Delayed fluorescence study on P*QA→ P+QA − charge separation energetics linked to protons and salt in reaction centers from Rhodobacter sphaeroides

The energetics of the first stable charge separated state, P⁺Q_A– relative to that of P–Q_A was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (H = – 818 ± 20 meV at pH 8) and the free energy gap between P–Q_A and P⁺Q_A– drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P⁺Q_A– state runs parallel to the (integrated) net proton uptake due to the PQ_A/P⁺Q_A– redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pK_a values as well as the light-induced pK_a changes of the protonatable groups of the protein. The observed dependence of the energetics of P⁺Q_A– on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt.     

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.     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

A thermoluminescence study of Photosystem II back electron transfer reactions in rice leaves – effects of salt stress

The recombination reactions of Photosystem II have been investigated in vivo in rice leaves by using the thermoluminescence (TL) emission technique. Excitation of dark-adapted leaf segments at 0 °C with different number of single turn-over flashes induced the appearance of complex TL glow curves. The mathematical analysis of these curves showed the existence of four TL components: B1-band (temperature maximum, t_max, at 24 °C, originating from S₃Q_B⁻ recombination), B2-band (t_max at 35 °C, from S₂Q_B⁻), AG-band (t_max at 46 °C) and C-band (t_max at 55 °C, from Tyr_D⁺Q_A⁻). Their contributions to the total TL signal were different depending on the number of flashes given. AG-band seems to reflect a special electron transfer from some unknown stroma donor to PS II. Q-band (t_max at 19 °C), originating from S₂Q_A⁻ recombination, was recorded after flashing samples incubated in the presence of DCMU. The recombination halftimes (t_1/2) at 20 °C of S₂Q_A⁻, S₃Q_B⁻, S₂Q_B⁻ and Tyr_D⁺Q_A⁻ were, respectively, 0.8 s, 48 s, 74 s and about 1 h. A sharp AG-band (t_max at 50 °C and t_1/2 of 210 s) could be also observed after illumination of leaves with far-red light and after a dark incubation period of whole plants. Incubation of leaf segments with 0.5 M NaCl abolished the inductions of AG-band by darkness and far-red illumination, significantly decreased Q-band intensity, whereas induced a strong increase in C-band intensity. The possible inhibition of S₂/S₃ formation and quinone oxidation by saline stress are discussed.     

Quantification of non-Q B -reducing centers in leaves using a far-red pre-illumination

An alternative approach to quantification of the contribution of non-Q_B-reducing centers to Chl a fluorescence induction curve is proposed. The experimental protocol consists of a far-red pre-illumination followed by a strong red pulse to determine the fluorescence rise kinetics. The far-red pre-illumination induces an increase in the initial fluorescence level (F_{25 μs}) that saturates at low light intensities indicating that no light intensity-dependent accumulation of Q_A⁻ occurs. Far-red light-dose response curves for the F_{25 μs}-increase give no indication of superimposed period-4 oscillations. F_{25 μs}-dark-adaptation kinetics following a far-red pre-pulse, reveal two components: a faster one with a half-time of a few seconds and a slower component with a half-time of around 100 s. The faster phase is due to the non-Q_B-reducing centers that re-open by recombination between Q_A⁻ and the S-states on the donor side. The slower phase is due to the recombination between Q_B⁻ and the donor side in active PS II reaction centers. The pre-illumination-induced increase of the F_{25 μs}-level represents about 4–5% of the variable fluorescence for pea leaves (∼2.5% equilibrium effect and 1.8–3.0% non-Q_B-reducing centers). For the other plant species tested these values were very similar. The implications of these values will be discussed.     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P₆₈₀⁺Q_A⁻ limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P₆₈₀⁺ by the secondary electron donor Tyrosine Z cannot occur because Y_Z is already oxidized. The 1.4 ms recombination is seen in Y_Z-less mutants of the cyanobacterium Synechocystis. However, the rate of P₆₈₀⁺Q_A⁻ recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P₆₈₀⁺ reduction by Y_Z were obtained, and the rate of the competing P₆₈₀⁺Q_A⁻ recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y_Z^ox slightly decreased the efficiency of excitation trapping but did not seem to accelerate P₆₈₀⁺Q_A⁻ recombination. The two P₆₈₀⁺Q_A⁻ lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

The functional sites of chlorophylls in D1 and D2 subunits of Photosystem II identified by pulsed EPR

The functional site of Chl_Z, an auxiliary electron donor to P680⁺, was determined by pulsed ELDOR applied to a radical pair of Y_D^• and Chlz⁺ in oriented PS II membranes from spinach. The radical-radical distance was determined to be 29.5 Å and its direction was 50° from the membrane normal, indicating that a chlorophyll on the D2 protein is responsible for the EPR Chlz⁺ signal. Spin polarized ESEEM (Electronin Spin Echo Envelop Modulation) of a ³Chl and Q_A⁻ radical pair induced by a laser flash was observed in reaction center D1D2Cytb₅₅₉ complex, in which Q_A was functionally reconstituted with DBMIB and reduced chemically. Q_A⁻ESEEM showed a characteristic oscillating time profile due to dipolar coupling with ³Chl. By fitting with the dipolar interaction parameters, the distance between ³Chl and Q_A⁻ was determined to be 25.9 Å, indicating that the accessory chlorophyll on the D1 protein is responsible for the ³Chl signal.     

Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks

A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S₂Q⁻_A state while that at 25°C is attributed to recombination of S₂Q_AQ⁻_B (and S₃Q_AQ⁻_B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S₂Q_AQ⁻_B and S₃Q_AQ⁻_B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to Q_A and thence to Q_B or Q⁻_B) results in predictable changes in the extent of flash-induced thermoluminescence.     

Reaction center light harvesting B875 complexes from Rhodocyclus gelatinosus: characterization and identification of quinones

Reaction center-B875 pigment-protein complexes were purified from Rhodocyclus gelatinosus. The proteic components consist of 7–8 polypeptides among which some were identified by their apparent molecular weights: the light harvesting B875 polypeptides and of 8 and 6 kDa, reaction center L (23 kDa), M (28 kDa) and H (34 kDa), cytochrome c (43 kDa). Four c-type hemes were found per reaction center. Flash-induced absorbance changes showed the presence of both Q_A and Q_B in the complex. Charge recombination times were determined to be: 1.16±0.2 (n=30) for P⁺Q_AQ_B ^- and 7–10 ms for P⁺Q_A ^- in presence of herbicides. From quinone analysis on one hand and kinetics of charge recombination on the other hand, we proposed that in the reaction center of Rhodocyclus gelatinosus Q_A is menaquinone 8 and Q_B is ubiquinone 8.     

Effect of the redox state of QB on electric field-induced charge recombination in Photosystem II

Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts (blebs) by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate Q_B was observed and the earlier failure to detect it is explained. The influence of the Q_B/Q_B ^– oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states S₂ and S₃, but not in the state S₄ (actually Z⁺S₃). The presence of the oxidized secondary electron donor, tyrosine Z⁺, appeared to prevent expression of the Q_B/Q_B ^– effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z⁺ and the oxygen evolving complex.Abbreviations BSA bovine serum albumin - EDTA ethylene-diaminetetraacetic acid - EL electroluminescence - FCCP carbonylcyanide p-trifluoromethyloxyphenyl-hydrazone - HEPESI 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - I primary electron acceptor - MOPS 3-(N-morpholino) propane sulfonic acid - P680 primary electron donor of Photosystem II - P700 primary electron donor of Photosystem I - Q_A and Q_B secondary and tertiary electron acceptors of Photosystem II - Z secondary electron donor (D1 Tyr 161)     

Acceptor side photoinhibition in PS II: On the possible effects of the functional integrity of the PS II donor side on photoinhibition of stable charge separation

Photoinhibition under aerobic and anaerobic conditions was analyzed in O₂-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680⁺ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced Q_A(Q_A ^-). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated Q_A (Q_AH₂). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680^*Q_A P680⁺Q_A ^-), is nearly the same in O₂-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the Q_AH₂-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of Q_A leading to Q_AH₂ formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.     

Thermodynamics of the charge recombination in photosystem II

The temperature dependence of the electric field-induced chlorophyll luminescence in photosystem II was studied in Tris-washed, osmotically swollen spinach chloroplasts (blebs). The system II reaction centers were brought in the state Z⁺P⁺-Q_A ^-Q_B ^- by preillumination and the charge recombination to the state Z⁺PQ_AQ_B ^- was measured at various temperatures and electrical field strengths. It was found that the activation enthalpy of this back reaction was 0.16 eV in the absence of an electrical field and diminished with increasing field strength. It is argued that this energy is the enthalpy difference between the states IQ_A ^- and I^-Q_A and accounts for about half of the free energy difference between these states. The redox state of Q_B does not influence this free energy difference within 150 s after the photoreduction of Q_A. The consequences for the interpretation of thermodynamic properties of Q_A are discussed.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - I intermediary electron acceptor - Mops 3-(N-morpholino)propanesulphonic acid - P (P₆₈₀) primary electron donor - PS II photosystem II - Q_A and Q_B first and second quinone electron acceptors - Tricine N-tris(hydroxymethyl)methylglycine - Tris tris-(hydroxymethyl)aminomethane - Z secondary electron donor Dedicated to Professor L.N.M. Duysens on the occasion of his retirement     

Dynamics of photosystem II heterogeneity in Dunaliella salina (green algae)

