochemistry in the dark was also biphasic and had a break point at 40 degrees C. The damage in the dark required high enthalpy change (DeltaH(++) = 64 kcal/mol for P and DeltaH(++) = 60 kcal/mol for B) and entropy increase (T x DeltaS(++) = 38 kcal/mol for P and T x DeltaS(++) = 34 kcal/mol for B at T = 300 K), whereas significantly smaller enthalpy change (DeltaH(++) = 21 kcal/mol for P and B and DeltaH(++) = 13 kcal/mol for H) and practically no (T x DeltaS(++) = -1 kcal/mol for P and B at T = 300 K) or small (T x DeltaS(++) = -9 kcal/mol for H at T = 300 K) entropy change was needed in the light. The thermodynamic parameters of activation reveal major steps common in the degradation of all bacteriochlorin pigments: ring opening reactions at C5 or C20 meso-bridges (or both) and breaking/removal of the phytyl chain. Their contribution in the degradation is probably reflected in the observed enthalpy/entropy compensation at an almost constant (DeltaG(++) = 22-26 kcal/mol at T = 300 K) free energy change of activation.     

Influence of cardiolipin on the functionality of the Q(a) site of the photosynthetic bacterial reaction center

The effect of cardiolipin on the functionality of the Q(A) site of a photosynthetic reaction center (RC) was studied in RCs from the purple non-sulfur bacterium Rhodobacter sphaeroides by means of time-resolved absorbance measurements. The binding of the ubiquinone-10 to the Q(A) site of the RC embedded in cardiolipin or lecithin liposomes has been followed at different temperatures and phospholipid loading. A global fit of the experimental data allowed us to get quite reliable values of the thermodynamic parameters joined to the binding process. The presence of cardiolipin does not affect the affinity of the Q(A) site for ubiquinone but has a marked influence on the rate of P+QA(-) --> PQA electron transfer. The P+QA(-) charge recombination kinetics has been examined in liposomes made of cardiolipin/lecithin mixtures and in detergent (DDAO) micelles doped with cardiolipin. The electron-transfer rate constant increases upon cardiolipin loading. It appears that the main effect of cardiolipin on the electron transfer can be ascribed to a destabilization of the charge-separated state. Results obtained in micelles and vesicles follow the same titration curve when cardiolipin concentration evaluated with respect to the apolar phase is used as a relevant variable. The dependence of the P+QA(-) recombination rate on cardiolipin loading suggests two classes of binding sites. In addition to a high-affinity site (compatible with previous crystallographic studies), a cooperative binding, involving about four cardiolipin molecules, takes place at high cardiolipin loading.     

Multichannel flash spectroscopy of the reaction centers of wild-type and mutant Rhodobacter sphaeroides: bacteriochlorophyllB-mediated interaction between the carotenoid triplet and the special pair

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light-induced spectral changes in the 350-1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870(+)QA(-), and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870(+)) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P870(+), and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 micros). The bleaching at 800 nm arises from the pigment interaction between P870(+) and the accessory bacteriochlorophylls on A and B branches (BA,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals--a sharp peak at 809 +/- 2 nm and a broad band at 870 nm--which decay at the same rate as the TmS spectrum of Car (ca 10 micros). The former is ascribed to the perturbation, by Car(+), of the absorption spectrum of BB; the latter, to the TmS spectrum of P870(+), a species that appears to be in approximate thermal equilibrium with Car(+). These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.     

Femtosecond spectroscopy of the primary charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation in the QY and Soret bands

The primary charge separation and electron-transfer processes of photosynthesis occur in the reaction center (RC). Isolated RCs of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus were studied at room temperature by using femtosecond transient absorption spectroscopy with selective excitation. Upon excitation in the Q(Y) absorbance band of the bacteriochlorophyll (BChl) dimer (P) at 865 nm, a 7.0 +/- 0.5 ps kinetic component was observed in the 538 nm region (Q(X) band of the bacteriopheophytin (BPheo)), 750 nm region (Q(Y) band of the BPheo), and 920 nm region (stimulated emission of the excited-state of P), indicating that this lifetime represents electron transfer from P to BPheo. The same time constant was also observed upon 740 nm or 800 nm excitation. A longer lifetime (300 +/- 30 ps), which was assigned to the time of reduction of the primary quinone, Q(A), was also observed. The transient absorption spectra and kinetics all indicate that only one electron-transfer branch is involved in primary charge separation under these excitation conditions. However, the transient absorption changes upon excitation in the Soret band at 390 nm reveal a more complex set of energy and electron-transfer processes. By comparison to studies on the RCs of the purple bacterium Rhodobacter sphaeroides, we discuss the possible mechanism of electron-transfer pathway dependence on excitation energy and propose a model of the Cf. aurantiacus RC that better explains the observed results.     

