Secondary pair charge recombination in photosystem I under strongly reducing conditions: temperature dependence and suggested mechanism.

Photoinduced electron transfer in photosystem I (PS I) proceeds from the excited primary electron donor P700 (a chlorophyll a dimer) via the primary acceptor A0 (chlorophyll a) and the secondary acceptor A1 (phylloquinone) to three [4Fe-4S] clusters, Fx, FA, and FB. Prereduction of the iron-sulfur clusters blocks electron transfer beyond A1. It has been shown previously that, under such conditions, the secondary pair P700+A1- decays by charge recombination with t1/2 approximately 250 ns at room temperature, forming the P700 triplet state (3P700) with a yield exceeding 85%. This reaction is unusual, as the secondary pair in other photosynthetic reaction centers recombines much slower and forms directly the singlet ground state rather than the triplet state of the primary donor. Here we studied the temperature dependence of secondary pair recombination in PS I from the cyanobacterium Synechococcus sp. PCC6803, which had been illuminated in the presence of dithionite at pH 10 to reduce all three iron-sulfur clusters. The reaction P700+A1- --> 3P700 was monitored by flash absorption spectroscopy. With decreasing temperature, the recombination slowed down and the yield of 3P700 decreased. In the range between 303 K and 240 K, the recombination rates could be described by the Arrhenius law with an activation energy of approximately 170 meV. Below 240 K, the temperature dependence became much weaker, and recombination to the singlet ground state became the dominating process. To explain the fast activated recombination to the P700 triplet state, we suggest a mechanism involving efficient singlet to triplet spin evolution in the secondary pair, thermally activated repopulation of the more closely spaced primary pair P700+A0- in a triplet spin configuration, and subsequent fast recombination (intrinsic rate on the order of 10(9) s(-1)) forming 3P700.     

Modulation of primary radical pair kinetics and energetics in photosystem II by the redox state of the quinone electron acceptor Q(A)

Time-resolved photovoltage measurements on destacked photosystem II membranes from spinach with the primary quinone electron acceptor Q(A) either singly or doubly reduced have been performed to monitor the time evolution of the primary radical pair P680(+)Pheo(-). The maximum transient concentration of the primary radical pair is about five times larger and its decay is about seven times slower with doubly reduced compared with singly reduced Q(A). The possible biological significance of these differences is discussed. On the basis of a simple reversible reaction scheme, the measured apparent rate constants and relative amplitudes allow determination of sets of molecular rate constants and energetic parameters for primary reactions in the reaction centers with doubly reduced Q(A) as well as with oxidized or singly reduced Q(A). The standard free energy difference DeltaG degrees between the charge-separated state P680(+)Pheo(-) and the equilibrated excited state (Chl(N)P680)* was found to be similar when Q(A) was oxidized or doubly reduced before the flash (approximately -50 meV). In contrast, single reduction of Q(A) led to a large change in DeltaG degrees (approximately +40 meV), demonstrating the importance of electrostatic interaction between the charge on Q(A) and the primary radical pair, and providing direct evidence that the doubly reduced Q(A) is an electrically neutral species, i.e., is doubly protonated. A comparison of the molecular rate constants shows that the rate of charge recombination is much more sensitive to the change in DeltaG degrees than the rate of primary charge separation.     

Oxidation-reduction thermodynamics of the acceptor quinone complex in whole-membrane fragments from Chloroflexus aurantiacus

Oxidation-reduction thermodynamic equilibria involving the quinone-acceptor complex have been examined in whole-membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to -50 mV at pH 8.2, in whole-membrane fragments it titrated at -210 mV (pH 8.0), with a pH dependence of -60 mV/pH up to a pK value of 9.3. o-Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o-phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ(-)B and free energy and kinetic relations between Q(-)AQB and QAQ(-)B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: (Formula: see text). We found that the charge recombination pathway of D+QAQ(-)B proceeds via the intermediate state D+Q(-)AQB, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product kAD-times the fraction of reaction centers in the Q-AQB state) with the observed recombination rate, kobsD+----D. The kinetic measurements were used to obtain the pH dependence (6.1 smaller than or equal to pH smaller than or equal to 11.7) of the free energy difference between the states Q(-)AQB and QAQ(-)B. At low pH (less than 9) QAQ(-)B is stabilized relative to Q(-)AQB by 67 meV, whereas at high pH Q(-)AQB is energetically favored. Both Q(-)A and Q(-)B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q(-)B provides the driving force for the forward electron transfer.     

