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Light-induced oxidation of the acceptor-side Fe(II) of Photosystem II by exogenous quinones acting through the QB binding site. I. Quinones, kinetics and pH-dependence
Abstract
We have recently shown by optical, EPR and Mössbauer spectroscopy that the high spin Fe(II) of the quinone-iron acceptor complex of Photosystem II can be oxidized by ferricyanide to high-spin Fe(III). The midpoint potential of the Fe(III)/Fe(II) couple is 370 mV at pH 7.5 and shows an approximate pH-dependence of −60 mV/pH unit. The iron was identified as being responsible for the high potential Photosystem II acceptor known as Q400, discovered by Ikegami and Katoh ((1975) Plant Cell Physiol. 14, 829–836) but until now not identified chemically. We establish here that QA and the oxidized Fe(III) are linked in series, with QA the first to be reduced in the primary charge separation of Photosystem II. At pH 7.5, an electron is then transferred from Q−A to Fe(III) with a of 25 μs, reforming QA Fe(II). The Fe(II) can also be oxidized to Fe(III) in oxygen-evolving thylakoid membranes through a photoreduction-induced oxidation in the presence of exogenous quinones, where . Single turnover illumination of the Photosystem II reaction center at 200 K, followed by warming to 0°C, results in photoreduction of these quinones to the semiquinone form which in turn oxidizes the Fe(II) to Fe(III). A second turnover of the reaction center reduces Fe(III) back to Fe(II). These reactions, similar to those reported by Zimmermann and Rutherford (Zimmermann, J.L. and Rutherford, A.W. (1986) Biochim. Biophys. Acta 851, 416–423) at room temperature, in work largely done in parallel, are summarized below: where QA and Qex are the primary quinone acceptor of Photosystem II and exogenous quinone, respectively. Detection of Fe(III) at g = 8 by EPR spectroscopy shows this signal to oscillate with period two upon successive turnovers of the Photosystem II reaction center. Different exogenous quinones give different EPR spectra for Fe(III), indicating that these bind close to the Fe binding site and modify the symmetry of the Fe(III) environment. A study of the pH-dependence of the light-induced oxidation of the Fe(III) by phenyl-p-BQ shows a pH-optimum at 6–7. The decline at higher pH is consistent with a pH-dependence of −60 mV/pH unit and −120 mV/pH unit, respectively, for redox couples Fe(III)/Fe(II) and Q−/QH2. The decline at lower pH was not foreseen and appears associated with a transformation of the quinone-iron environment from that showing a Q−AFe(II) EPR resonance of g = 1.9 at high pH to one at g = 1.84 below pH 6.5. The latter form appears not to support light-induced oxidation of the Fe(II) by exogenous quinones.
... 12.6 ± 2.5 1.6 ± 0.4 2.1 ± 0.4 410 ± 40 277 [33] 62 [33] 2,5-DMBQ 15.6 ± 2.7 2.5 ± 0.5 6.2 ± 1.6 0.38 ± 0.15 810 ± 90 180 [31,34] -66 [34] 2,6-DMBQ 15.5 ± 2.8 2.1 ± 0.4 7.4 ± 1.9 0.53 ± 0.21 880 ± 90 174 [33] -80 [32,34] DQ 2.0 ± 0.5 < 0.4 n.d. < 0.2 a > 1000 b 52 [33] -254 [33] 2,6- [31] -140 [36] BQ ~ 0 ~ 0 n.d. ...
... 12.6 ± 2.5 1.6 ± 0.4 2.1 ± 0.4 410 ± 40 277 [33] 62 [33] 2,5-DMBQ 15.6 ± 2.7 2.5 ± 0.5 6.2 ± 1.6 0.38 ± 0.15 810 ± 90 180 [31,34] -66 [34] 2,6-DMBQ 15.5 ± 2.8 2.1 ± 0.4 7.4 ± 1.9 0.53 ± 0.21 880 ± 90 174 [33] -80 [32,34] DQ 2.0 ± 0.5 < 0.4 n.d. < 0.2 a > 1000 b 52 [33] -254 [33] 2,6- [31] -140 [36] BQ ~ 0 ~ 0 n.d. ...
... 12.6 ± 2.5 1.6 ± 0.4 2.1 ± 0.4 410 ± 40 277 [33] 62 [33] 2,5-DMBQ 15.6 ± 2.7 2.5 ± 0.5 6.2 ± 1.6 0.38 ± 0.15 810 ± 90 180 [31,34] -66 [34] 2,6-DMBQ 15.5 ± 2.8 2.1 ± 0.4 7.4 ± 1.9 0.53 ± 0.21 880 ± 90 174 [33] -80 [32,34] DQ 2.0 ± 0.5 < 0.4 n.d. < 0.2 a > 1000 b 52 [33] -254 [33] 2,6- [31] -140 [36] BQ ~ 0 ~ 0 n.d. ...
Among all the chemical and biotechnological strategies implemented to extract energy from oxygenic photosynthesis, several concern the use of intact photosynthetic organisms (algae, cyanobacteria…). This means rerouting (fully or partially) the electron flow from the photosynthetic chain to an outer collecting electrode generating thus a photocurrent. While diverting photosynthetic electrons from living biological systems is an encouraging approach, this strategy is limited by the need to use an electron shuttle. Redox mediators that are able to interact with an embedded photosynthetic chain are rather scarce. In this respect, exogenous quinones are the most frequently used. Unfortunately, some of them also act as poisoning agents within relatively long timeframes. It thus raises the question of the best quinone. In this work, we use a previously reported electrochemical device to analyze the performances of different quinones. Photocurrents (maximum photocurrent, stability) were measured from suspensions of Chlamydomonas reinhardtii algae/quinones by chronoamperometry and compared to parameters like quinone redox potentials or cytotoxic concentration. From these results, several quinones were synthesized and analyzed in order to find the best compromise between bioelectricity production and toxicity.
... Redox mechanism of activation is also not supported by experimental results rather convincingly. DCBQ has redox potential E m = 309 mV (Petrouleas and Diner 1987), whereas FSC redox potential is probably significantly lower. For example, redox potential of Fe(III)-fructose complex is about − 150 mV at pH 6.5 (Charley et al. 1963), i.e., FSC most likely cannot oxidize the reduced DCBQ. ...
... For example, redox potential of Fe(III)-fructose complex is about − 150 mV at pH 6.5 (Charley et al. 1963), i.e., FSC most likely cannot oxidize the reduced DCBQ. Moreover, PPBQ with similar E m = 279-290 mV (Petrouleas and Diner 1987) does not interact with FSC (Table 2). FSC cannot also oxidize the DCPIPH 2 (for DCPIP/DCPIPH 2 couple E m = 130/145 mV (Petrova et al. 2018) as we observed mixing the solutions of DCPIPH 2 and FSC. ...
Effect of water-soluble and stable sucrose-bound iron oxyhydroxide nanoparticles [Fe[III] sucrose complex (FSC)] on the efficiency of electron transport in the photosystem II membranes was studied. FSC significantly increases (by a factor 1.5) the rate of light-induced oxygen evolution in the presence of alternative electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ). Without DCBQ, FSC only slightly (5%) provides the oxygen evolution. Electron transport supported by pair DCBQ + FSC is inhibited by diuron. Maximum of stimulating effect was recorded at Fe(III) concentration 5 µM. In the case of another benzoquinone electron acceptor (2-phenyl-p-benzoquinone and 2,3-dimethyl-p-benzoquinone) and 2,6-dichlorophenolindophenol, stimulating effect of FSC was not observed. Incubation of PSII membranes at different concentrations with FSC is accompanied by binding of Fe(III) by membrane components but only about 50% of iron can be extracted by membranes from Fe(III) solution at pH 6.5. This result implies the heterogeneity of FSC solution in a buffer. The heterogeneity depends on pH and decreases with its rising. At pH around 9.0 Fe(III), sucrose solution is homogeneous. The study of pH effect has shown that stimulation of electron transport is induced only by iron cations which can be bound by membranes. Not extractable iron pool cannot activate electron transfer from oxygen-evolving complex to DCBQ.
... As shown in Fig. 2B, addition of the electron acceptor DMBQ produces much stronger oscillations in O 2 yield in both Sr-grown and Cagrown cultures, extending over 10 and 7 cycles, respectively. Titrations with DMBQ were done to determine the optimal concentration (250 μM), in a manner observed for all other cell types that we and others have investigated [29,31,41]. DMBQ introduces some photoinactivation seen as a decreasing slope. ...
... Measurements at the growth temperature (45°C , Table 2B) show there is a general decrease in misses, while backward transitions are somewhat increased (Ca) or unchanged (Sr). Double-hits are only observed in Ca-grown cells with DMBQ added and quite substantial (11.6%), indicating that Sr suppresses oxidation of the non-heme iron substantially relative to Ca [29,31,41] [42]. Measurement at growth temperature reveals the initial dark S-state populations in the Srgrown culture retain a significant fraction (5%) of WOCs in the S3 state with DMBQ added, but not in the S2 state, consistent with the slower decay of S3 (Table 1). ...
Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5–8 × slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45 °C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S = 5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S = 1/2 form.
... It can be assumed that such an AEA in the used pair of AEA-s can be each of these agents or one of them: DCBQ or FeCN. If we compare the potentials of substances oxidized by the Cu(II) cation: ascorbate E m,7 = +60 mV [77]; 1,5-diphenylcarbazide E m,7 = −300 mV [78]; glutathione E m,7 = −240 mV [79]; NADH E m,7 = −320 mV [77]; DCBQ E m,7 = +309 mV [80] and FeCN E m,7 = +420 mV [81], it is obvious that such an AEA can be DCBQ and not FeCN. On the other hand, it was shown that the redox potential of Cu(II) in the complex could differ from the reduction potential of the Cu(II) Cu(I) aqua couple (164 mV) [82] and be significantly more positive [83], for example, including a number of copper proteins: Rusticyanin (bacteria) 680 mV; Plastocyanin (plants) 370 mV; Amicyanin (bacteria) 294 mV; Stellacyanin (plants) 285 mV; and Azurin (bacteria) 276 mV [84]. ...
The effects of the novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide complex) on the photosystem II (PSII) activity of PSII membranes isolated from spinach were studied. The absence of photosynthetic oxygen evolution by PSII membranes without artificial electron acceptors, but in the presence of [CuL2]Br2, has shown that it is not able to act as a PSII electron acceptor. In the presence of artificial electron acceptors, [CuL2]Br2 inhibits photosynthetic oxygen evolution. [CuL2]Br2 also suppresses the photoinduced changes of the PSII chlorophyll fluorescence yield (FV) related to the photoreduction of the primary quinone electron acceptor, QA. The inhibition of both characteristic PSII reactions depends on [CuL2]Br2 concentration. At all studied concentrations of [CuL2]Br2, the decrease in the FM level occurs exclusively due to a decrease in Fv. [CuL2]Br2 causes neither changes in the F0 level nor the retardation of the photoinduced rise in FM, which characterizes the efficiency of the electron supply from the donor-side components to QA through the PSII reaction center (RC). Artificial electron donors (sodium ascorbate, DPC, Mn2+) do not cancel the inhibitory effect of [CuL2]Br2. The dependences of the inhibitory efficiency of the studied reactions of PSII on [CuL2]Br2 complex concentration practically coincide. The inhibition constant Ki is about 16 µM, and logKi is 4.8. As [CuL2]Br2 does not change the aromatic amino acids’ intrinsic fluorescence of the PSII protein components, it can be proposed that [CuL2]Br2 has no significant effect on the native state of PSII proteins. The results obtained in the present study are compared to the literature data concerning the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes.
... Fluorescence measurements of different PSII complexes versus an increasing DCBQ concentration, ranging from the millimolar to the nanomolar range, are shown in Figure 4. Titration of increasing concentrations of an electron acceptor to PSII decreased the fluorescence values measured at each acceptor concentration. Plastoquinone analogues, such as DCBQ, produce their fluorescence quenching effect by oxidizing Fe(II) through the QB binding pocket [38]. Oxidation occurs via an interaction with D1 His215 [35], a residue involved in coordinating the non-heme iron [6]. ...
Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.
... Kok's model has been extended during recent years in order to account for additional observations. These extensions include additional S i states below the S 0 state (S −1 , S −2 , S − 3 , S − 4 and S − 5 ) that are observed after reduction of the Mn 4 CaO 5 cluster with small water soluble reductants like NH 2 OH and NH 2 NH 2 [53][54][55], a special double hit in the first flash observed in samples with oxidized non-heme iron [49,56,57], and the loss of active centers during a flash train, which has been either accounted for by the introduction of an inactivated state S ε (accessed with the probability ε from all S i states) [58,59] or by an activity (dampening) parameter d [51,53]. Furthermore, first attempts were made to arrive at dynamic models that include the back reactions of S 2 and S 3 with Y D during the dark-times between flashes, either by explicitly including the reactions [51,60,61], or by introducing an empiric δ parameter [62]. ...
The oxygen-evolving complex (OEC) in photosystem II catalyzes the oxidation of water to molecular oxygen. Four decades ago, measurements of flash-induced oxygen evolution have shown that the OEC steps through oxidation states S0, S1, S2, S3 and S4 before O2 is released and the S0 state is reformed. The light-induced transitions between these states involve misses and double hits. While it is widely accepted that the miss parameter is S state dependent and may be further modulated by the oxidation state of the acceptor side, the traditional way of analyzing each flash-induced oxygen evolution pattern (FIOP) individually did not allow using enough free parameters to thoroughly test this proposal. Furthermore, this approach does not allow assessing whether the presently known recombination processes in photosystem II fully explain all measured oxygen yields during Si state lifetime measurements. Here we present a global fit program that simultaneously fits all flash-induced oxygen yields of a standard FIOP (2Hz flash frequency) and of 11-18 FIOPs each obtained while probing the S0, S2 and S3 state lifetimes in spinach thylakoids at neutral pH. This comprehensive data treatment demonstrates the presence of a very slow phase of S2 decay, in addition to the commonly discussed fast and slow reduction of S2 by YD and QB(-), respectively. Our data support previous suggestions that the S0→S1 and S1→S2 transitions involve low or no misses, while high misses occur in the S2→S3or S3→S0 transitions.
... 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), 2,6-dichloro-1,4-benzo-quinone (DCBQ, E m = +319 mV vs SHE, pH 7, determined via cyclic voltammetry in a three electrode system with a platinum mesh working electrode, a platinum counter electrode and an Ag/AgCl reference) and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1benzopyran-4-on (quercetin, E m = +331 mV vs SHE, pH 7, determined as described above for DCBQ), phenyl-p-benzoquinone (PpBQ) (E m = +279 mV vs SHE, pH 7 [43]) and additional chemicals were purchased from Sigma Aldrich. Nanostructured TiO 2 on conductive ITO glass were obtained from Solaronix S. A., Aubonne, Switzerland (20 nm particle size, 250-500 nm layer thickness) and used as electrodes for photocurrent generation. ...
We have investigated the nature of the photocurrent generated by Photosystem II (PSII), the water oxidizing enzyme, isolated from Thermosynechococcus elongatus, when immobilized on nanostructured titanium dioxide on an indium tin oxide electrode (TiO2/ITO). We investigated the properties of the photocurrent from PSII when immobilized as a monolayer versus multilayers, in the presence and absence of an inhibitor that binds to the site of the exchangeable quinone (QB) and in the presence and absence of exogenous mobile electron carriers (mediators). The findings indicate that electron transfer occurs from the first quinone (QA) directly to the electrode surface but that the electron transfer through the nanostructured metal oxide is the rate-limiting step. Redox mediators enhance the photocurrent by taking electrons from the nanostructured semiconductor surface to the ITO electrode surface not from PSII. This is demonstrated by photocurrent enhancement using a mediator incapable of accepting electrons from PSII. This model for electron transfer also explains anomalies reported in the literature using similar and related systems. The slow rate of the electron transfer step in the TiO2 is due to the energy level of electron injection into the semiconducting material being below the conduction band. This limits the usefulness of the present hybrid electrode. Strategies to overcome this kinetic limitation are discussed.
... 190,231,233 The Q A − → Q B electron transfer taking place at the PSII acceptor side, also in the case of anaerobic purple bacteria, shows a similar blockage upon freezing. The influence of the latter reaction correlates well with the protein dynamics, as was shown by Moßbauer spectroscopy 234 and neutron scattering. 235 Figure 7 presents a comparison between the temperature dependence of the protein dynamics and that of the thermal blockage of both transitions involving the S i states, as well as the reoxidation of Q A − by Q B within the membrane fragments of PSII. ...
All cyanobacteria, algae, and plants use a similar water-oxidizing catalyst for water oxidation. This catalyst is housed in Photosystem II, a membrane-protein complex that functions as a light-driven water oxidase in oxygenic photosynthesis. Water oxidation is also an important reaction in artificial photosynthesis because it has the potential to provide cheap electrons from water for hydrogen production or for the reduction of carbon dioxide on an industrial scale. The water-oxidizing complex of Photosystem II is a Mn-Ca cluster that oxidizes water with a low overpotential and high turnover frequency number of up to 25-90 molecules of O2 released per second. In this Review, we discuss the atomic structure of the Mn-Ca cluster of the Photosystem II water-oxidizing complex from the viewpoint that the underlying mechanism can be informative when designing artificial water-oxidizing catalysts. This is followed by consideration of functional Mn-based model complexes for water oxidation and the issue of Mn complexes decomposing to Mn oxide. We then provide a detailed assessment of the chemistry of Mn oxides by considering how their bulk and nanoscale properties contribute to their effectiveness as water-oxidizing catalysts.
