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Deactivation kinetics and temperature dependence of the S-state transitions in the oxygen-evolving system of Photosystem II measured by EPR spectroscopy
Abstract
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.
... Oxygen evolves during the S 3 to (S 4 )S 0 transition, the S 4 being a transient state[1,2]and references therein. The transitions S 0 –S 1 , S 2 –S 3 and S 3 –S 0 have half-inhibition temperatures of $230 K[3,4], while the S 1 to S 2 transition proceeds at temperatures as low as 135–140 K[3,4]or even lower [5 and our unpublished observations]. Tyrosine Z (TyrZ) of Photosystem II mediates electron transfer between the primary electron donor in PSII (P 680 ), a specialized chlorophyll (chl) moiety, and the Mn 4 CaO 5 cluster during the S-state transitions of the OEC in a proton-coupled reaction. ...
... Oxygen evolves during the S 3 to (S 4 )S 0 transition, the S 4 being a transient state[1,2]and references therein. The transitions S 0 –S 1 , S 2 –S 3 and S 3 –S 0 have half-inhibition temperatures of $230 K[3,4], while the S 1 to S 2 transition proceeds at temperatures as low as 135–140 K[3,4]or even lower [5 and our unpublished observations]. Tyrosine Z (TyrZ) of Photosystem II mediates electron transfer between the primary electron donor in PSII (P 680 ), a specialized chlorophyll (chl) moiety, and the Mn 4 CaO 5 cluster during the S-state transitions of the OEC in a proton-coupled reaction. ...
... These have been discussed in[17]and refined in subsequent studies[18]. While all intermediates can be trapped at temperatures well below the half-inhibition temperature for the respective transitions, induction of the S[3,4]. Preliminary evidence for the trapping of the former intermediate in untreated samples at these elevated temperatures has already been published[19]. ...
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.
... (Kok et al. 1970;Forbush et al. 1971;Shen 2015;Vinyard and Brudvig 2017). S 0 is the most reduced state, whereas S 1 , S 2 and S 3 states represent sequentially higher oxidation states (Kok et al. 1970;Forbush et al. 1971;Styring and Rutherford 1988). S 1 is the dominant state in the dark, whereas S 2 and S 3 are metastable states that decay back to S 1 in a few minutes at room temperature (Forbush et al. 1971;Styring and Rutherford 1988). ...
... S 0 is the most reduced state, whereas S 1 , S 2 and S 3 states represent sequentially higher oxidation states (Kok et al. 1970;Forbush et al. 1971;Styring and Rutherford 1988). S 1 is the dominant state in the dark, whereas S 2 and S 3 are metastable states that decay back to S 1 in a few minutes at room temperature (Forbush et al. 1971;Styring and Rutherford 1988). S 4 is a transient state that spontaneously progresses to S 0 concomitant with the release of O 2 (Goussias et al. 2002;Haumann et al. 2005). ...
Photosystem II (PSII) has a number of hydrogen-bonding networks connecting the manganese cluster with the lumenal bulk solution. The structure of PSII from Thermosynechococcus vulcanus (T. vulcanus) showed that D1-R323, D1-N322, D1-D319 and D1-H304 are involved in one of these hydrogen-bonding networks located in the interfaces between the D1, CP43 and PsbV subunits. In order to investigate the functions of these residues in PSII, we generated seven site-directed mutants D1-R323A, D1-R323E, D1-N322R, D1-D319L, D1-D319R, D1-D319Y and D1-H304D of T. vulcanus and examined the effects of these mutations on the growth and functions of the oxygen-evolving complex. The photoautotrophic growth rates of these mutants were similar to that of the wild type, whereas the oxygen-evolving activities of the mutant cells were decreased differently to 63–91% of that of the wild type at pH 6.5. The mutant cells showed a higher relative activity at higher pH region than the wild type cells, suggesting that higher pH facilitated proton egress in the mutants. In addition, oxygen evolution of thylakoid membranes isolated from these mutants showed an apparent decrease compared to that of the cells. This is due to the loss of PsbU during purification of the thylakoid membranes. Moreover, PsbV was also lost in the PSII core complexes purified from the mutants. Taken together, D1-R323, D1-N322, D1-D319 and D1-H304 are vital for the optimal function of oxygen evolution and functional binding of extrinsic proteins to PSII core, and may be involved in the proton egress pathway mediated by YZ.
... S 0 is the most reduced state while S 4 is the most oxidised state [6]. The S 1 state is dominant in samples kept in the dark whereas S 2 and S 3 decay back to S 1 in just a few minutes at room temperature [8,9]. The S 0 → S 1 , S 1 → S 2 , S 2 → S 3 transitions are activated by photochemical oxidation of P 680 + via Try Z , which in turn oxidises the OEC [10]. ...
... S 4 is a transient state, while S 0 up to S 3 are called metastable states. The metastable states can be freeze trapped by rapid freezing after exposing PSII samples to appropriate number of laser flashes [9]. Water freely exchanges with the Kok cycle up to S 3 , followed by the final oxidation reaction, which converts two water molecules into di-oxygen. ...
Characterizing the photosystem II (PSII) sample, continuous wave electron paramagnetic resonance (CW-EPR) simulations of the S2 ML signal at X-band frequencies was our focus. This can help increase our understanding of how the manganese (Mn) atoms in the catalytic site of the PSII magnetically interact using ML signals. It can also be used to further the understanding of possible water-splitting mechanisms in the oxygen-evolving complex (OEC). The question that remains is how much does each manganese (Mn) ion contribute to the ML signal through its hyperfine interactions in the OEC? Currently, there are two proposals for the average oxidation states of the Mn ions, denoted the ‘high’ oxidation paradigm (HOP) and the ‘low’ oxidation paradigm (LOP). Majority of PSII researchers favour the HOP. Various experiments have been conducted to investigate the two alternative oxidation states, including EPR (Jin et al. in Phys Chem Chem Phys 16(17):7799–7812, https://doi.org/10.1039/c3cp55189j, 2014; Baituti in Hyperfine Interact 238(1), https://doi.org/10.1007/s10751-017-1440-8, 2017; Ioannidis et al. in Biochemistry, https://doi.org/10.1021/bi060520s, 2006). The S2 ML EPR signal simulation using the 55Mn hyperfine coupling constants, with one very large, one medium, and two small hyperfine values, fits the experimental data. The Mn1 has a large hyperfine coupling, which agrees well with earlier data by Jin et al. [19]. Three large fractional anisotropy observed on three Mn centers (Mn1,3,4), suggests the presence of three MnIII ions, and Mn2 center is likely to be MnIV ion, hence favouring the LOP (MnIII MnIV MnIII MnIII).