Water Activity Regulates the Q(A)(-) to Q(B) electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides

We report on the effects of water activity and surrounding viscosity on electron transfer reactions taking place within a membrane protein: the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides. We measured the kinetics of charge recombination between the primary photoxidized donor (P(+)) and the reduced quinone acceptors. Water activity (aW) and viscosity (eta) have been tuned by changing the concentration of cosolutes (trehalose, sucrose, glucose, and glycerol) and the temperature. The temperature dependence of the rate of charge recombination between the reduced primary quinone, Q(A)(-), and P(+) was found to be unaffected by the presence of cosolutes. At variance, the kinetics of charge recombination between the reduced secondary quinone (Q(B)(-)) and P(+) was found to be severely influenced by the presence of cosolutes and by the temperature. Results collected over a wide eta-range (2 orders of magnitude) demonstrate that the rate of P(+)Q(B)(-) recombination is uncorrelated to the solution viscosity. The kinetics of P(+)Q(B)(-) recombination depends on the P(+)Q(A)(-)Q(B) <--> P(+)Q(A)Q(B)(-) equilibrium constant. Accordingly, the dependence of the interquinone electron transfer equilibrium constant on T and aW has been explained by assuming that the transfer of one electron from Q(A)(-) to Q(B) is associated with the release of about three water molecules by the RC. This implies that the interquinone electron transfer involves at least two RC substates differing in the stoichiometry of interacting water molecules.     

Afterglow thermoluminescence measured in isolated chloroplasts

The thermoluminescence afterglow (AG) measured in plant leaves originates from the S(2)/S(3)Q(B)(-) charge pair recombination in photosystem II (PSII) initiated by reverse electron flow from stromal reductants to PQ and then to the Q(B) site in PSII centers that are in the S(2)/S(3)Q(B) state. In this study, we show that this luminescence, absent in isolated thylakoid membranes, can be measured in intact chloroplasts that retain their stromal content including the electron acceptor pool (oxidized ferredoxin/NADP(+)) of photosystem I. The properties of the chloroplasts AG emission is similar to the AG in leaves in terms of temperature maximum, period-four modulation, far-red light stimulation, and antimycin A inhibition.     

Spectroscopic study of trypsin, heat and triton X-100-induced denaturation of the chlorophyll-binding protein CP43

Trypsin-, heat- and Triton X-100-induced denaturation of CP43, the core antenna complex of photosystem II purified from spinach, has been investigated using absorption, fluorescence and circular dichroism spectroscopy. Triton X-100 was found to bring about considerable dissolution of pigments from the protein to the monomeric state in solution and destruction of the interactions among the chlorophyll, carotene and protein. Heat induced significant unfolding of the protein secondary structure and loss of excitonic interactions of the pigments, but no apparent dissolution of the pigments from CP43. Trypsin caused structural changes in the extrinsic part of the protein but no change of the native state of the pigments. Trypsin, heat and Triton X-100 treatments increased the light sensitivity of chlorophyll in CP43 to different extents. The results suggest that the protein and beta-carotene can protect the chlorophyll from light-induced destruction in CP43.     