Energetics of Photosystem II charge recombination in Acaryochloris marina studied by thermoluminescence and flash-induced chlorophyll fluorescence measurements

We studied the charge recombination characteristics of Photosystem II (PSII) redox components in whole cells of the chlorophyll (Chl) d-dominated cyanobacterium, Acaryochloris marina, by flash-induced chlorophyll fluorescence and thermoluminescence measurements. Flash-induced chlorophyll fluorescence decay was retarded in the mus and ms time ranges and accelerated in the s time range in Acaryochloris marina relative to that in the Chl a-containing cyanobacterium, Synechocystis PCC 6803. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, which blocks the Q(B) site, the relaxation of fluorescence decay arising from S(2)Q(A)(-) recombination was somewhat faster in Acaryochloris marina than in Synechocystis PCC 6803. Thermoluminescence intensity of the so called B band, arising from the recombination of the S(2)Q(B)(-) charge separated state, was enhanced significantly (2.5 fold) on the basis of equal amounts of PSII in Acaryochloris marina as compared with Synechocystis 6803. Our data show that the energetics of charge recombination is modified in Acaryochloris marina leading to a approximately 15 meV decrease of the free energy gap between the Q(A) and Q(B) acceptors. In addition, the total free energy gap between the ground state and the excited state of the reaction center chlorophyll is at least approximately 25-30 meV smaller in Acaryochloris marina, suggesting that the primary donor species cannot consist entirely of Chl a in Acaryochloris marina, and there is a contribution from Chl d as well.     

P+QA- and P+QB- charge recombinations in Rhodopseudomonas viridis chromatophores and in reaction centers reconstituted in phosphatidylcholine liposomes. Existence of two conformational states of the reaction centers and effects of pH and o-phenanthroline

The P+QA- and P+QB- charge recombination decay kinetics were studied in reaction centers from Rhodopseudomonas viridis reconstituted in phosphatidylcholine bilayer vesicles (proteoliposomes) and in chromatophores. P represents the primary electron donor, a dimer of bacteriochlorophyll; QA and QB are the primary and secondary stable quinone electron acceptors, respectively. In agreement with recent findings for reaction centers isolated in detergent [Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54-65] the P+QA- decay kinetics were biphasic (kfast and kslow). Arrhenius plots of the kinetics were linear, in agreement with the hypothesis of a thermally activated process (probably via P+I-; I is the first electron acceptor, a bacteriopheophytin) for the P+QA- charge recombination. Similar activation free energies (delta G) for this process were found in chromatophores and in proteoliposomes. Significant pH dependences of kfast and kslow were observed in chromtophores and in proteoliposomes. In the pH range 5.5-11, the pH titration curves of kfast and kslow were interpreted in terms of the existence of three protonable groups, situated between I- and QA-, which modulate the free energy difference between P+I- and P+QA-. In proteoliposomes, a marked effect of o-phenanthroline was observed on two of the three pKs, shifting one of them by more than 2 pH units. On the basis of recent structural data, we suggest a possible interpretation for this effect, which is much smaller in Rhodobacter sphaeroides. The decay kinetics of P+QB- were also biphasic. Marked pH dependences of the rate constants and of the relative proportions of both phases were also detected for these decays. The major conclusion of this work comes from the biphasicity of the P+QB- decay kinetics. We had suggested previously that biphasicity of the P+QA- charge recombination in Rps. viridis comes from nonequilibrium between protonation states of the reaction centers due to comparable rates of the protonation events and charge recombination. This hypothesis does not hold since the P+QB- decays occur on a time scale (tau approximately 300 ms at pH 8) much longer than protonation events. This leads to the conclusion that kfast and kslow (for both P+QA- and P+QB-) are related to conformational states of the reaction centers, existing before the flash. In addition, the fast and slow decays of P+QB- are related to those measured for P+QA-, via the calculations of the QA-QB in equilibrium QAQB- apparent equilibrium constants, K2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutational control of bioenergetics of bacterial reaction center probed by delayed fluorescence