... There is also the more flexible ligation of a bicarbonate ion that can either be bidentate (reduced Fe 2+ ) or monodentate (oxidized Fe 3+ ) in nature (19). The redox potential of the Fe 3+ /Fe 2+ couple is too high for be involved in the electron transfer from Q A to Q B , but exogenous electron acceptors like PPBQ in their semiquinone form can oxidize this iron (20). When this occurs, the resulting Fe 3+ is rapidly reduced by Q A - (21). ...
... This poised all centers to the S 1 state. The samples were subsequently supplemented with 1 mM of an exogenous electron acceptor, either di-chloro-p-benzoquinone (DCBQ), or Phenyl-p-benzoquinone (PpBQ),[22,23]. ...
We report the trapping of two metalloradical intermediates corresponding to the transitions S2 to S3 and S3 to S0 of the oxygen evolving complex of photosystem II, in preparations containing methanol, at temperatures near that of half inhibition of the respective S-state transitions. The first intermediate, with an EPR width of 160 G, is assigned to S2Yz(•), based on its similarity to the one previously characterized after trapping at 10 K. The second with a splitting of ∼100 G is tentatively assigned to S3Yz(•). The S3Yz(•) EPR signal is weaker than the S2Yz(•) one, and both are stable at cryogenic temperatures.
... Actually, the redox potential of the NHI is too high for a participation in native ET between the quinones. However, it could be demonstrated that certain exogenous quinones, when brought into the semiquinone form by a normal turnover of the RC (i.e., by light-induced ET via Q À A ), can oxidize the iron, which in turn can be re-reduced by Q À A ( Ono et al. 1986;Petrouleas and Diner 1987 and Joliot 1981), which simply could be the NHI. Experimental evidence for oxidation of the NHI in bRC has not been reported yet. ...
Photosystem II (PSII), the light-driven water:plastoquinone (PQ) oxidoreductase of oxygenic photosynthesis, contains a nonheme iron (NHI) at its electron acceptor side. The NHI is situated between the two PQs QA and QB that serve as one-electron transmitter and substrate of the reductase part of PSII, respectively. Among the ligands of the NHI is a (bi)carbonate originating from CO2, the substrate of the dark reactions of oxygenic photosynthesis. Based on recent advances in the crystallography of PSII, we review the structure of the NHI in PSII and discuss ideas concerning its function and the role of bicarbonate along with a comparison to the reaction center of purple bacteria and other enzymes containing a mononuclear NHI site.
... The pH-dependence of its redox potential (60 mV/pH-unit) implies the uptake of one proton per electron [32,33]. Accordingly, its rereduction upon the first flash in some tens of microseconds [34] causes extra proton uptake in a long-term dark-adapted sample, which is absent upon the following flashes. Under repetitive dark adaptation, on the other hand, the time interval between flash groups (25 s) was too short for the oxidation of the non-haem iron. ...
By exposing dark-adapted Photosystem II core particles to a series of light flashes, we aimed at kinetic resolution of proton release during the four steps of water oxidation. The signal-to-noise ratio was improved by averaging under repetitive dark adaptation. The previously observed kinetic damping of pH-transients by particle aggregation was prevented by detergent. The complicating superimposition of protolytic events at the donor side (water oxidation) and at the acceptor side (quinone oxido-reduction) was unravelled by characterizing the rate constants of electron and proton transfer at the acceptor side (QA− · nH+ + DCBQ → QA + DCBQ− + nH+: k = 1.7 · 106 M−1 //2 DCBQ− + 2H+ → DCBQ + DCBQH2: k = 4 · 108 M−1 s−1). Contrasting with the pronounced period of four oscillations of the oxygen-evolving centre, the extent of proton release was practically constant. The apparent half-rise time of the stepped acidification was shortened upon lowering of the pH (250 μs at pH 7.5, 70 μs at pH 6.0 and 12 μs at pH 5.2). This kinetic behaviour was independent of the nature and the concentration of the added pH-indicator. We conclude that this reflects the protolysis of several electrostatically interacting acids at the surface of the protein in response to a new positive charge on YZ+, and persisting upon electron transfer from the manganese cluster to YZ+.
Photosystem II (PSII) is a multi‐subunit protein‐pigment complex with diverse redox‐active cofactors, which enabled the biological availability of O2 on Earth. The substrate specificity and the underlying redox chemistry of the Mn4CaO5 catalytic center are investigated using alternate substrates such as small molecules (ammonia and methanol) and halides (Cl‐, Br‐, I‐) instead of the natural substrate water. Changes in the kinetic profiles of steady‐state O2 evolution and of dichlorophenolindophenol (DCIP) photochemical reduction by PSII as well as the detection of modified sites by proteomic analysis implied the possibility of alternate substrate photooxidation. Of particular interest is the role of two chlorides bound close to the putative water channels in the native system. The mutation of D2‐K317 to alanine is believed to impair the binding of a catalytically relevant chloride, eliminating the chloride requirement for water oxidation catalysis. The efficiency of small molecule photooxidation by the OEC is enhanced by the mutated D2‐K317A PSII complex without the competition from chloride. These results provide insight into the role of bound chloride in native PSII as a filter for enhancing the selectivity of water oxidation. The design principles for PSII may be extended to new strategies for developing highly selective catalysts.
Chloride (Cl-) is essential for O2 evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P+QA-), rates of water oxidation steps (S-states), rate of proton evolution, and flash O2 yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O2 evolution rate (-20 %) and proton release (-36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S2 → S3 → S0') slow with increasing light intensity and during O2/water exchange (S0' → S0 → S1), while the non-protonogenic S1 → S2 transition is kinetically unaffected. As flash rate increases in Cl- cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br-. The Cl- advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H3O+Cl- in dry water channels vs. separated Br- and H+ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br- cultures for both proton evolution and less PSII charge recombination. In Br- cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br- with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.
Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone (PQ) molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with similar molecular structures to PQ can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95-2.10 Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site, and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII, and also provide information for the design of more efficient electron acceptors.
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The occurrence of a wide variety of metal-carbon bonds in living organisms, ranging from bacteria to humans, is only recently recognized. Of course, the historical examples are the B12 coenzymes containing cobalt-carbon bonds, but now such bonds are also known for nickel, iron, copper, and other transition metal ions. There is no other comparable book; MILS-6, written by 17 experts, summarizes the most recent insights into this fascinating topic.
Crystals of Photosystem II (PSII) contain the most homogeneous copies of the water-oxidizing reaction center where O2 is evolved (WOC). However, few functional studies of PSII operation in crystals have been carried out, despite their widespread use in structural studies. Here we apply oximetric methods to determine the quantum efficiency and lifetimes of intermediates of the WOC cycle as a function of added electron acceptors (quinones and ferricyanide), both aerobically and anaerobically. PSII crystals exhibit the highest quantum yield of O2 production yet observed of any native or isolated PSII (61.6%, theoretically 59,000 μmol O2/mg Chl/h). WOC cycling can be sustained for thousands of turnovers using an irreversible electron acceptor (ferricyanide). Simulations of the catalytic cycle identify four distinct photochemical inefficiencies in both PSII crystals and dissolved PSII cores that are nearly the same. The exogenous acceptors equilibrate with the native plastoquinone acceptor at the QB (or QC) site(s), for which two distinct redox couples are observable that regulate flux through PSII. Flux through the catalytic cycle of water oxidation is shown to be kinetically restricted by the QAQB two-electron gate. The lifetimes of the S2 and S3 states are greatly extended (especially S2) by electron acceptors and depend on their redox reversibility. PSII performance can be pushed in vitro far beyond what it is capable of in vivo. With careful use of precautions and monitoring of populations, PSII microcrystals enable the exploration of WOC intermediates and the mechanism of catalysis.
Plants, algae, and some bacteria convert solar energy into chemical energy by using photosynthesis. In light of the current energy environment, many research strategies try to take benefits from photosynthesis in order to generate usable photobioelectricity. Among all the developed strategies for transferring electrons from the photosynthetic chain to an outer collecting electrode, we recently implemented a method at preparative scale (high surface electrode) based on a Chlamydomonas reinhardtii green algae suspension in presence of exogenous quinones as redox mediators. While giving rise to interesting performances (10-60 µA.cm-2) during one hour, such a device appears to result in a slow decrease of the recorded photocurrent. In this paper, we wish to analyze and understand this gradual fall in performance in order to limit this issue in future applications. We thus first show that this kind of degradation could be related to over-irradiation conditions or side-effects of quinone depending on experimental conditions. We therefore built an empirical model involving a kinetic quenching induced by quinone incubation which is globally consistent with the experimental data provided by fluorescence measurements achieved after dark incubation of algae in the presence of quinones.