The photophysics of monomeric bacteriochlorophylls c and d and their derivatives: properties of the triplet state and singlet oxygen photogeneration and quenching

Measurements of pigment triplet-triplet absorption, pigment phosphorescence and photosensitized singlet oxygen luminescence were carried out on solutions containing monomeric bacteriochlorophylls (Bchl) c and d, isolated from green photosynthetic bacteria, and their magnesium-free and farnesyl-free analogs. The energies of the pigment triplet states fell in the range 1.29-1.34 eV. The triplet lifetimes in aerobic solutions were 200-250 ns; they increased to 280 +/- 70 microseconds after nitrogen purging in liquid solutions and to 0.7-2.1 ms in a solid matrix at ambient or liquid nitrogen temperatures. Rate constants for quenching of the pigment triplet state by oxygen were (2.0-2.5) x 10(9) M-1 s-1, which is close to 1/9 of the rate constant for diffusion-controlled reactions. This quenching was accompanied by singlet oxygen formation. The quantum yields for the triplet state formation and singlet oxygen production were 55-75% in air-saturated solutions. Singlet oxygen quenching by ground-state pigment molecules was observed. Quenching was the most efficient for magnesium-containing pigments, kq = (0.31-1.2) x 10(9) M-1 s-1. It is caused mainly by a physical process of singlet oxygen (1O2) deactivation. Thus, Bchl c and d and their derivatives, as well as chlorophyll and Bchl a, combine a high efficiency of singlet oxygen production with the ability to protect photochemical and photobiological systems against damage by singlet oxygen.     

Redox state of the photosynthetic electron transport chain in wild-type and mutant leaves of Arabidopsis thaliana: Impact on photosystem II fluorescence

In addition to the photosynthetic linear electron transport, several alternative electron transport routes exist in thylakoids of higher plants. The plastoquinone (PQ) pool acts as a common electron carrier in these pathways. In the cyclic electron flow around photosystem I (PSI), reduced ferredoxin is used by the ferredoxin-quinone reductase (FQR) to reduce the PQ pool. Chlororespiratory pathway consists in the reduction of the PQ pool by the NAD(P)H dehydrogenase (NDH). These alternative pathways and their role in photosynthesis are still not fully understood. In the present study, the accumulation kinetics of quinone acceptors was measured by fluorescence induction in leaves of Arabidopsis thaliana wild-type and mutants altered in alternative electron pathways after various light- and dark-adaptation conditions. Results show that NDH activity can be probed by fluorescence induction during light-to-dark transition of plants. Also, the activity of FQR pathway did not affect directly the FI kinetics. However, the accumulation kinetics of reduced PQ under actinic light was dependant on the redox state of PSI acceptors prior to illumination.     

非均一交联高分子网络的结构表征

均一交联高分子网络在良溶剂和劣溶剂中平衡溶胀比Q和Q_0的比值依从Q/Q_0=1+k(Q-1)的关系,k是一个与溶剂性质和网络种类有关的恒定常数.当k已知时,测定非均一网络在良、劣两种溶剂中溶胀比Q~*和Q_0~*,依 Z=k(Q~*-1)(Q_0~*-1)/[(1-k)(Q~*-1)-(Q_0~*-1)],可得非均一因子Z及其他网络结构参数.用此方法研究了自由基引发的苯乙烯-二乙烯基苯无规共聚网络,所得结果符合聚合和网络形成机理的预计.     

Excited-state proton transfer through water bridges and structure of hydrogen-bonded complexes in 1H-pyrrolo[3,2-h]quinoline: adiabatic time-dependent density functional theory study

Proton transfer reaction is studied for 1H-pyrrolo[3,2-h]quinoline-water complexes (PQ-(H(2)O)(n), n = 0-2) in the ground and the lowest excited singlet states at the density functional theory (DFT) level. Cyclic hydrogen-bonded complexes are considered, in which water molecules form a bridge connecting the proton donor (pyrrole NH group) and acceptor (quinoline nitrogen) atoms. To understand the effect of the structure and length of water bridges on the excited-state tautomerization in PQ, the potential energy profile of the lowest excited singlet state is calculated adiabatically by the time-dependent DFT (TDDFT) method. The S(0) --> S(1) excitation of PQ is accompanied by significant intramolecular transfer of electron density from the pyrrole ring to the quinoline fragment, so that the acidity of the N-H group and the basicity of the nitrogen atom of the quinoline moiety are increased. These excited-state acid-base changes introduce a driving force for the proton transfer reaction. The adiabatic TDDFT calculations demonstrate, however, that the phototautomerization requires a large activation energy in the isolated PQ molecule due to a high energy barrier separating the normal form and the tautomer. In the 1:1 cyclic PQ-H(2)O complex, the energy barrier is dramatically reduced, so that upon excitation of this complex the tautomerization can occur rapidly in one step as concerted asynchronous movements of the two protons assisted by the water molecule. In the PQ-(H(2)O)(2) solvate two water molecules form a cyclic bridge with sterically strained and unfavorable hydrogen bonds. As a result, some extra activation energy is needed for initiating the proton dislocation along the longer hydrogen-bond network. The full tautomerization in this complex is still possible; however, the cooperative proton transfer is found to be highly asynchronous. Large relaxation and reorganization of the hydrogen-bonded water bridge in PQ-(H(2)O)(2) are required during the proton translocation from the pyrrole NH group to the quinoline nitrogen; this may block the complete tautomerization in this type of solvate.     