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from Q_A to plastoquinone (Q_B-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from Q_A to Q_B (Q_B-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of Q_B-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 E m^-2s^-1), dark incubation induces an increase (half-time 45 min) in the Q_B-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of Q_B-nonreducing centers to 25%. In cells grown under low illumination intensity (30 µE m^–2s^–1), dark incubation elicits no change in the relative concentration of Q_B-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the Q_B-nonreducing pool size and a concomitant increase in the Q_B-reducing pool size. These and other results are explained in terms of a pool of Q_B-nonreducing centers existing in a steady-state relationship with Q_B-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane of D. salina. It is proposed that Q_B-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the Q_B-nonreducing pool to maintain a constant pool size of Q_B-nonreducing centers in the thylakoid membrane.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F_o non-variable fluorescence yield - F_pl intermediate fluorescence yield plateau level - F_max maximum fluorescence yield - F_i mitial fluorescence yield increase from F_o to F_pl(F_pl-F_o) - F_v total variable fluorescence yield (F_max-F_o) - DCMU dichlorophenyl-dimethylurea     

Recovery of photosystem I and II activities during re-hydration of lichen<Emphasis Type="Italic"> Hypogymnia physodes</Emphasis> thalli

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700⁺ and the reduced acceptor F_X of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700⁺ reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q_A^–, the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.Abbreviations A₀ and A₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - Chl a Chlorophyll a - F_m Maximal level of chlorophyll fluorescence when all PSII centers are closed - F_o Minimal level of fluorescence when all PSII centers are open after dark adaptation - FR Far-red - F_v Variable fluorescence (=F_m–F_o) - F_X, F_A, and F_B Iron–sulfur centers - MT pulse Multiple-turnover pulse - PS Photosystem - P700 Reaction center chlorophyll of PSI - Q_A Primary quinone acceptor of PSII - Q_B Secondary quinone acceptor of PSII - ST pulse Single-turnover pulse     

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S₂₍₃₎Q_B ^– (B band), S₂Q_A ^– (Q band), Tyr-D⁺Q_A ^– (C band) and Tyr-Z⁺(His⁺)Q_A ^– (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S₂₍₃₎Q_B ^– recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DT desiccation tolerant - PS II Photosystem II - TL thermoluminescence - P₆₈₀ reaction center Chl of PS II - Q_A and Q_B puinone electron acceptors of PS II - S₀,...,S₄ the redox states of the water-oxidizing complex - Tyr-Z and Tyr-D redox-active tyrosine electron donors of PS II     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II

Chlorophyll a fluorescence induction (FI) is widely used as a probe for studying photosynthesis. On illumination, fluorescence emission rises from an initial level O to a maximum P through transient steps, termed J and I. FI kinetics reflect the overall performance of photosystem II (PSII). Although FI kinetics are commonly and easily measured, there is a lack of consensus as to what controls the characteristic series of transients, partially because most of the current models of FI focus on subsets of reactions of PSII, but not the whole. Here we present a model of fluorescence induction, which includes all discrete energy and electron transfer steps in and around PSII, avoiding any assumptions about what is critical to obtaining O J I P kinetics. This model successfully simulates the observed kinetics of fluorescence induction including O J I P transients. The fluorescence emission in this model was calculated directly from the amount of excited singlet-state chlorophyll in the core and peripheral antennae of PSII. Electron and energy transfer were simulated by a series of linked differential equations. A variable step numerical integration procedure (ode15s) from MATLAB provided a computationally efficient method of solving these linked equations. This in silico representation of the complete molecular system provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the O J I P kinetics and fluorescence emission at these points. Simulations based on this model showed that J corresponds to the peak concentrations of Q _A ⁻ Q_B (Q_A and Q_B are the first and second quinone electron acceptor of PSII respectively) and Q _A ⁻ Q _B ⁻ and I to the first shoulder in the increase in concentration of Q _A ⁻ Q _B ²⁻ . The P peak coincides with maximum concentrations of both Q _A ⁻ Q _B ²⁻ and PQH₂. In addition, simulations using this model suggest that different ratios of the peripheral antenna and core antenna lead to differences in fluorescence emission at O without affecting fluorescence emission at J, I and P. An increase in the concentration of Q_B-nonreducing PSII centers leads to higher fluorescence emission at O and correspondingly decreases the variable to maximum fluorescence ratio (F _v/F _m).     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Effect of hydrogen bonds on the energetics of macromolecules in the course of electron transfer

For a model system consisting of a bacteriochlorophyll dimer (P) and a primary quinone with the nearest environment (Q_A), which are the electron donor and acceptor in the recombination reaction in the Rhodobacter spheroides reaction center, the energies of states P⁺Q _A ^? and PQ_A have been calculated at several stable conformations of Q_A that differ in the positions of the proton involved in the hydrogen bond. It is shown that the position of the proton has a considerable influence on the energy of vertical transition P⁺Q _A ^? → PQ_A.