Photosystem II (PSII) is conserved in all oxygenic photosynthetic organisms and is important for its unique ability to use energy from light to split water, generate molecular oxygen in the Earth’s atmosphere and drive electrons into the photosynthetic electron transport chain by reducing the plastoquinone (PQ) pool in the thylakoid membrane. The focus of this chapter is on alternative electron-transfer pathways on the acceptor side of PSII. Upon close examination of the literature there is evidence of exogenous electron acceptors that are reduced directly by the primary PQ electron acceptor (QA), bypassing the canonical terminal PQ-reduction (QB) site. These herbicide-insensitive electron-acceptor molecules include but are not limited to ferricyanide, synthetic cobalt coordination complexes, and cytochrome c. We also discuss experimental treatments to PSII such as cation exchange and herbicide treatment that have been shown to alter the redox midpoint potential (Em) of QA and impact electron transfer from QA to QB. The results described in this chapter provide a platform for understanding how electrons generated in PSII by photochemical water oxidation can be extracted from the electron-acceptor side of PSII for energy applications.
Plants or algae take many benefits from oxygenic photosynthesis by converting solar energy into chemical energy through the synthesis of carbohydrates from carbon dioxide and water. However, the overall yield of this process is rather low (about 4% of the total energy available from sunlight is converted into chemical energy). This is the principal reason why recently many studies have been devoted to extraction of photosynthetic electrons in order to produce a sustainable electric current. Practically, the electron transfer occurs between the photosynthetic organism and an electrode and can be assisted by an exogenous mediator, mainly a quinone. In this regard, we recently reported on a method involving fluorescence measurements to estimate the ability of different quinones to extract photosynthetic electrons from a mutant of Chlamydomonas reinhardtii. In the present work, we used the same kind of methodology to establish a zone diagram for predicting the most suitable experimental conditions to extract photoelectrons from intact algae (quinone concentration and light intensity) as a function of the purpose of the study. This will provide further insights into the extraction mechanism of photosynthetic electrons using exogenous quinones. Indeed fluorescence measurements allowed us to model the capacity of photosynthetic algae to donate electrons to an exogenous quinone by considering a numerical parameter called "open center ratio" which is related to the Photosystem II acceptor redox state. Then, using it as a proxy for investigating the extraction of photosynthetic electrons by means of an exogenous quinone, 2,6-DCBQ, we suggested an extraction mechanism that was globally found consistent with the experimentally extracted parameters.
The reaction center of Photosystem II contains an iron atom which is likely to be located between the acceptor-side quinones QA and QB, in analogy with the structure in photosynthetic purple bacteria. The iron, which is probably not directly involved in the electron transfer between the quinones, can be oxidized by strong oxidants, such as ferricyanide and high-potential semiquinones in plants (but not in purple bacteria) and may then be observed by EPR (1,2). This offers an additional possibility to probe the local environment of the primary iron-quinone acceptor other than that provided by the reduced complex, QA-Fe, which is EPR detectable in plants as well as in bacteria. Measurements, utilizing the sensitivity of EPR to changes in the ligand geometry around the Fe(III) ion, have detected distortions in the protein environment when quinone acceptor analogs or acceptor-side inhibitors bind (2,3). In this communication we report on EPR studies of the acceptor side in the cyanobacterium Anacystis nidulans which allow comparisons with the quinone-iron complex in plants.
The primary charge separation of the PS II reaction center is electron transfer from P680 to a pheophytin molecule and reduced pheophytin is oxidized by the first bound plastoquinone QA.
Mossbauer spectroscopy was applied, for the first time, to study the interaction of copper ions with the non-heme iron and the heme iron of cytochrome bssg in photosystem II thylakoids isolated from a Chlamydomonas reinhardtii photosystem I minus mutant. We showed that copper ions oxidize the heme iron and change its low spin state into a high spin state. This is probably due to deprotonation of the histidine coordinating the heme. We also found that copper preserves the non-heme iron in a low spin ferrous state, enhancing the covalence of iron bonds as compared to the untreated sample. We suggest that a disruption of hydrogen bonds stabilizing the quinone-iron complex by Cu 2+ is the mechanism responsible for a new arrangement of the binding site of the non-heme iron leading to its more "tense" structure. Such a diamagnetic state of the non-heme iron induced by copper results in a magnetic decoupling of iron from the primary quinone acceptor. These results indicate that Cu does not cause removal of the non-heme iron from its binding site. The observed Cu2+ action on the non-heme iron and cytochrome bsss is similar to that previously observed for α-tocopherol quinone.
Photosystem II (PS II) is a pigment-protein complex in thylakoid membranes from all oxygenic photosynthetic organisms (cyanobacteria and photosynthetic eukaryotes). It catalyzes the light-induced reduction of plastoquinone by water through a number of redox reactions. The electron transport chain in PS II is composed of various protein-bound components, which are held in close proximity and suitable orientation with respect to each other by the protein environment, so that a rapid and efficient electron transport is feasible. The PS II complex consists of at least five integral membrane proteins in the thylakoid (together forming the PS II “core complex”), in addition to several peripheral proteins. Many of these polypeptides interact with one or more components of the electron transport chain or with light-harvesting pigments, such that these protein ligands can fulfill a specific function in PS II.
The possibility to determine the difference spectra Δεi+1jλ of each univalent redox step Si→Si+1(i=0,...3) of the water-oxidizing enzyme system was analyzed by theoretical calculations and by measurements of 320 nm absorption changes induced by a train of saturating laser flashes (FWHM:7 ns) in PS II membrane fragments. It was found: a) Lipophilic quinones complicate the experimental determination of optical changes due the Si-state transitions because they lead to an additional binary oscillation probably caused by a reductant-induced oxidation of the Fe2+ at the PS II acceptor side. b) In principle, a proper separation can be achieved at sufficiently high K3[Fe(CN)6] concentrations. c) An unequivocal deconvolution into the difference spectra Δεi+1jλ of flash train-induced optical changes which are exclusively due to Si-state transitions is impossible unless the Kok parameters α, β and [Si]0 can be determined by an independent method.
Measurements of the oxygen yield induced by a flash train reveals, that in thylakoids and PS II membrane fragments Si is the stable state of dark adapted samples even at alkaline pH (up to pH=9). However, in PS II membrane fragments at pH>7.7 the misses probability α markedly increases, in contrast to the properties of intact thylakoids. Based on these data the possibility is discussed that an equilibrium exists of two types of S2-states with different properties.
Inhibition of electron flow from H2O to methylviologen by 3-(3′4′ dichlorophenyl)-1,1 dimethyl urea (DCMU), yields a biphasic curve — an initial high sensitivity phase and a subsequent low sensitivity phase. The two phases of electron flow have a different pH dependence and differ in the light intensity required for saturation.Preincubation of chloroplasts with ferricyanide causes an inhibition of the high sensitivity phase, but has no effect on the low sensitivity phase. The extent of inhibition increases as the redox potential during preincubation becomes more positive. Tris-treatment, contrary to preincubation with ferricyanide, affects, to a much greater extent, the low sensitivity phase.Trypsin digestion of chloroplasts is known to block electron flow between Q
A and Q
B, allowing electron flow to ferricyanide, in a DCMU insensitive reaction. We have found that in trypsinated chloroplasts, electron flow becomes progressively inhibited by DCMU with increase in pH, and that DCMU acts as a competitive inhibitor with respect to [H+]. The sensitivity to DCMU rises when a more negative redox potential is maintained during trypsin treatment. Under these conditions, only the high sensitivity, but not the low sensitivity phase is inhibited by DCMU.The above results indicate the existence of two types of electron transport chains. One type, in which electron flow is more sensitive to DCMU contains, presumably Fe in a Q
A Fe complex and is affected by its oxidation state, i.e., when Fe is reduced, it allows electron flow to Q
B in a DCMU sensitive step; and a second type, in which electron transport is less sensitive to DCMU, where Fe is either absent or, if present in its oxidized state, is inaccessible to reducing agents.
A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q400 oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q400 did not seem to be associated with the other phases.
The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 μM ferricyanide or after Cyt b-559 photooxidation in the presence of 2 μM CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 μM DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.
We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.
It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQA complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.
Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.
The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQA complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the QB site.
Photosystem II cyclic electron transport was investigated at low pH in spinach thylakoids and PS II preparations from the cyanobacteriumPhormidium laminosum. Variable fluorescence (Fv) quenching at a very low light intensity was examined as an indicator of cyclic electron flow. A progressive quenching of Fv was observed as the pH was lowered; however, this was shown to be mainly due to an inhibition of oxygen evolution. Cyclic electron flow in the uninhibited centres was estimated to occur at a rate comparable to or smaller than 1 μ mole O2 mg Chl−1 h−1 in the pH range 5.0 to 7.8.