The free energy gap between the metastable charge separated state P⁺Q_A⁻ and the excited bacteriochlorophyll dimer P* was measured by delayed fluorescence of the dimer in mutant reaction center proteins of the photosynthetic bacterium Rhodobacter sphaeroides. The mutations were engineered both at the donor (L131L, M160L, M197F and M202H) and acceptor (M265I and M234E) sides. While the donor side mutations changed systematically the number of H-bonds to P, the acceptor side mutations modified the energetics of Q_A by altering the van-der-Waals and electronic interactions (M265IT) and H-bond network to the acidic cluster around Q_B (M234EH, M234EL, M234EA and M234ER). All mutants decreased the free energy gap of the wild type RC (~ 890 meV), i.e. destabilized the P⁺Q_A⁻ charge pair by 60–110 meV at pH 8. Multiple modifications in the hydrogen bonding pattern to P resulted in systematic changes of the free energy gap. The destabilization showed no pH-dependence (M234 mutants) or slight increase (WT, donor-side mutants and M265IT above pH 8) with average slope of 10–15 meV/pH unit over the 6–10.5 pH range. In wild type and donor-side mutants, the free energy change of the charge separation consisted of mainly enthalpic term but the acceptor side mutants showed increased entropic (even above that of enthalpic) contributions. This could include softening the structure of the iron ligand (M234EH) and the Q_A binding pocket (M265IT) and/or increase of the multiplicity of the electron transfer of charge separation in the acceptor side upon mutation.     

Calculated protein and proton motions coupled to electron transfer: electron transfer from QA- to QB in bacterial photosynthetic reaction centers.

Reaction centers from Rhodobacter sphaeroides were subjected to Monte Carlo sampling to determine the Boltzmann distribution of side-chain ionization states and positions and buried water orientation and site occupancy. Changing the oxidation states of the bacteriochlorophyll dimer electron donor (P) and primary (QA) and secondary (QB) quinone electron acceptors allows preparation of the ground (all neutral), P+QA-, P+QB-, P0QA-, and P0QB- states. The calculated proton binding going from ground to other oxidation states and the free energy of electron transfer from QA-QB to form QAQB- (DeltaGAB) compare well with experiment from pH 5 to pH 11. At pH 7 DeltaGAB is measured as -65 meV and calculated to be -80 meV. With fixed protein positions as in standard electrostatic calculations, DeltaGAB is +170 meV. At pH 7 approximately 0.2 H+/protein is bound on QA reduction. On electron transfer to QB there is little additional proton uptake, but shifts in side chain protonation and position occur throughout the protein. Waters in channels leading from QB to the surface change site occupancy and orientation. A cluster of acids (GluL212, AspL210, and L213) and SerL223 near QB play important roles. A simplified view shows this cluster with a single negative charge (on AspL213 with a hydrogen bond to SerL233) in the ground state. In the QB- state the cluster still has one negative charge, now on the more distant AspL210. AspL213 and SerL223 move so SerL223 can hydrogen bond to QB-. These rearrangements plus other changes throughout the protein make the reaction energetically favorable.     

Free energy change accompanying electron transfer from P870 to quinone in Rhodopseudomonas sphaeroides chromatophores

Delayed fluorescence of chromatophores of Rhodopseudomonas sphaeroides was measured to estimate the standard free energy change accompanying the electron transfer from the bacteriochlorophyll dimer (P) to the primary acceptor quinone (Q_A). The chromatophores emitted delayed fluorescence with a lifetime of about 60 ms in the presence of o-phenanthroline. By comparing the intensity of the delayed fluorescence with that of the prompt fluorescence, the standard free energy of the P⁺Q_A^? radical pair was evaluated. It was about 0.87 eV below the level of excited singlet state, P1Q_A, or 0.51 eV above the ground state, PQ_A, independent of pH.     

Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier

The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range. The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse. The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6). The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV. This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45.     