... It has been reported that the half-inhibition temperature for the S 1 -to-S 2 transition is 130−140 K, while, for other transitions, the temperatures are 220−235 K ( Figure 1D). 19 At 190 K, the S 1to-S 2 transition can occur, but the other S-state transitions are blocked. At this temperature, YZ can be oxidized by P 680 + , but subsequently, YZ• fails to oxidize the OEC. ...
... 33,34,46 These data also provide the first evidence for an interaction between the S 2 n(H 2 O)H 3 O + internal water cluster (W n + ) and YZ• PCET reactions. 19 The red arrow indicates the transition studied here. ...
A redox-active tyrosine, YZ (Y161 in the D1 polypeptide), is essential in photosystem II (PSII), which conducts photosynthetic oxygen evolution. On each step of the light-driven oxygen evolving reaction, YZ radical is formed by a chlorophyll cation radical. YZ radical is then reduced by a Mn4CaO5 cluster in a proton coupled electron transfer (PCET) reaction. YZ is hydrogen bonded to His190-D1 and to water molecules in a hydrogen-bonding network, involving calcium. This network is sensitive to disruption with ammonia and to removal and replacement of calcium. Only strontium supports activity. Here, we use electron paramagnetic resonance (EPR) spectroscopy to define the influence of ammonia treatment, calcium removal, and strontium/barium substitution on YZ radical PCET at two pH values. A defined oxidation state of the metal cluster (S2) was trapped by illumination at 190 K. The net reduction and protonation of YZ radical via PCET were monitored by EPR transients collected after a 532 nm laser flash. At 190 K, YZ radical cannot oxidize the Mn4CaO5 cluster and decays on the seconds time scale by recombination with QA(-). The overall decay half-time and biexponential fits were used to analyze the results. The reaction rate was independent of pH in control, calcium-reconstituted PSII (Ca-PSII). At pH 7.5, the YZ radical decay rate decreased in calcium-depleted (CD-PSII) and barium/strontium-reconstituted PSII (Ba-PSII, Sr-PSII), relative to Ca-PSII. At pH 6.0, the YZ radical decay rate was not significantly altered in CD-PSII and Sr-PSII but decreased in Ba-PSII. A two-pathway model, involving two competing proton donors with different pKa values, is proposed to explain these results. Ammonia treatment decreased the YZ decay rate in Ca-PSII, Sr-PSII, and CD-PSII, consistent with a reaction that is mediated by the hydrogen-bonding network. However, ammonia treatment did not alter the rate in Ba-PSII. This result is interpreted in terms of the large ionic radius of barium and the elevated pKa of barium-bound water, which are expected to disrupt hydrogen bonding. In addition, evidence for a functional interaction between the S2 protonated water cluster (Wn(+)) and the YZ proton donation pathway is presented. This interaction is proposed to increase the rate of the YZ PCET reaction.
... In principle, the same situation should occur in S 1 Y D OX thylakoids, but here the sample contains 40-50% Q B − due to the pre-flash protocol, thus making a description of the decay with a single average rate sufficiently good (since k 2(s) / k 2(vs) ≈ 4, Table 1). A very slow S 2 decay of similar rate (k 21(vs) ≈ 0.0035 s −1 at 18°C) was described previously when the S 2 decay in PSII membrane fragments was studied by EPR in presence of external electron acceptors [83]. Our data strongly reinforce this earlier report and show that the very slow decay occurs also in untreated thylakoids. ...
... In contrast, our data set provides no conclusive evidence for a very slow decay of the S 3 state; under all circumstances, we found the rates for the slow and the very slow phases to be indistinguishable. This discrepancy to the data by Styring and Rutherford [83] was likely due to experimental constraints. In the S 1 Y D samples the fast reduction of S 3 dominated, making it difficult to accurately determine the (very) slow decay rate, and in the S 1 Y D OX sample an about equal mix of Q B and Q B − may lead, as in case of S 2 (see above), to the observation of an apparent single decay phase of average rate. ...
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.
... The S 2 and S 3 states are reduced, in the seconds to minutes time scales, to S 1 by charge recombination with electrons from the electron acceptor side (Q B − , Q B H 2 ) or by electron donation from tyrosine Y D (see Figs. 3 and 5b). The S 0 state, on the other hand, is oxidized to S 1 , which occurs in the 10's of min time scale by electron transfer to the oxidized form of Y D , which is an unusually stable neutral radical, abbreviated as Y D ox or Y D • (Styring and Rutherford 1988;Vass and Styring 1991;Messinger et al. 1993;Messinger andRenger 1994, 2008;Isgandarova et al. 2003). ...
Photosynthetic water oxidation by Photosystem II (PSII) is a fascinating process because it sustains life on Earth and serves as a blue print for scalable synthetic catalysts required for renewable energy applications. The biophysical, computational, and structural description of this process, which started more than 50 years ago, has made tremendous progress over the past two decades, with its high-resolution crystal structures being available not only of the dark-stable state of PSII, but of all the semi-stable reaction intermediates and even some transient states. Here, we summarize the current knowledge on PSII with emphasis on the basic principles that govern the conversion of light energy to chemical energy in PSII, as well as on the illustration of the molecular structures that enable these reactions. The important remaining questions regarding the mechanism of biological water oxidation are highlighted, and one possible pathway for this fundamental reaction is described at a molecular level.
... A High oxidation state model B Low oxidation state model fraction of PSII centers (double hits) but to the extremely long S 2 and S 3 state lifetimes in the presence of PPBQ that led to an incomplete S 1 state synchronization within the 60 min darkadaptation period after the preflash employed to oxidize tyrosine D (40)(41)(42)(43)(44). ...
Knowledge of the manganese oxidation states of the oxygen-evolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S2 state of this cluster. This signal implies an overall oxidation state of either Mn(III) 3 Mn(IV) or Mn(III)Mn(IV) 3 for the S2 state. These 2 competing assignments are commonly known as “low oxidation (LO)” and “high oxidation (HO)” models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster’s oxidation states.