Coupling of electron and proton transport in photosynthetic membranes: molecular mechanism

Using the method of Modified Neglect of Diatomic Overlap (MNDO), the electronic structure of plastoquinol (PQH(2)) and plastoquinone (PQ) in neutral, singly (PQ(-)) and doubly (PQ(2-)) reduced states is studied. The conformational analysis performed on these molecules shows that in the lowest energy conformation, the angle between the first link of the tail backbone and the ring plane of neutral and singly reduced PQ and plastoquinol is nearly the same and differs by 15 degrees from that of doubly reduced PQ. Nevertheless, for all states of plastoquinone and for plastoquinol, the total energy changes by less than 0.2 eV when the studied angle is varied from 0 degrees to 180 degrees. As in Rhodobacter sphaeroides, the oxygen of the PQ ring is reported to form a hydrogen bond with a nitrogen in the ring of Histidine (His) L 190. The energy of the PQ-His complex was calculated for different redox states of PQ and for several values of the distance between the molecules (N-O distance from 0.2 to 0.5 nm). For every considered complexes, the total energy dependence on the proton position on the line connecting the N and O atoms was determined, to see if the hydrogen bond is formed. It is shown that for only singly reduced PQ this dependence has a symmetric two-well form, i.e. the hydrogen bond is formed. For neutral and doubly reduced PQ, the curve is also two-well but asymmetric, so that the proton is bound to His or to PQ, correspondingly.On the basis of these results, we propose the following scheme of electron-proton coupling. Negatively charged oxygens of PQ form H-bonds with proton donor groups of the surrounding protein and fix PQ in its pocket. While the negative charges of oxygens increase after quinone reduction, protons shift to PQ oxygens and form strong hydrogen bonds with them. Upon second PQ reduction, protons are torn away from surrounding amino acids and form covalent bonds with the quinol. Resulting PQH(2) detaches from its binding place and is replaced by a neutral PQ. The lacking protons on amino acids in the Q(B) pocket are replaced by a step-by-step transfer from the stroma bulk through the proton channels.     

Excited state dynamics in photosynthetic reaction center and light harvesting complex 1

Key to efficient harvesting of sunlight in photosynthesis is the first energy conversion process in which electronic excitation establishes a trans-membrane charge gradient. This conversion is accomplished by the photosynthetic reaction center (RC) that is, in case of the purple photosynthetic bacterium Rhodobacter sphaeroides studied here, surrounded by light harvesting complex 1 (LH1). The RC employs six pigment molecules to initiate the conversion: four bacteriochlorophylls and two bacteriopheophytins. The excited states of these pigments interact very strongly and are simultaneously influenced by the surrounding thermal protein environment. Likewise, LH1 employs 32 bacteriochlorophylls influenced in their excited state dynamics by strong interaction between the pigments and by interaction with the protein environment. Modeling the excited state dynamics in the RC as well as in LH1 requires theoretical methods, which account for both pigment-pigment interaction and pigment-environment interaction. In the present study we describe the excitation dynamics within a RC and excitation transfer between light harvesting complex 1 (LH1) and RC, employing the hierarchical equation of motion method. For this purpose a set of model parameters that reproduce RC as well as LH1 spectra and observed oscillatory excitation dynamics in the RC is suggested. We find that the environment has a significant effect on LH1-RC excitation transfer and that excitation transfers incoherently between LH1 and RC.