Singlet oxygen production in photosynthesis

A photosynthetic organism is subjected to photo-oxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by beta-carotene, alpha-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Protein/lipid interaction in the bacterial photosynthetic reaction center: phosphatidylcholine and phosphatidylglycerol modify the free energy levels of the quinones

The role of characteristic phospholipids of native membranes, phosphatidylcholine (PC), phosphatidylglycerol (PG), and cardiolipin (CL), was studied in the energetics of the acceptor quinone side in photosynthetic reaction centers of Rhodobacter sphaeroides. The rates of the first, k(AB)(1), and the second, k(AB)(2), electron transfer and that of the charge recombination, k(BP), the free energy levels of Q(A)(-)Q(B) and Q(A)Q(B)(-) states, and the changes of charge compensating protein relaxation were determined in RCs incorporated into artificial lipid bilayer membranes. In RCs embedded in the PC vesicle, k(AB)(1) and k(AB)(2) increased (from 3100 to 4100 s(-1) and from 740 to 3300 s(-1), respectively) and k(BP) decreased (from 0.77 to 0.39 s(-1)) compared to those measured in detergent at pH 7. In PG, k(AB)(1) and k(BP) decreased (to values of 710 and 0.26 s(-1), respectively), while k(AB)(2) increased to 1506 s(-1) at pH 7. The free energy between the Q(A)(-)Q(B) and Q(A)Q(B)(-) states decreased in PC and PG (DeltaG degrees (Q)A-(Q)B(-->)(Q)A(Q)B- = -76.9 and -88.5 meV, respectively) compared to that measured in detergent (-61.8 meV). The changes of the Q(A)/Q(A)(-) redox potential measured by delayed luminescence showed (1) a differential effect of lipids whether RC incorporated in micelles or vesicles, (2) an altered binding interaction between anionic lipids and RC, (3) a direct influence of PC and PG on the free energy levels of the primary and secondary quinones probably through the intraprotein hydrogen-bonding network, and (4) a larger increase of the Q(A)/Q(A)(-) free energy in PG than in PC both in detergent micelles and in single-component vesicles. On the basis of recent structural data, implications of the binding properties of phospholipids to RC and possible interactions between lipids and electron transfer components will be discussed.     

The effect of an applied electric field on the charge recombination kinetics in reaction centers reconstituted in planar lipid bilayers.

Reaction Centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides were incorporated in planar bilayers made from monolayers derived from liposomes reconstituted with purified RCs. The photocurrents associated with the charge recombination process between the reduced primary quinone (QA-) and the oxidized bacteriochlorophyll donor (D+) were measured as a function of voltage (-150 mV less than V less than 150 mV) applied across the bilayer. When QA was the native ubiquinone (UQ) the charge recombination was voltage independent. However, when UQ was replaced by anthraquinone (AQ), the recombination time depended on the applied voltage V according to the relation tau = 8.5 X 10(-3) eV/0.175S. These results were explained by a simple model in which the charge recombination from UQ- proceeds directly to D+ while that from AQ occurs via a thermally activated intermediate state, D+I-QA, where I is the intermediate acceptor. The voltage dependence arises from an electric field induced change in the energy gap, delta G0, between the states D+I-QA and D+IQA-. This model is supported by the measured temperature dependence of the charge recombination time, which for RCs with AQ gave a value of delta G0 = 340 +/- 20 meV. In contrast, delta G0 for RCs with UQ as the primary acceptor, is sufficiently large (approximately 550 meV) so that even in the presence of the field, the direct pathway dominates. The voltage dependence shows that the electron transfer from I- to QA is electrogenic. From a quantitative analysis of the voltage dependence on the recombination rate it was concluded that the component of the distance between I and QA along the normal to the membrane is about one-seventh of the thickness of the membrane. This implies that the electron transfer from I to Q contributes at least one-seventh to the potential generated by the charge separation between D+ and QA-.     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

Effects of mutations near the bacteriochlorophylls in reaction centers from Rhodobacter sphaeroides.

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.     

The balance between primary forward and back reactions in bacterial photosynthesis.