... Tyr Z % → S 3 Tyr Z transition happens [57]. Secondly, the temperature threshold at which the S 2 LS to S 3 transition is blocked at pH 6.5, that is ≈ 230-240 K [59], corresponds to the temperature at which the S 2 LS Tyr Z to S 2 HS Tyr Z transition is inhibited at high pH [47], a situation supposed to be close to that for S 2 LS Tyr Z % to S 2 ...
The Mn 4 CaO 5 -cluster in Photosystem II advances through five oxidation states, S 0 to S 4 , before water is oxidized and O 2 is generated. The S 2 -state exhibits either a low-spin, S = 1/2 (S 2LS ), or a high-spin state, S = 5/2 (S 2HS ). Increasing the pH favors the S 2HS configuration and mimics the formation of Tyr Z [rad] in the S 2LS -state at lower pH values (Boussac et al. Biochim. Biophys. Acta 1859 (2018) 342). Here, the temperature dependence of the S 2HS to S 3 transition was studied by EPR spectroscopy at pH 8.6. The present data strengthened the involvement of S 2HS as a transient state in the S 2LS Tyr Z [rad] → S 2HS Tyr Z [rad] → S 3 Tyr Z transition. Depending on the temperature, the S 2HS progresses to S 3 states exhibiting different EPR properties. One S 3 -state with a S = 3 signal, supposed to have a structure with the water molecule normally inserted in S 2 to S 3 transition, can be formed at temperatures as low as 77 K. This suggests that this water molecule is already bound in the S 2HS state at pH 8.6. The nature of the EPR invisible S 3 state, formed down to 4.2 K from a S 2HS state, and that of the EPR detectable S 3 state formed down to 77 K are discussed. It is proposed that in the S 2LS to S 3 transition, at pH < 8.6, the proton release (Sugiura et al. Biochim. Biophys. Acta 1859 (2018) 1259), the S 2LS to S 2HS conversion and the binding of the water molecule are all triggered by the formation of Tyr Z [rad].
... (iii) The temperature of the experiment (~235 K) blocks the re-reduction from the OEC. The reaction cycle of the OEC 29 is strongly inhibited at 230 K [30][31][32] . If we assume that the OEC remains blocked in its S2 state, even at pH 6-7.5, the re-reduction rate would slow down to 250 ns 33 . ...
The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect allows for increase of signal and sensitivity in magic-angle spinning (MAS) NMR experiments. The effect occurs in photosynthetic reaction centers (RC) proteins upon illumination and induction of cyclic electron transfer. Here we show that the strength of the effect allows for observation of the cofactors forming the spin-correlated radical pair (SCRP) in isolated proteins, in natural photosynthetic membranes as well as in entire plants. To this end, we measured entire selectively 13C isotope enriched duckweed plants (Spirodela oligorrhiza) directly in the MAS rotor. Comparison of 13C photo-CIDNP MAS NMR spectra of photosystem II (PS2) obtained from different levels of RC isolation, from entire plant to isolated RC complex, demonstrates the intactness of the photochemical machinery upon isolation. The SCRP in PS2 is structurally and functionally very similar in duckweed and spinach (Spinacia oleracea). The analysis of the photo-CIDNP MAS NMR spectra reveals a monomeric Chl a donor. There is an experimental evidence for matrix involvement, most likely due to the axial donor histidine, in the formation of the SCRP. Data do not suggest a chemical modification of C-13^1 carbonyl position of the donor cofactor.
... The S 2 (III, IV, IV, IV) and S 3 (IV, IV, IV, IV) states decay to the S 1 state within seconds to minutes via reduction by the reduced form of tyrosine Y D RED (D2-Tyr160) or recombination with Q B − (Diner 1977;Rutherford et al. 1982;Robinson and Crofts 1983;Rutherford and Inoue 1984;Vermaas et al. 1984Vermaas et al. , 1988Nugent et al. 1987). In addition, a very slow decay was reported, but the electron donor for this phase was not yet identified (Styring and Rutherford 1988). By contrast, the S 0 state is slowly oxidized to the S 1 state by the long-lived Y D OX radical, but is stable if Y D is reduced (Styring and Rutherford 1987;Vass and Styring 1991;Messinger and Renger 1993). ...
Photosynthetic water oxidation is catalyzed by the oxygen-evolving complex (OEC) in photosystem II (PSII). This process is energetically driven by light-induced charge separation in the reaction center of PSII, which leads to a stepwise accumulation of oxidizing equivalents in the OEC (Si states, i = 0–4) resulting in O2 evolution after each fourth flash, and to the reduction of plastoquinone to plastoquinol on the acceptor side of PSII. However, the Si-state advancement is not perfect, which according to the Kok model is described by miss-hits (misses). These may be caused by redox equilibria or kinetic limitations on the donor (OEC) or the acceptor side. In this study, we investigate the effects of individual S state transitions and of the quinone acceptor side on the miss parameter by analyzing the flash-induced oxygen evolution patterns and the S2, S3 and S0 state lifetimes in thylakoid samples of the extremophilic red alga Cyanidioschyzon merolae. The data are analyzed employing a global fit analysis and the results are compared to the data obtained previously for spinach thylakoids. These two organisms were selected, because the redox potential of QA/QA⁻ in PSII is significantly less negative in C. merolae (Em = − 104 mV) than in spinach (Em = − 163 mV). This significant difference in redox potential was expected to allow the disentanglement of acceptor and donor side effects on the miss parameter. Our data indicate that, at slightly acidic and neutral pH values, the Em of QA⁻/QA plays only a minor role for the miss parameter. By contrast, the increased energy gap for the backward electron transfer from QA⁻ to Pheo slows down the charge recombination reaction with the S3 and S2 states considerably. In addition, our data support the concept that the S2 → S3 transition is the least efficient step during the oxidation of water to molecular oxygen in the Kok cycle of PSII.
... Axel Bergmann measured time correlated fluorescence spectra of the light harvesting complex, LHCII, with, essentially, a major emission band at 681 nm, which had a 4 ns decay time at 277 K. The steady state absorption spectrum shows several peaks and shoulders implying channeling of excitation energy to a common (Styring and Rutherford 1988) k H /k D (Karge et al. 1997) i = 0 emitter pool. The transient absorption changes with 120 fs excitation, measured by Theiss, then allowed the determination of energy transfer times in LHCII and in CP29 and CP47 ). ...