The quantum yeeld of oxygen production is known to decrease at low pH and has been taken to indicate cyclic electron flow (Crofts and Horton (1991) Biochim Biophys Acta 1058: 187–193). However, a direct all-or-none inhibition of oxygen production at low pH has also been reported (Meyer et al. (1989) Biochim Biophys Acta 974: 36–43). We have analysed the effects of light intensity on the rates of oxygen evolution in order to calculate ΦU, the quantum yield of open and uninhibited centres. ΦU was found to be constant over a broad pH range, and by using ferricyanide and phenyl-p-benzoquinone as electron acceptors the maximum possible rate of cyclic electron transport was equivalent to no more than 1 μmole O2 mg Chl−1 h−1. The rate was no greater when the acceptor was adjusted to provide the most favourable conditions for cyclic flow.
We have investigated interactions of various p-benzoquinones with The QB-binding domain in oxygen-evolving PS II particles isolated from Synechococcus elongatus by two different methods. First, rates of oxygen evolution were determined in the presence of various concentrations of benzoquinones and the two kinetic parameters, Km and Vmax, were estimated from double reciprocal plots. The Vmax value increased with increasing hydrophobicity of quinone molecules, suggesting that the hydrophobicity of acceptor molecules is an important factor affecting the terminal limiting step of quinone reduction. The Km values agreed with the binding constants of the QB site for corresponding benzoquinones which were determined by the second spectrophometric method. Thus, the first method offers a simple and convenient procedure to estimate the affinities of quinone acceptors to the QB site. In the second method, fractions of the QB domains occupied by 3(3,4-dichlorophenyl)-1,1-dimethylurca (DCMU) were determined by measuring the magnitude of the QA−QB− to QAQB− transition. DCMU reduced the magnitude of QA− oxidation by 50% at 60 nM. The inhibition was significantly reversed on addition of benzoquinones, indicating that quinone molecules bind to the QB domain in competition with DCMU and, once bound, serve as electron acceptors of QA. The binding constants of the QB site for benzoquinones were estimated from quinone-induced changes in the concentration of DCMU required to occupy 50% of the QB domains. No evidence was obtained to indicate the occurrence of oxidation of the iron (II) located near QA and QB under the assay conditions, which otherwise would affect the estimation of the binding constant. The relationship between the binding affinity and the molecular structure of benzoquinones is discussed.
We have examined the orientation dependence of two EPR signals associated with the iron of the ferroquinone complex of Photosystem II. The first signal with g values (4.09, 3.95 and approx. 2.0) results from the reversible interaction of the ferrous ion with NO (Petrouleas et al. (1990) Biochim. Biophys. Acta 1015, 131–140). Studies of oriented spinach BBY membranes treated with NO show that gx lies on the membrane plane, gy is oriented at 30° and gz is oriented at 60° with respect to the membrane plane. The latter, most likely, indicates the approximate direction of the Fe-NO bond. Since NO displaces CO2/HCO−3 upon binding (Diner et al. (1990) Biochim. Biophys. Acta 1015, 141–149), the latter physiological ligand is probably well above the membrane plane containing the iron. This places CO2/HCO−3 at an approximately homologous position with the bacterial glutamate ligand (Deisenhofer and Michel (1989) EMBO J. 8, 2149–2169); it is not known, however, whether CO2/HCO−3 binds as a monodentate or bidentate ligand. The second signal with characteristic g values in the region 5.6–8.1 results from the oxidized non-heme iron (Fe3+) [3]. The orientation results show that the gx = 8.1 and gz = 5.6 resonances have both maxima in the plane of the membrane. The gy axis is, therefore, perpendicular to the plane of the membrane and runs along the homologous direction of the bacterial twofold symmetry axis. Based on the strong effect that molecules bound at the QB binding site have on the x-y plane anisotropy () but not on the axial field strength, D, and assuming basic similarities with the bacterial reaction center, it is suggested that, the x-y plane is defined by the two oxygen ligands and the two nitrogens from the histidine imidazoles which have contacts with the QA and QB site. The gz-axis lies then along the N-Fe-N direction defined by the nitrogen ligands from the other two histidines. An unusual finding is that the process of membrane orientation (slow drying of the membranes) induces an axial signal, gxy = 6, in a fraction of centers. The axial plane of the latter is perpendicular to the membrane in agreement with the orientation results of the more rhombic signals. A possible arrangement of the nitrogen and oxygen ligands in the equatorial plane (vertical to the membrane) as to impose pseudo-C3 symmetry is discussed.
Flash-induced absorption changes at 295 nm and oxygen flash yields were measured in Photosystem II-enriched membranes and thylakoids from spinach. In the presence of formate and reduced 2,6-dichloro-t p-benzoquinone (DCBQ), the normal period-4 oscillation in the oxygen-evolving mechanism is shifted one flash, a maximum appearing on the 4th rather than the 3rd flash. Analysis of the flash-induced sequence shows that in 90–100% of the centers, the oxygen evolving complex is present in the most reduced state (S0), rather than in the singly oxidized state (S1) as is normally the case. An electron donation to the oxidized states S2 or S3 during the flash sequence by reduced tyrosine ‘YD’ cannot account for the data unless the rate of this reaction is dramatically accelerated by formate. Rather, it appears that the normally dark-stable S1 state of the oxygen-evolving mechanism is modified by formate so that it becomes reducible to S0. The reduced form of DCBQ is an efficient mediator to promote the shift to S0. As in other instances where a chemical reduction of S1 is achieved, the ‘formal S0’ state caused by the formate/DCBQ treatment is not spectroscopically identical with the S0 state occurring under illumination. Incubation at high redox potential (550 mV) did not reverse the S-state shift caused by the treatment with formate and DCBQ at lower potential, but induced a distortion of the absorption change sequence that could indicate an enhanced yield of oxygen on the second flash.
The nonheme iron of the photosystem II reaction center was converted to its low-spin state (S = 0) by treatment with CN-. This allowed the study of the plastoquinone, Q(A)(-) anion radical by electron spin-echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of the ESEEM data of Q(A)(-) in N-14- and N-15-labeled PSII demonstrates the existence of a protein nitrogen nucleus coupled to the Q(A)(-). The N-14 coupling is characterized by a quadrupolar coupling constant e(2)qQ/4h = 0.82 MHz, an asymmetry parameter eta = 0.45, and hyperfine coupling constant A similar to 2.1 MHz. The N-15 hyperfine coupling is characterized by T = 0.41 MHz and alpha(iso) similar to 3.3 MHz. The possible origins of the nitrogen hyperfine coupling are discussed in terms of the amino acids thought to be close to the Q(A)(-) in PSII. Based on a comparison of the N-14 ESEEM with N-14-NQR and N-14-ESEEM data from the literature, the most likely candidate is the amide nitrogen of the peptide backbone of Ala261 of the polypeptide D2, although the indole nitrogen of Trp254 and the imino nitrogen of His215 of D2 also remain candidates.
The decay kinetics for the S2 and S3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S2- and S3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S3 decays via S2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S0 to S1 220–225 K, for S1 to S2 135–140 K, for S2 to S3 230 K and for the S3-to-S0 transition 235 K.
The chloroplast grana margins of spinach thylakoids were isolated by sonication and aqueous-two-phase partitioning and their electron transport properties examined. Photosystem II and I electron transport activities were measured and compared to the appressed and non-appressed grana core and stroma lamellae, respectively, as well as to whole thylakoids. The results show that the PS II complexes in the grana margins are of the PS IIβ subtype with respect to antenna size, but are QB reducing with respect to the acceptor side properties, while the PS I centers in the grana margins have slightly larger antennae as compared to the PS I centers in the stroma lamellae and are more like the PS Iα centers located in the grana domain. The ability to reduce ferredoxin and NADP+ was also tested and it was found that the grana margin membrane fraction was unable to reduce ferredoxin, even in the presence of added artificial electron donors. The stroma lamellae and whole thylakoid fractions both reduced ferredoxin at high rates. However, the grana margins could catalyze the reduction of NADP+ when supplied with the necessary components (ferredoxin, ferredoxin:NADP+ oxidoreductase, and an electron source). The results suggest that the PS I populations located in the margins of the grana domain are functionally different from the PS I centers located in the stroma lamellae. The data support a model whereby the PS I centers in the grana are primarily involved in non-cyclic electron transport, while the PS I centers located in the stroma lamellae are capable of participating in both cyclic and non-cyclic electron transport.