The temperature dependence of the bacteriochlorophyll fluorescence and reaction center triplet yield in while cells of Rhodopseudomonas sphaeroides strain 2.4.1 and of the magnetic field-induced fluorescence increase are calculated, taking into account rate constants of losses in the antenna system and of charge separation and recombination in the reaction center. Triplet and singlet yield after recombination in the reaction center are described by the radical pair mechanism. Good fits of the theoretically calculated temperature dependence with published experimental results could be obtained, assuming that ks, the rate constant for recombination of the charges on the primary donor P+ and the reduced intermediate acceptor I- to the lowest excited singlet state P*I of the reaction center bacteriochlorophyll, is temperature-dependent via the Boltzmann factor Kso exp(-delta E/kT), where delta E is the energy difference between P*I and P+I- and kso is the frequency factor. kg and/or kt, the rate constants for recombination to the singlet ground and triplet states, respectively, were assumed to be temperature-independent, or temperature-dependent via their exothermicity factors ki = CiT-1/2 exp(-Ei/kT) with i = g, t. Depending on the particular choice for the temperature dependence of kg and kt, best fits were obtained for delta E = 45-75 meV and recombination rate constants at 300 K of ks = 0.4-0.8 ns-1, kg = 0.08-0.12 ns-1, and kt = 0.3-0.5 ns-1. The model predicts a lifetime of the radical pair P+I- that is somewhat larger than that of delayed fluorescence; a magnetic field increases both.     

Study of QB- stabilization in herbicide-resistant mutants from the purple bacterium Rhodopseudomonas viridis

The pH dependences of the rate constants of P+QB- (kBP) and P+QA- (kAP) charge recombination decays have been studied by flash-induced absorbance change technique, in chromatophores of three herbicide-resistant mutants from Rhodopseudomonas (Rps.) viridis, and compared to the wild type. P, QA, and QB are the primary electron donor and the primary and the secondary quinone acceptors, respectively. The triazine resistant mutants T1 (Arg L217----His and Ser L223----Ala), T3 (Phe L216----Ser and Val M263----Phe), and T4 (Tyr L222----Phe), all mutated in the QB binding pocket of the reaction center, have previously been characterized (Sinning, I., Michel, H., Mathis, P., & Rutherford, A. W. (1989) Biochemistry 28, 5544-5553). The pH dependence curves of kBP in T4 and the wild type are very close. This confirms that the sensitivity toward DCMU of T4 is mainly due to a structural rearrangement in the QB pocket rather than to a change in the charge distribution in this part of the protein. In T3, a 6-fold increase of kAP is observed (kAP = 4200 +/- 300 s-1 at pH 8) compared to that of the wild type (kAP = 720 +/- 50 s-1 at pH 8). We propose that the Val M263----Phe mutation induces a free energy decrease between P+QA- and P+I- (delta G zero IA) (I is the primary electron acceptor) of about 49 meV. The very different pH dependence of kAP in T3 suggests a substantial change in the QA pocket. The 2.5 times increase of kAP above pH 9.5 in the wild type is no longer detected in T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.

The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.     

Changes in the redox potential of primary and secondary electron-accepting quinones in photosystem II confer increased resistance to photoinhibition in low-temperature-acclimated Arabidopsis

Exposure of control (non-hardened) Arabidopsis leaves for 2 h at high irradiance at 5 degrees C resulted in a 55% decrease in photosystem II (PSII) photochemical efficiency as indicated by F(v)/F(m). In contrast, cold-acclimated leaves exposed to the same conditions showed only a 22% decrease in F(v)/F(m). Thermoluminescence was used to assess the possible role(s) of PSII recombination events in this differential resistance to photoinhibition. Thermoluminescence measurements of PSII revealed that S(2)Q(A)(-) recombination was shifted to higher temperatures, whereas the characteristic temperature of the S(2)Q(B)(-) recombination was shifted to lower temperatures in cold-acclimated plants. These shifts in recombination temperatures indicate higher activation energy for the S(2)Q(A)(-) redox pair and lower activation energy for the S(2)Q(B)(-) redox pair. This results in an increase in the free-energy gap between P680(+)Q(A)(-) and P680(+)Pheo(-) and a narrowing of the free energy gap between primary and secondary electron-accepting quinones in PSII electron acceptors. We propose that these effects result in an increased population of reduced primary electron-accepting quinone in PSII, facilitating non-radiative P680(+)Q(A)(-) radical pair recombination. Enhanced reaction center quenching was confirmed using in vivo chlorophyll fluorescence-quenching analysis. The enhanced dissipation of excess light energy within the reaction center of PSII, in part, accounts for the observed increase in resistance to high-light stress in cold-acclimated Arabidopsis plants.