Gernot Renger (October 23, 1937–January 12, 2013), one of the leading biophysicists in the field of photosynthesis research, studied and worked at the Max-Volmer-Institute (MVI) of the Technische Universität Berlin, Germany, for more than 50 years, and thus witnessed the rise and decline of photosynthesis research at this institute, which at its prime was one of the leading centers in this field. We present a tribute to Gernot Renger’s work and life in the context of the history of photosynthesis research of that period, with special focus on the MVI. Gernot will be remembered for his thought-provoking questions and his boundless enthusiasm for science.
... 114 It is assumed that there is at least one breakpoint in the reaction coordinate of the trigger process(es), as blockage of the sequences of eqs 11 and 12 for the S i -state transitions is observed below threshold temperatures. 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. ...
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.
... Specically, while long dark adapted PSII samples are expected to be predominantly in the S1 state, shorter dark adapted samples represent a 75% : 25% mix of the S 1 and S 0 states, respectively. 14 S-state synchronization requires either long-term dark adaptation or a pre-illumination sequence described by Styring and Rutherford,174,175 which relies on fast S 2 and S 3 deactivation to S 1 and slow S 0 oxidation by the tyrosine D residue. Without further controls it is unclear into which regime the XFEL dataset belongs, or that the kinetics of S-state synchronization are identical for the partially dehydrated crystal preparation and those previously measured in solution samples. ...
A central question in biological water splitting concerns the oxidation states of the manganese ions that comprise the oxygen-evolving complex of photosystem II. Understanding the nature and order of oxidation events that occur during the catalytic cycle of five S
i
states (i = 0-4) is of fundamental importance both for the natural system and for artificial water oxidation catalysts. Despite the widespread adoption of the so-called "high-valent scheme"-where, for example, the Mn oxidation states in the S2 state are assigned as III, IV, IV, IV-the competing "low-valent scheme" that differs by a total of two metal unpaired electrons (i.e. III, III, III, IV in the S2 state) is favored by several recent studies for the biological catalyst. The question of the correct oxidation state assignment is addressed here by a detailed computational comparison of the two schemes using a common structural platform and theoretical approach. Models based on crystallographic constraints were constructed for all conceivable oxidation state assignments in the four (semi)stable S states of the oxygen evolving complex, sampling various protonation levels and patterns to ensure comprehensive coverage. The models are evaluated with respect to their geometric, energetic, electronic, and spectroscopic properties against available experimental EXAFS, XFEL-XRD, EPR, ENDOR and Mn K pre-edge XANES data. New 2.5 K 55Mn ENDOR data of the S2 state are also reported. Our results conclusively show that the entire S state phenomenology can only be accommodated within the high-valent scheme by adopting a single motif and protonation pattern that progresses smoothly from S0 (III, III, III, IV) to S3 (IV, IV, IV, IV), satisfying all experimental constraints and reproducing all observables. By contrast, it was impossible to construct a consistent cycle based on the low-valent scheme for all S states. Instead, the low-valent models developed here may provide new insight into the over-reduced S states and the states involved in the assembly of the catalytically active water oxidizing cluster.
... The damping is due to an unusually rapid deactivation of the higher S-states S 2 and S 3. Figure 5B shows the relative amplitude of AATR at 295 nm on the third flash as a function of the delay time between flash 2 and 3 (squares) or 1 and 2 (circles), reflecting the decay of S 3 and of S 2, respectively. The observed half-times of 1 and 3 s are much shorter than in PS II membrane fragments (Styring and Rutherford 1988) and agree with the times reported by Gleiter et al. (1993) for a similar core preparation. The incomplete recovery after 2 flashes may reflect inactivation, consistent with the decreased amplitude of the oscillation of AAsT measured after 30 s (Fig. 3A). ...
Flash-induced redox reactions in spinach PS II core particles were investigated with absorbance difference spectroscopy in the UV-region and EPR spectroscopy. In the absence of artificial electron acceptors, electron transport was limited to a single turnover. Addition of the electron acceptors DCBQ and ferricyanide restored the characteristic period-four oscillation in the UV absorbance associated with the S-state cycle, but not the period-two oscillation indicative of the alternating appearance and disappearance of a semiquinone at the QB-site. In contrast to PS II membranes, all active centers were in state S1 after dark adaptation. The absorbance increase associated with the S-state transitions on the first two flashes, attributed to the Z(+)S1→ZS2 and Z(+)S2→ZS3 transitions, respectively, had half-times of 95 and 380 μs, similar to those reported for PS II membrane fragments. The decrease due to the Z(+)S3→ZS0 transition on the third flash had a half-time of 4.5 ms, as in salt-washed PS II membrane fragments. On the fourth flash a small, unresolved, increase of less than 3 μs was observed, which might be due to the Z(+)S0→ZS1 transition. The deactivation of the higher S-states was unusually fast and occurred within a few seconds and so was the oxidation of S0 to S1 in the dark, which had a half-time of 2-3 min. The same lifetime was found for tyrosine D(+), which appeared to be formed within milliseconds after the first flash in about 10% inactive centers and after the third and later flashes by active centers in Z(+)S3.
... These estimates may be compared with corresponding values of 4.3 s for $2 and 1.5 s for $3 obtained from oxygen flash dependence data (Gleiter et al. 1993), and 3 s for $2 and 1 s for $3 obtained from 295 nm absorption measurements (van Leeuwen et al. 1993). All of these values are much smaller than those previously reported (Styring and Rutherford 1988) for the S-state deactivation kinetics in PS II membrane fragments. ...
The kinetics of P680+ reduction in oxygen-evolving spinach Photosystem II (PS II) core particles were studied using both repetitive and single-flash 830 nm transient absorption. From measurements on samples in which PS II turnover is blocked, we estimate radical-pair lifetimes of 2 ns and 19 ns. Nanosecond single-flash measurements indicate decay times of 7 ns, 40 ns and 95 ns. Both the longer 40 ns and 95 ns components relate to the normal S-state controlled Yz → P680+ electron transfer dynamics. Our analysis indicates the existence of a 7 ns component which provides evidence for an additional process associated with modified interactions involving the water-splitting catalytic site. Corresponding microsecond measurements show decay times of 4 μs and 90 μs with the possibility of a small component with a decay time of 20–40 μs. The precise origin of the 4 μs component remains uncertain but appears to be associated with the water-splitting center or its binding site while the 90 μs component is assigned to P680+-QA
− recombination. An amplitude and kinetic analysis of the flash dependence data gives results that are consistent with the current model of the oxygen-evolving complex.