Photosystem II (PS II) heterogeneity during photoinhibition at 4°C and subsequent recovery at 20°C was investigated in spinach leaves and chloroplasts. The population of inactive, QB-nonreducing centers was estimated by means of fluorescence induction in the presence of ferricyanide and was only 5–10% of the total PS II population in the untreated material, when assuming that the fluorescence yield from all PS II units was identical. No significant changes in this population of QB-nonreducing centers after photoinhibition treatment at 4°C of spinach leaves or chloroplasts was found. During subsequent recovery of the inhibited leaves in low light at room temperature no changes in this QB-nonreducing pool could be detected, while electron transport measured in the presence of methylviologen or 1,4-benzoquinone and the yield of variable fluorescence recovered in 6–8 h. During photoinhibitory irradiation of the leaves and thylakoids, the PS IIα population became strongly inhibited while PS IIβ was inhibited to a lesser extent. The inhibition treatment did not affect the photochemical efficiency of the remaining PS IIα and PS IIβ centers, but diminished the cooperativity between the PS IIα centers. In leaves, a small population of inhibited PS II centers seemed to have an impairment of the donor side, which disappeared during recovery in low light but not in darkness, concomitant with recovery of electron transport and variable fluorescence. No significant changes in the QB-nonreducing PS II, PS IIα and PS IIβ populations or electron transport activities occurred when the inhibited leaves were transferred to room temperature in darkness. In conclusion, no evidence for a repair cycle that proceeds via QB-nonreducing centers could be obtained. However, we cannot exclude the possibility that a dynamics of PS II heterogeneity was concealed by synchronous turnover of different populations of PS II. Our electron transport data show that rate measurements with different quinone acceptors cannot be used to discriminate between effects of photoinhibition on ‘active’ and QB-nonreducing centers.
The presence of erogenous quinones PPBQ, DCBQ and DMQ in inside-out thylakoids alters the oscillation pattern with period 4 of the flash-induced fluorescence yield. The results can be interpreted by assuming that at ambient temperature PPBQ replaces PQ in 100% of the PS II centers detected by fluorescence, DMQ and DCBQ being bound to 40–60% of these centers only. The evaluation of the percentage of double hits in a 3 μs flash allows estimation of the electron-transfer rate from Q-A to Fe3+, which varies from 9 μs (PPBQ) to 20μs (DMQ).
In a unicellular cyanobacterium, the mobile fraction of phycobilisome (PBS) was found to be maximum at a particular redox value of QA (i.e., 0.52). An upward or downward shift in the redox value leads to a decrease in this mobile fraction of PBS. Furthermore, the regulatory effect of the redox state of QA on PBS mobility was found to be independent of the effect exerted by the plastoquinone pool. These findings indicate for the first time that PBS mobility is regulated by the QA redox state in cyanobacteria. A possible working mechanism underlying this control is discussed.
We have examined the effects of a number of carboxylate anions on the iron-quinone complex of Photosystem II (PS II). Typical effects are the following. In the state Q−AFe2+ oxalate enhances significantly the g = 1.84 EPR resonance while, for example, glycolate and glyoxylate suppress it. The anions have variable effects on the iron midpoint potential. Formate and oxalate raise significantly the Em of the iron. Glycolate lowers the Em significantly and the Em shows a weak pH dependence. In the presence of glycolate the native plastosemiquinone () can oxidise the iron. Glyoxylate also lowers the Em, but the Em shows a greater pH dependence than with glycolate but still weaker than the −60 mV/pH unit of the untreated iron. The Fe3+ EPR spectra are characterised by small but distinct shifts, while in addition an unusual resonance at close to g = 4.3 is observed. These as well as the temperature dependence of the spectra are analysed by a spin-Hamiltonian model. Comparison with competition studies in the companion paper indicates that the anions bind as iron ligands displacing bicarbonate.
We have studied the EPR signal in PS II from Phormidium laminosum with a g-value of 1.66, which we assign to an interaction between the semiquinones of Qa and Qb and the non-heme iron. 77 K illumination of samples from dark-poised redox titrations show the rise of the signal has a midpoint potential (Em) of about +60 mV, and it is lost with an Em of about −10 mV. Under the same conditions, the rise of the g = 1.9 signal from Q·−a-Fe2+ in the dark was found to be about +10-mV. The g = 1.66 signal can also be formed with a high yield by first illuminating dark-adapted PS II particles at 293 K, followed by a short period of darkness at 273 K and subsequent illumination at 77 K. We have measured the effect on signal yield of varying the period of darkness following 293 K illumination. Over 60% of the maximum signal size is seen after 1 min darkness, and increases further over 2 h. In these samples a signal attributed to Q·−b-Fe2+ is seen prior to 77 K illumination. Confirmation of the presence of Q·t-b was obtained by reductant-linked oxidation of the non-haem iron using phenyl-para-benzoquinone (PPBQ). Samples treated with the Qb-analogue tribromotoluquinone (TBTQ) give a modified EPR signal. We propose (i) that Qb is preserved in PS II preparations from P. laminosum; (ii) that Qb-semiquinone can be readily formed and trapped by freezing; and (iii) the g = 1.66 signal arises from a coupling between the primary and secondary plastosemiquinones and the non-haem iron.
Substitution of lanthanides for Ca2+ in Photosystem II results in an inhibition of oxygen-evolution activity. In addition to blocking electron transport from the Mn-complex to TyrZ+, the presence of the lanthanides also affects electron transport from TyrZ to P680+. This latter effect depends on two factors: (i) The ionic radius of the trivalent lanthanide ion; lanthanide ions with an ionic radius smaller than that of Ca2+, exert a more pronounced inhibitory effect. (ii) The pH of the system; although the presence of lanthanides has a strong influence on the reduction of P680+ by TyrZ at pH 6.0, no such effect is observed at pH 7.5. A pH titration has attributed the pH effect to an amino acid residue with a pK of 6.5.
The electron spin resonance spectrum of P680+ has been measured in photosystem II membranes at room temperature under repetitive flash excitation by using gated integration techniques. Oxygen evolution was inhibited in the samples used in these experiments, and the lifetime of the radical is extended into the 150-200-μs range. Three different treatments were used that allowed us to determine the spectral characteristics of P680+ when the paramagnetic species YZ+ was also present. These results were compared to the P680+ spectral properties that we measured under conditions in which YZ was in its reduced, diamagnetic form. With Tris-inactivated membranes, where YZ+ but not manganese was present, only a low P680+ signal amplitude could be measured, which precluded an accurate determination of the line width. With NaCl-washed membranes and membranes treated with K3Fe(CN)6, in which YZ+ and manganese were both present during the measurement, the field-modulated P680+ spectrum is 8.9 G wide. This is 1 G wider than the spectrum measured when YZ remains reduced, as happens in membranes inhibited with NH2OH. The broadening of the P680+ spectrum that occurs when its immediate donor is oxidized is attributed to a magnetic dipole-dipole interaction between P680+ and YZ+. The extent of broadening allows us to estimate that the center-to-center distance between the two radicals is 10-15 Å.
Time-resolved ESR has been used to study electron-transfer reactions in oxygen-evolving photosystem II membrane fragments. The exogenous acceptor dichlorobenzoquinone (DCBQ) is reduced by photosystem II; the ESR spectrum of the resulting DCBQ radical overlaps the center but not the wings of the ESR spectra of the endogenous tyrosine radicals YD+ and YZ+. Here YZ+ denotes the species that is involved in electron transfer between the reaction center chlorophyll, P680, and the manganese-containing, oxygen-evolving complex, and YD+ denotes the stable photosystem II radical. By using appropriate magnetic fields, we recorded kinetic transients of YZ+ under repetitive flash conditions with DCBQ present. We also used 1 mM K3Fe(CN)6 as an exogenous acceptor when recording kinetic traces of YZ+, although at concentrations above 5 mm we observe an additional signal that could be due to P680+. The kinetic traces of YZ+ obtained with DCBQ or 1 mM K3Fe(CN)6 are similar and show two phases. The slower phase has a half-time of 1.2 ms and corresponds to the reduction of YZ+ by the S3 state; the faster phase reflects reduction of YZ+ by both S1 and S2. By using flowing, dark-adapted PSII membranes, we resolved the YZ+S1 reaction (t1/2 = 100 μs) on the first flash and found it to be significantly faster than the YZ+S2 reaction (t1/2 = 300 μs) which occurs on the second flash. A high-resolution ESR spectrum of YZ+ in O2-evolving PSII membranes was obtained with gated integration techniques and found to be similar to the spectra of YD+ and of Yz+ in inhibited membranes. Thus, the magnetic interaction between spins on YZ+ and the manganese in the oxygen-evolving complex broadens the YZ+ spectrum negligibly. These results support the idea that a single electron carrier, YZ, operates between P680+ and the manganese ensemble in the oxygen-evolving complex and functions on all four S-state transitions.