In Photosystem II, light-induced water splitting occurs via the S state cycle of the CaMn4O5-cluster. To understand the role of various possible conformations of the CaMn4O5-cluster in this process, the temperature dependence of the S1 → S2 and S2 → S3 state transitions, induced by saturating laser flashes, was studied in spinach photosystem II membrane preparations under different conditions. The S1 → S2 transition temperature dependence was shown to be much dependent on the type of the cryoprotectant and presence of 3.5% methanol, resulting in the variation of transition half-inhibition temperature by 50 K. No similar effect was observed for the S2 → S3 state transition, for which we also show that both the low spin g = 2.0 multiline and high spin g = 4.1 EPR configurations of the S2 state advance with similar efficiency to the S3 state, both showing a transition half-inhibition temperature of 240 K. This was further confirmed by following the appearance of the Split S3 EPR signal. The results are discussed in relevance to the functional and structural heterogeneity of the water oxidizing complex intermediates in photosystem II.
D1-Tyr161 (TyrZ) forms a low-barrier H-bond with D1-His190 and functions as a redox-active group in photosystem II. When oxidized to the radical form (TyrZ-O•), it accepts an electron from the oxygen-evolving Mn4CaO5 cluster, facilitating an increase in the oxidation state (Sn; n = 0–3). Here we investigated the mechanism of how TyrZ-O• drives proton-coupled electron transfer during the S2 to S3 transition using a quantum mechanical/molecular mechanical approach. In response to TyrZ-O• formation and subsequent loss of the low-barrier H-bond, the ligand water molecule at the Ca2+ site (W4) reorients away from TyrZ and donates an H-bond to D1-Glu189 at Mn4 of Mn4CaO5 together with an adjacent water molecule. The H-bond donation to the Mn4CaO5 cluster triggers the release of the proton from the lowest pKa site (W1 at Mn4) along the W1…D1-Asp61 low-barrier H-bond, leading to protonation of D1-Asp61. The interplay of the two low-barrier H-bonds, involving the Ca2+ interface and forming the extended Grotthuss-like network [TyrZ…D1-His190]-[Mn4CaO5]-[W1…D1-Asp61], rather than the direct electrostatic interaction, is likely a basis of the apparent long-distance interaction (11.4 Å) between TyrZ-O• formation and D1-Asp61 protonation.
In photosystem II (PSII), one-electron oxidation of the most stable oxidation state of the Mn4CaO5 cluster (S1) leads to formation of two distinct states, the open-cubane S2 conformation [Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV)] with low spin and the closed-cubane S2 conformation [Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III)] with high spin. In electron paramagnetic resonance (EPR) spectroscopy, the open-cubane S2 conformation exhibits a g=2 multiline signal. However, its protonation state remains unclear. Here we investigated the protonation state of the open-cubane S2 conformation by calculating exchange couplings in the presence of the PSII protein environment and simulating the pulsed electron-electron double resonance (PELDOR). When a ligand water molecule, which forms an H-bond with D1-Asp61 (W1), is deprotonated at dangling Mn4(IV), the first-exited energy (34 cm-1) in manifold spin excited states aligns with the observed value in temperature-dependent pulsed EPR analyses, and the PELDOR signal is best reproduced. Consequently, the g=2 multiline signal observed in EPR corresponds to the open-cubane S2 conformation with the deprotonated W1 (OH−).
Existence of the alternative charge separation pathway in Photosystem II under the far-red light was induced photochemistry in Photosystem II was proposed by us on the basis of induced electron transfer reactions at cryogenic temperature 5 K. Here we extend these studies to the higher temperature range of 77-295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of cytochrome b559 and chlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730-750 nm) spectral regions. Temperature dependence of the S2 state multiline signal induction after single flash at 730-750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data show that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in PSII with localization of hole on the ChlD1 molecule.
Photosynthesis stores solar light as chemical energy and efficiency of this process is highly important. The electrons required for CO2 reduction are extracted from water in a reaction driven by light-induced charge separations in the Photosystem II reaction center and catalyzed by the CaMn4O5-cluster. This cyclic process involves five redox intermediates known as the S0-S4 states. In this study, we quantify the flash-induced turnover efficiency of each S state by electron paramagnetic resonance spectroscopy. Measurements were performed in photosystem II membrane preparations from spinach in the presence of an exogenous electron acceptor at selected temperatures between -10 °C and +20 °C and at flash frequencies of 1.25, 5 and 10 Hz. The results show that at optimal conditions the turnover efficiencies are limited by reactions occurring in the water oxidizing complex, allowing the extraction of their S state dependence and correlating low efficiencies to structural changes and chemical events during the reaction cycle. At temperatures 10 °C and below, the highest efficiency (i.e. lowest miss parameter) was found for the S1 → S2 transition, while the S2 → S3 transition was least efficient (highest miss parameter) over the whole temperature range. These electron paramagnetic resonance results were confirmed by measurements of flash-induced oxygen release patterns in thylakoid membranes and are explained on the basis of S state dependent structural changes at the CaMn4O5-cluster that were determined recently by femtosecond X-ray crystallography. Thereby, possible "molecular errors" connected to the e - transfer, H+ transfer, H2O binding and O2 release are identified.
The temperature dependence of the formation of the g ~ 5 S2 state electron paramagnetic resonance (EPR) signal in photosystem II (PSII) was investigated. The g ~ 5 signal was produced at an illumination above 200 K. The half inhibition temperature of the formation of the g ~ 5 EPR signal was approximately 215 K. The half inhibition temperature is close to that of the transition from the S2 state-to-S3 state in the untreated PSII, and not to that of the transition from S1 state -to-S2 state in the untreated PSII. The upshift of the half inhibition temperature of the transition from the S1 state -to-S2 state (g ~ 5) reflects the structural change upon transition from the S1 state to the S2 state. The activation energy of the g ~ 5 state formation was estimated as 40.7 ± 4.4 kJ/mol, which is comparable to the reported activation energy for the S2 formation in the untreated PSII. The activation enthalpy and entropy were estimated to be 39.0 ± 4.4 kJ/mol and − 103 ± 19 J/mol K at 210 K, respectively. Based on these parameters, the formation process of the g ~ 5 state is discussed in this study.
The reactivity of the S3 and S2 states towards NO and NH2OH was studied and compared using the period-4 oscillations in the F0-value induced by a train of single turnover Xenon flashes spaced 100 ms apart to monitor the reaction kinetics. The flash frequency also determined the time resolution of the assay, i.e. 100 ms. The S2 and S3-states were created by one and two single turnover pre-flashes, respectively. The NO concentration-dependence of the S3-decay indicated that at low NO-concentrations an S2-state was formed as an intermediate, whereas at higher concentrations a seemingly monophasic decay to the S1-state was observed. The sigmoidal concentration dependence indicated that a fast interaction of the S3-state with (at least) two NO-molecules is necessary for the fast S3 to S1 decay (τ ~0.4 s at 1.2 mM NO). The pH-dependence of the S3-decay suggests that a protonation reaction (pK ~6.9) is involved in the S3 to S1 decay. At intermediate NO-concentrations the protonation is only partially rate limiting, since the pH effect is more pronounced at high compared to intermediate NO-concentrations. A comparison of the reactivity of NO and hydroxylamine suggests that hydroxylamine reacts more efficiently with the S1 and S2 states, whereas NO reacts more efficiently with the S3-state. Based on our present knowledge of the oxygen evolving complex a possible reaction mechanism is proposed for the interaction between NO and the S3 state.
[FeFe]-hydrogenases are nature's blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H2 fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe]H), cysteine-linked to a diiron site ([2Fe]H) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (μCO) in the oxidized protein (Hox). Recently, the debate on the structure of reduced H-cluster states was intensified by the assignment of new species under cryogenic conditions. We investigated temperature effects (4-280 K) in infrared (IR) and X-ray absorption spectroscopy (XAS) data of [FeFe]-hydrogenases using fit analyses and quantum-chemical calculations. IR data from our laboratory and literature sources were evaluated. At ambient temperatures, reduced H-cluster states with a bridging hydride (μH-, in Hred and Hsred) or with an additional proton at [4Fe]H (Hred') or at the distal iron of [2Fe]H (Hhyd) prevail. At cryogenic temperatures, these species are largely replaced by states that hold a μCO, lack [4Fe]H protonation, and bind an additional proton at the adt nitrogen (HredH
+
and HsredH
+
). XAS revealed the atomic coordinate dispersion (i.e., the Debye-Waller parameter, 2σ2) of the iron-ligand bonds and Fe-Fe distances in the oxidized and reduced H-cluster. 2σ2 showed a temperature dependence typical for the so-called protein-glass transition, with small changes below ∼200 K and a pronounced increase above this "breakpoint". This behavior is attributed to the freezing-out of larger-scale anharmonic motions of amino acid side chains and water species. We propose that protonation at [4Fe]H as well as ligand rearrangement and μH- binding at [2Fe]H are impaired because of restricted molecular mobility at cryogenic temperatures so that protonation can be biased toward adt. We conclude that a H-cluster with a μCO, selective [4Fe]H or [2Fe]H protonation, and catalytic proton transfer via adt facilitates efficient H2 conversion in [FeFe]-hydrogenase.
In photosystem II (PSII), photosynthetic water oxidation occurs at the O2-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S0 - S4) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S2 to S1 transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7 - 2.7x slower rate of charge recombination with QA_ relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an Ea of 5.87 ± 1.15 kcal mol⁻¹ for decay back to the S1 state, compared to 0.80 ± 0.13 kcal mol⁻¹ for the wild-type S2 state. In addition, we find that ammonia does not affect the S2-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S2 state of D1-S169A PSII. The altered S2 state of D1-S169A PSII provides insight into the S2➔S3 state transition.
We develop a rapid “stroboscopic” fluorescence induction method, using the fast repetition rate fluorometry (FRRF) technique, to measure changes in the quantum yield of light emission from chlorophyll in oxygenic photosynthesis arising from competition with primary photochemical charge separation (P680* ➔ P680⁺QA⁻). This method determines the transit times of electrons that pass through PSII during the successive steps in the catalytic cycle of water oxidation/O2 formation (S states) and plastoquinone reduction in any oxygenic phototroph (in vivo or in vitro). We report the first measurements from intact living cells, illustrated by a eukaryotic alga (Nannochloropsis oceanica). We demonstrate that S state transition times depend strongly on the redox state of the PSII acceptor side, at both QB and the plastoquinone pool which serve as the major locus of regulation of PSII electron flux. We provide evidence for a kinetic intermediate S3′ state (lifetime 220 μs) following formation of S3 and prior to the release of O2. We compare the FRRF-detected kinetics to other previous spectroscopic methods (optical absorbance, EPR, and XES) that are applicable only to in vitro samples.
Natural photosynthesis is the only working system of solar energy storage that operates on a global scale. The water-oxidation reaction, carried out by the protein complex photosystem II (PSII), is the key reaction that initiates photosynthesis. However, the detailed mechanism of this reaction is yet to be fully understood. In this article, we review the current knowledge of how the essential components in PSII, including the oxygen-evolving complex and its surrounding environment, take part in the water-oxidation reaction, and finally present proposals for the water-oxidation mechanism.
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.
Oxygenic photosynthesis in nature occurs via water splitting catalyzed by the oxygen evolving complex (OEC) of photo-system II. To split water, the OEC cycles through a sequence of oxidation states (Si, i = 0-4), the structural mechanism of which is not fully understood under physiological conditions. We monitored the OEC in visible light-driven water-splitting action by using in-situ, aqueous-environment surface-enhanced Raman scattering (SERS). In the unexplored low-frequency region of SERS, we found dynamic vibrational signatures of water binding and splitting. Specific snapshots in the dynamic SERS correspond to intermediate states in the catalytic cycle, as determined by density functional theory and isotopologue comparisons. We assign the previously ambiguous protonation configuration of the S0-S3 states and propose a structural mechanism of the OEC’s catalytic cycle. The findings address unresolved questions about photosynthetic water splitting and introduce spatially-resolved, low-frequency SERS as a chemically sensitive tool for interrogating homogeneous catalysis in operando.
This chapter describes the principles of solar energy exploitation by water splitting. The most delicate part of the overall process is the oxidation of two water molecules leading to O2 formation and H+ release. The chapter focuses on problems of this reaction and how it is performed in the multimeric photosystem II (PSII) core complex of the photosynthetic apparatus. It shows that the living organisms can use the enormous potential of highly specialized biopolymers (i.e., proteins) in tuning energetics and kinetics of the cofactors as the functional sites of biological catalysis. Of special relevance for oxidative water splitting, taking place at a Mn4OxCa cluster, is an optimized coupling between electron and proton transfer steps. The chapter considers the possibility of developing functionalized synthetic polymer matrices for both binding the catalytic sites and simultaneously providing a spatial separation of oxidative and reductive pathways of light-induced water splitting.