The iron quinone-complex of the reaction centers of photosystem II and the purple non-sulphur photosynthetic bacteria contains two quinones, QA and QB connected in series with respect to electron transfer, and separated by a non-heme iron coordinated by amino acid residues. It is the site of inhibition of many of the common photosynthetic herbicides, which act by displacing QB from the center. The complex is responsible for reducing QB to QBH2 in two successive one-electron photo acts. OBH2 dissociates from the center, to be replaced by a new QB molecule and reduces the following membrane-bound electron-transfer complex (cytochrome b6/for b/c1). The energetic, kinetics and mechanism of complex function are reviewed here. Recent crystallographic, spectroscopic and molecular biological evidence has produced a considerable quantity of structural information on this complex. These data have given a less formal and more molecular view of how the complex functions. They have also revealed fundamental differences between the photo system II and bacterial complexes, particularly with respect to the coordination of the iron and its chemistry. The comparative anatomy of the complexes is reviewed and its implications for function discussed.
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
The effects of ferricyanide on Photosystem II reactions have been investigated by measurements of microsecond and millisecond prompt fluorescence and microsecond-delayed fluorescence in dark-adapted chloroplasts: (1) Titrations using ferri-ferrocyanide mixtures on: (a) the fast phase of the increase in fluorescence yield observed during a xenon flash, and (b) the normalised area above the millisecond fluorescence induction curve for chloroplasts inhibited by DCMU, showed a pH dependent mid point potential of 400 mV at pH 7.0 which varied by approx. -60 mV/pH unit between pH 6 and 8.5. (2) A saturating laser flash induced a fluorescence increase (as monitored by a weak measuring beam) of only 50% of that reached following a second flash in chloroplasts preincubated with ferricyanide and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prior to illumination. In the absence of ferricyanide, the fluorescence level reached after a single flash was initially close to that measured after a second flash (although the level subsequently declined). (3) The initial amplitude of the microsecond-delayed fluorescence excited by a single laser flash was diminished in chloroplasts dark-adapted with ferricyanide. In the presence of DCMU and ferricyanide, the amplitude was also diminished for the first flash of a series, but subsequently enhanced above the level obtained in chloroplasts in the presence of DCMU alone. (4) The above effects were not seen if DCMU was added to the chloroplasts before ferricyanide, or if the period of incubation with ferricyanide was much less than 4 min. (5) These results suggested the presence of a second acceptor Q2, with Em7 = 400 mV and n = 1, before the DCMU block in Photosystem II. There is 0.35--1 equivalent of the acceptor per reaction centre, and its reduction occurs within less than 5 mus. The role of the acceptor in double turnovers of the photochemistry during a single flash and its likely operating redox potential are discussed.
Electron paramagnetic resonance of spinach chloroplasts given a series of laser flashes, n = 0, 1,..., 6, at room temperature and rapidly cooled to -140 degrees C reveals a signal possessing at least 16 and possibly 21 or more hyperfine lines when observed below 35 K. The spectrum is consistent with a pair of antiferromagnetically coupled Mn ions, or possibly a tetramer of Mn ions, in which Mn(III) and Mn(IV) oxidation states are present. The intensity of this signal peaks on the first and fifth flashes, suggesting a cyclic change in oxidation state of period 4. The multiline signal produced on the first flash is not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is abolished by agents that influence the state of bound manganese, such as incubation with alkaline Tris, or dithionite, and by extraction with cholate detergent in the presence of ammonium sulfate. These results indicate that the paramagnetic signal is monitoring oxidation state changes in the enzyme involved in oxidation of water.
Chloroplasts were submitted to a sequence of saturating short flashes and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor (B−) present was estimated from the DCMU-induced increase in fluorescence in the dark caused by the reaction: QB− Q−B. By varying the time interval between the preillumination and the mixing, the time course of B− reoxidation by externally added benzoquinone was investigated. It was found that benzoquinone oxidizes B− in a bimolecular reaction, and does not interact directly with Q−. When a sufficient delay after the preillumination was allowed in order to let benzoquinone reoxidize B− before the injection of DCMU, the fluorescence increase caused by one subsequent flash fired in the presence of DCMU was followed by a fast decay phase (). The amplitude of this phase was proportional to the amount of B− produced by the preillumination. This fast decay was observed only after the first flash in the presence of DCMU. These results are interpreted by assuming a binding of the singly reduced benzoquinone to Photosystem II where it acts as an efficient, DCMU-insensitive, secondary (exogenous) acceptor.
Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q−AFe. o-Phenanthroline slightly broadens the Q−AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q−AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q−AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin− signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b559 to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.
Using a theory of electron transfers which takes cognizance of reorganization of the medium outside the inner coordination shell and of changes of bond lengths inside it, relations between electrochemical and related chemical rate constants are deduced and compared with the experimental data. A correlation is found, without the use of arbitrary parameters. Effects of weak complexes with added electrolytes are included under specified conditions. The deductions offer a way of coordinating a variety of data in the two fields, internally as well as with each other, and a way of predicting results in one field from those in another. For example, the rate of oxidation or reduction of a series of related reactants by one reagent is correlated with that of another and with that of the corresponding electrochemical oxidation-reduction reaction, under certain specified conditions. These correlations may also provide a test for distinguishing an electron from an atom transfer mechanism.
The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.
The ferrous ion associated with the electron acceptors in Photosystem II can be oxidized by the unstable semiquinone form of certain high-potential quinones (phenyl-p-benzoquinone, dimethylbenzoquinone and benzoquinone) which are used as electron acceptors. In a flash sequence, alternating oxidation of the iron by the photoreduced semiquinone on odd-numbered flashes is followed by photoreduction of the iron on even-numbered flashes. These reactions are detected by monitoring EPR signals arising from Fe3+. The oxidation of the iron can also occur in the frozen state (−30°C) indicating that the high-potential quinone can occupy the QB site. The reaction also takes place when the exogenous quinone is added in the dark to samples in which QB is already in the semiquinone form. The inhibitors of electron transfer between QA− and QB, DCMU and sodium formate, block the photoreductant-induced iron oxidation. It is suggested that the iron oxidation takes place through the QB site. This unexpected photochemistry occurs under experimental conditions routinely used in studies of Photosystem II. Some previously reported phenomena can be reinterpreted on the basis of these new data.
Four representative inhibitors of Photosystem II (PS II) Q−A to QB electron transfer were shown to bind, at high concentrations, to PS II reaction centers having the acceptor-side non-heme iron in the Fe(III) state. Three of the inhibitors studied, DCMU, o-phenanthroline and dinoseb, modified the EPR spectrum of the Fe(III) relative to that obtained by ferricyanide oxidation in the absence of inhibitor. o-Phenanthroline gave particularly axial symmetry, while DCMU and dinoseb gave more rhombic configurations. The herbicide inhibitor, atrazine and its analogue, terbutryn, had no effect. The dissociation constants for inhibitor binding to reaction centers in the Fe(III) state were measured directly and also estimated from shifts in the midpoint potential of the Fe(III)/Fe(II) couple and were shown to increase by factors of approx. 100, approx. 10 and 10–15 for DCMU (pH 7.5), atrazine (pH 7.0) and o-phenanthroline (pH 7.0), respectively, upon oxidation of the iron. Atrazine and o-phenanthroline, which induce the smallest changes in the midpoint potential of the Fe(III)/Fe(II) couple, were shown to inhibit light-induced oxidation of the Fe(II) by phenyl-p-BQ, described in the preceding paper (Petrouleas, V. and Diner, B.A. (1987) Biochim. Biophys. Acta 893, 126–137). The extent of inhibition was much greater than would be predicted from a simple shift in the midpoint potential for Fe(III)/Fe(II) and we conclude that phenyl-p-BQ and the other quinones, which show light-induced oxidation, act through the QB binding site. It is also argued that reduction and oxidation of the iron by ferro- and ferricyanide, respectively, occur through this site. The effects of these inhibitors and of various quinones on the Fe(III) environment are discussed with reference to the known contact points between the protein and o-phenanthroline and terbutryn in the QB binding pocket of Rhodopseudomonas viridis reaction centers (Michel, H., Epp, O. and Deisenhofer, J. (1986) EMBO J. 5, 2445–2451). The Fe(III) EPR spectrum is thus a new and sensitive probe of the contact points at which molecules bind to the QB binding site.
Photosystem II particles from spinach, containing about 80 chlorophyll a molecules per reaction center, have been investigated with picosecond absorbance-difference spectroscopy. The 35 ps excitation pulse at 532 nm produced absorbance changes due to the formation of singlet excited antenna chlorophyll a and to the primary-charge separation in the reaction centers. The appearance of excited chlorophyll a was accompanied by the bleaching of the ground state Qy absorption band and by the formation of a rather flat absorption band in the region 550–900 nm. At high flash intensity its average lifetime was found to be several tens of picoseconds. In the reaction center charge separation was observed between the primary electron donor P-680 and pheophytin a. Reduction of pheophytin a was accompanied by an absorbance increase between 640 and 675 nm and a bleaching around 685 nm. Electron transfer to a secondary acceptor occurred with a time constant of 250–300 ps. If this secondary acceptor was reduced chemically, the primary radical pair decayed by charge recombination in about 2 ns.
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q−AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin− signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin− signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.
Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl−1.h−1). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680+ is particularly well preserved in the preparation, as judged by P680+ reduction kinetics, the detection by EPR of Signal IILT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).
Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe2+ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe2+ has low symmetry, with zero-field splitting parameters of |D1|=7.0±1.3 cm−1 and |E1|=1.7±0.5 cm−1 and for the redox state Q1−Fe2+Q2−. The rhombic character of the ligand field is increased in the redox state Q1Fe2+Q−2, where . This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe2+. There exists an electron-spin exchange interaction between Fe2+ and Q−1 and Q−2, having magnitudes |J1|=0.12±0.03 cm−1 and , respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe2+ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe2+ and Q−1 and Q−2. Selective line-broadening of some of the EPR transitions, involving Q− coupling to the magnetic sublevels of the Fe2+ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 Ȧ between Fe2+ and Q−1, thus placing Q1 outside the immediate coordination shell of Fe2+.
The decay of fluorescence yield following each of a series of saturating laser flashes has been used to monitor the kinetics of reoxidation of the primary acceptor of Photosystem II under conditions of varied redox potential. 1.1. In dark-adapted chloroplasts, a damped binary oscillation as a function of flash number was observed in the kinetics of the decay of the fluorescence yield. The decay was faster on odd than on even-numbered flashes.2.2. In the presence of low concentrations of 1,4-benzoquinone, the oscillation was more marked, and over the range approx 200–350 mV, independent of redox potential. The decay following flash 1 under these conditions had a half-time of approx. 200–400 μs. The decay following flash 2 was decelerated; the initial rate was up to 10-fold slower than after flash 1.3.3. We suggest that the kinetics following a single flash reflect the rate of the reaction Q−B → QB−, and following the second flash, Q−B− → QB2−. Benzoquinone at low concentrations oxidises a residual fraction of B− which is usually reduced in the dark before the flash sequence.4.4. A faster component in the decay () following the first flash titrated in over the range Eh > 350 mV. The binary oscillation was still apparent but delayed by one flash.5.5. We discuss the relative redox potentials of the couples and , and the role of the component which titrates in at Eh > 350 mV.
The primary events of photosynthetic light reactions excel in efficiency and speed. They occur in highly organized protein-pigment aggregates that have been well characterized, both chemically and functionally. The recent structural characterization at the atomic level of some important components of the primary photosynthetic processes in bacteria helps our understanding of the underlying physical principles.
Measurements of the area bounded by the variable fluorescence induction curve and the maximum fluorescence yield as a function of redox potential led I. Ikegami and S. Katoh ((1973) Plant Cell Physiol. 14, 829–836) to propose the existence of a high-potential electron acceptor, Q400 (Em7.8 = 360 mV), associated with Photosystem II (PS II). We have generated the oxidized form of this acceptor (Q+400) using ferricyanide and other oxidants in thylakoid membranes isolated from a mutant of Chlamydomonas reinhardtii lacking Photosystem I and the cytochrome complex. Q+400 was detected by a decrease in the extent of reduction of the primary quinone electron acceptor, QA, in a low-intensity light flash exciting PS II reaction centers only once. EPR measurements in the presence of Q+400 indicated the presence of new signals at g = 8, 6.4 and 5.5. These disappeared upon illumination at 200 K or upon reduction with ascorbate. Mössbauer absorption attributed to the Fe2+ of the QA-Fe2+ acceptor complex of PS II disappeared upon addition of ferricyanide due to the formation of Fe3+. The Fe2+ signal was restored by subsequent addition of ascorbate. All of these spectroscopic signals show similar pH-dependent (n = 1) midpoint potentials (approx. −60 mV / pH unit) and an Em7.5 = 370 mV. We assign the EPR signals to the Fe3+ state of the quinone-iron acceptor. Electron transfer to the Fe3+ is responsible for the decrease in QA reduction upon single-hit flash excitation. The properties of the redox couple are consistent with those of and we conclude that the iron of the QA-Fe acceptor complex is responsible for this species.
Using dark adapted isolated spinach chloroplasts and sequences of brief saturating flashes the correlation of the uptake and release of protons with electron transport from Photosystem II to Photosystem I were studied. The following observations and conclusions are reported: (1) Flash-induced proton uptake shows a weak, damped binary oscillation, with maxima occurring after the 2nd, 4th, etc. flashes. The damping factor is comparable to that observed in the O2 flash yield oscillation and therefore explained by misses in Photosystem II. (2) On the average and after a steady state is reached, each flash (i.e. each reduction of Q) induces the uptake of 2H+ from outside the chloroplasts. (3) Flash induced proton release inside the chloroplast membrane shows a strong damped binary oscillation with maximum release occurring also after the 2nd, 4th, etc. flashes. (4) This phenomenon is correlated with the earlier reported binary oscillations of electron transport [2] and shows that both electrons and protons are transported in pairs between the photosystems. (5) In two sequential flashes 4H+ from the outside of the thylakoid and 2e- from water are accumulated at a binding site B. Subsequently, the two electrons are transferred to non-protonated acceptors in Photosystem I (probably plastocyanin and cytochrome f) and the 4H+ are released inside the thylakoid. (6) It is concluded that a primary proton transporting site and/or energy conserving step located between the photosystems is being observed.
The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)
The complete absorption difference spectrum of the primary electron acceptor of Photosystem II has been measured at room temperature in subchloroplast fragments prepared with deoxycholate. The shape and amplitude of the spectrum indicate that the primary reaction involves the reduction of one bound plastoquinone molecule per reaction center to its semiquinone anion. In addition two small absorbance band shifts occur near 545 (C550) and 685 nm, which may be due to an influence of the semiquinone on the absorption spectrum of a reaction center pigment.
1. The mechanisms by which p-benzoquinol and its derivatives reduce cytochrome c in solution have been investigated. 2. The two major reductants are the species QH- (anionic quinol) and Q.- (anionic semiquinone). A minor route of electron transfer from the fully protonated QH2 species can also occur. 3. The relative contributions of these routes to the overall reduction rate are governed by pH, ionic strength and relative reactant concentrations. 4. For a series of substituted p-benzoquinols, the forward rate constant, k1, of the anionic quinol-mediatd reaction is related to the midpoint potential of the QH-/QH. couple involved in the rate-limiting step, as predicted by the theory of Marcus for outer-sphere electron transfer reactions in a bimolecular collision process. 5. A mechanism for the biological quinol oxidation reactions in mitochondria and chloroplasts is proposed based upon the findings with these reactions in solution.
The characteristics of double hitting in Photosystem II charge separation and oxygen evolution in algae and chloroplasts were investigated with saturating excitation flashes of 3 microseconds, 300 ns and 5 ns duration. Two types of double hitting or advancement in S-states were found to occur in oxygen evolution: a non-photochemical type found even with 5 ns flashes and a photochemical type seen only with microsecond-long flashes, which have extensive tails. The non-photochemical type, occurring with a probability of about 3%, is sensitive to the physiological condition of the sample, and is only present in algae or chloroplast samples that have been freshly prepared. In chloroplasts incubated with ferricyanide, a 3-fold increase in double advancement of S-states is observed with xenon-flash illumination but not with 300 ns or 5 ns laser illumination. However, double turnovers in Photosystem II reaction center charge separation are large with xenon flash or 300 ns laser illumination but not with 5 ns laser illumination. This indicates that quite different kinetic processes are involved in double advancement in S-states for oxygen evolution and double turnovers in charge separation. Various models of the Photosystem II reaction center are discussed. Also, based on experiments with chloroplasts incubated with ferricyanide, an unique solution to the oxygen S-state distribution in the dark suggested by Thibault (Thibault, P. (1978) C.R. Acad. Sci. Paris 287, 725-728) can be rejected.
Measurements of chlorophyll fluorescence have been used to monitor electron transfer from Q (the primary electron acceptor of photosystem II) to B (the bound quinone which serves as the secondary acceptor) in chloroplasts isolated from atrazine-susceptible and atrazine-resistant pigweed chloroplasts. The Q− → B electron transfer was at least 10-fold slower in the plastids from resistant plants. Binary oscillations in the rate of Q− decay after a series of flashes were of opposite phase in the two types. The data are interpreted to indicate that the apoprotein of B is altered in the photosytem II complex of the two types of plants—this is correlated to altered binding affinity of herbicides to this component and may be related to altered redox properties of the bound quinone cofactor.
On illuminating chloroplasts with “short-wavelength” monochromatic light that supports oxygen evolution, spectral evidence was obtained for a new photoreactive chloroplast component, provisionally designated C550, which shows a reversible decrease of absorbance with a maximum at 550 mμ. The light-induced absorbance changes in C550 have been separated from those due to cytochromes in the same spectral region.
The light-induced decrease of absorbance in C550 appears to be independent of temperature, persisting even at -189° and is therefore likely to be linked to the primary light reaction associated with oxygen evolution in photosynthesis.