Photosynthetic water splitting, localized in photosystem II, is the source of atmospheric oxygen and possible alternative energy source. It is therefore important to understand the related processes which influence the efficiency of water splitting. We have employed kinetic models of photosystem II to study deactivation processes of higher S-states (redox states of water splitting) in the dark. Our analysis of spinach samples, treated or untreated by electron acceptor phenylparabenzoquinone (PPBQ) indicated an unknown mechanism, decay, related to S\(_{2,3}\)-state deactivation. We concluded that: (1) S\(_3\)-state decay occurs independently on the PPBQ treatment, i.e., independently on the redox state of the acceptor side of photosystem II, (2) S\(_2\)-state decay can be fully described by S\(_2\)-\(Q^{-}_{A,B}\) charge recombination, neglected in previous models, and (3) the mechanism of S\(_3\)-state decay can be explained by the involvement of slow cooperation within photosystem II dimer between S\(_3\)PSIIa and S\(_{3,2,1}\)PSIIb in higher plants. Finally, the slow cooperation is able to explain experimental data both from PPBQ-free and PPBQ treated samples.
Internal water is known to play a catalytic role in several enzymes. In photosystem II (PSII), water is the substrate. To oxidize water, the PSII Mn4CaO5 cluster or oxygen evolving center (OEC) cycles through five oxidation states, termed Sn states. As reaction products, molecular oxygen is released, and protons are transferred through a ~25 Å hydrogen-bonded network from the OEC to the thylakoid lumen. Previously, it was reported that a broad infrared band at 2880 cm-1 is produced during the S1-to-S2 transition and accompanies flash-induced, S state cycling at pH 7.5. Here, we report that when the S2 state is trapped by continuous illumination under cryogenic conditions (190 K), an analogous 2740/2900 cm-1 band is observed. The frequency depended on the sodium chloride concentration. This band is unambiguously assigned to a normal mode of water by D216O and H218O solvent exchange. Its large, apparent H218O isotope shift, ammonia sensitivity, frequency, and intensity support assignment to a stretching vibration of a hydronium cation, H3O+, in a small, protonated internal water cluster, nH2O(H3O+). Water OH stretching bands, which may be derived from the hydration shell of the hydronium ion, are also identified. Using the 2740 cm-1 infrared marker, the results of calcium depletion and strontium reconstitution on the protonated water cluster are found to be pH dependent. This change is attributed to protonation of an amino acid side chain and a possible change in nH2O(H3O+) localization in the hydrogen-bonding network. These results are consistent with an internal water cluster functioning as a proton acceptor and an intermediate during the S1-to-S2 transition. Our experiments demonstrate the utility of this infrared signal as a novel functional probe in PSII.
The Magnetically-Split Spectra Of The Metalloradical Intermediates S0YZ • And S2,Yz • Collapse Above ∼100 K To The Unperturbed Spectrum Of YZ • (Zahariou Et Al. 2007). We Studied The Spectrum Of YZ •As Well As Its Decay Kinetics Above 100 K In Various S States. In This Report The Intermediate S2YZ •Is Examined. Its Unperturbed Spectrum Appears Distinct From That Of The Stable YD • Radical. The Spectrum Of YZ • Was Simulated By Varying The Rotational Conformation Of The Phenoxyl Ring And The Spin Density p On Carbon C1. The Latter Can Be Related To The Strength Of The Hydrogen Bond Between Tyrosine And Its Base Partner (Presumably D1 His190).
We studied the temperature dependence of the S1YZ •, S2YZ •, S0YZ •(+MeOH) and S2YZ •(+MeOH) metalloradical EPR signals in the temperature range 4.2–240 K, using slow and rapid scans. As the temperature increases the spectra narrow progressively and above 100 K collapse to a “25 G” signal somewhat broader than “signal II”. The spectra do not reach saturation at microwave powers up to 100 mW over the entire temperature range, and the signal intensity multiplied by temperature remains approximately constant. The narrowing of the SnYz • EPR signals is consisted with an increase of the Mn spin-lattice relaxation rate, with increasing temperature. We conclude that the broad EPR signals observed at low temperatures and the narrow signals at elevated temperatures are manifestations of the same intermediate SnYz • (n = 0, 1, 2) and the high temperature spectrum is due to Tyr Z• unperturbed by the magnetic interaction with Mn.
An introduction to electron magnetic resonance (EMR) with applications in biophysical studies is presented at the level of nonspecialist or beginning graduate student. The first half of the chapter briefly introduces the resonance phenomenon, a typical EMR spectrum and its interpretation, and describes fundamental applications of electron resonance spectroscopy in free radical research, identification and characterization of metalloproteins and reaction intermediates, spin probes, and imaging. The second half of the chapter describes the magnetochemical origins of resonance spectroscopy and the steps that have led to modern EMR techniques.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
Electron transfer from the reduced tyrosine YD and cytochrome b559 (Cyt b559) to the S2 and S3 states of photosystem II was investigated at the temperature of 195 K. Electron transfer reactions were followed by measuring EPR signals of tyrosine YD·, oxidized Cyt b559 and the S2-state multiline signal. Long term incubation (∼90 days) at 195 K causes decay of the majority of S2 centers up to ∼40% of initial value, while in this time scale the intensity of YD· radical increases less than 10%. Samples advanced to S3 state demonstrates an increasing behavior of the S2-state multiline signal intensity in the beginning of incubation (∼20 days) and slow decay up to 40% of maximal amplitude during further incubation of the samples. Similarly to the S2 sample, small increase in YD· radical signal was observed during the S3 decay. However, in both types of samples prepared in S2 and S3 states after 90 days of incubation the signal of oxidized Cyt b559 is increased from 45%–50% up to 100% maximal intensity. The results obtained in this study support the conclusion of our early investigations which claimed the reduced Cyt b559 as electron source for the S2 and S3 states.
The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (YZ). Cl(-) is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca(2+)/Sr(2+) exchange and Cl(-)/I(-) exchange perturb the proportions of centres showing high (S=5/2) and low spin (S=1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S=3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ(•))' split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S=5/2) form of S2 shows a marked heterogeneity with a g=4.3 form generated at low temperature that converts to a relaxed form at g=4.9 at higher temperatures. The high spin g=4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180K whereas the low spin (S=1/2) S2-state can only advance to the S3 state at temperatures≥235K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at≥235K but not at 198K. Since both S2 configurations are formed at 198K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s).
Copyright © 2015. Published by Elsevier B.V.
We have used the technique of thermoluminescence (TL) to investigate high-light-induced chlorophyll fluorescence quenching phenomena in barley leaves, and have shown it to be a powerful tool in such investigations. TL measurements were taken from wild-type and chlorina f2 barley leaves which had been dark-adapted or exposed to 20 min illumination of varying irradiance or given varying periods of recovery following strong irradiance. We have found strong evidence that there is a sustained trans-thylakoid ΔpH in leaves following illumination, and that this ΔpH gives rise to quenching of chlorophyll fluorescence which has previously been identified as a slowly-relaxing component of antenna-related protective energy dissipation; we have identified a state of the PS II reaction centre resulting from high light treatments which is apparently able to perform normal charge separation and electron transport but which is `non-photochemically' quenched, in that the application of a light pulse of high irradiance cannot cause the formation of a high fluorescent state; and we have provided evidence that a transient state of the PS II reaction centre is formed during recovery from such high light treatments, in which electron transport from Q_A to Q_B is apparently impaired.
We have investigated the electron transfer from reduced tyrosine Y D (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S 2 state multiline EPR signal and the EPR signal from Y D· were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD· signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD·S1 centers, via the intermediate YD·S2 state. The early increase of the S2 state multiline signal involves electron transfer from Y Dred to the S3 state resulting in formation of YD·S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b 559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S 2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.
Earlier mass spectrometric measurements, in which oxygen evolution was measured following short saturating light flashes, indicated that with a time resolution of about 30 s no form of bound water and/or an oxidation product exists up to the redox state S3 of the oxygen evolving center (R. Radmer and O. Ollinger, 1986, FEBS Lett 195: 285-289; K.P. Bader, P. Thibault and G.H. Schmid, 1987, Biochim Biophys Acta 893: 564-571). In the present study, isotope exchange experiments with H2 (18)O were performed under different experimental conditions. We found: a) the isotope exchange pattern is virtually the same at both pH 6.0 and 7.8, although marked structural changes of the PS II donor side are inferred to take place within this pH-range (Renger G., Messinger J. and Wacker U., 1992, Research in Photosynthesis, II: 329-332); b) injection of H2 (18)O at about 0°C gives rise to mass ratios of the evolved oxygen which markedly deviate from the theoretically expected values of complete isotope scrambling; and c) rapid injection of H2 (18)O into samples with high population of S1 and S2 and subsequent illumination with three and two flashes, respectively, spaced by a dark time of only 1.5 ms lead to similar (18)O-labeling of the evolved oxygen. Based on the published data on the interaction with redox active amines, possible pathways of substrate exchange in the water oxidase are discussed.
The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59-63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O2 evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O2 evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn(2+) and Ca(2+), while Cl(-) addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S3→(S4)→S0 transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH4Cl-suggesting that NH3 might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.
Redox events that occur in photosynthetic O2-evolving centers in NaCl/EDTA-washed PS II membranes were investigated by means of low temperature EPR and thermoluminescence. The following results have been obtained: (i) In the washed membranes, O2 centers could maintain the S2 state more than 3 h in darkness at room temperature. This dark-stable S2 was modified as seen by a multiline EPR signal with reduced hyperfine line spacing and also by a thermoluminescence band with upshifted peak temperature. (ii) This modified S2 state had an abnormally long life of at 20°C, and its appearance required the presence of EDTA in the medium. On addition of exogenous Ca2+, the modified S2 was converted in darkness to normal S2, and then decayed rapidly to be undetectable. (iii) On illuminating this modified S2, an EPR signal centering at aroung g = 2 was newly induced at no expense of the dark-stable EPR multiline signal. This EPR signal was accompanied by a new thermoluminescence band peaking at around 5°C, suggesting the presence of a new redox component whose oxidized form is capable of providing a positive charge for thermoluminescence in place of Mn. (iv) This new component was efficiently oxidized by illumination at −5°C but much less at −60°C, showing a half-inhibition temperature at around −40°C. (v) Addition of various divalent cations in place of Ca2+ variously affected both thermoluminescence glow peaks arising from the dark-stable S2 or from the new redox component, suggesting a cation-species-dependent modulation of the redox properties of both components. (vi) Both of these two thermoluminescence bands showed no dependency on flash number, suggesting interruption of further oxidation beyond their respective abnormal states. On addition of Ca2+, all these abnormal properties were abolished and normal period-four flash pattern was restored. These abnormal properties of the redox events in NaCl/EDTA-washed PS II membranes were discussed in relation to the demand for exogenous Ca2+ in recovery of normal properties.
Wateroxidation in Photosystem II is dependent on a particular amino acid residue, TyrosineZ. This is a redox intermediate in steady state oxygen evolution and transfers electrons from the water splitting CaMn4cluster to the central chlorophyll radical P680+. This Perspective discusses the functional principles of TyrosineZ as a proton-coupled redox active link, as well as mechanistic studies of synthetic model systems and implications for artificial photosynthesis. Experimental studies of temperature dependence and kinetic isotope effects are important tools to understand these reactions. We emphasize the importance of proton transfer distance and hydrogen bond dynamics that are responsible for variation in the rate of PCET by several orders of magnitude. The mechanistic principles discussed and their functional significance are not limited to tyrosine and biological systems, but are important to take into account when constructing artificial photosynthetic systems. Of particular importance is the role of proton transfer management in water splitting and solar fuel catalysis.
In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.
A major breakthrough in our understanding of how plants oxidize water to molecular O(2) was the discovery by P. Joliot and co-workers that the O(2) yield per flash, in a series of light flashes, oscillates with a periodicity of 4. This led to the concept by B. Kok and co-workers that these reactions involve accumulation of four positive charges in independent "O(2)-evolving centers," which undergo a series of changes in their redox state (the so-called S states). In the present paper, we have applied optical techniques (such as thermoluminescence and delayed light emission, both discovered by W. Arnold and co-workers) to monitor charge storage on the O(2)-evolving system in leaves from higher plants. We observed a period of four oscillations in both thermoluminescence and delayed light emission, with maxima on flashes 2 and 6, establishing a relationship with the charge accumulation process in photosynthesis. These measurements provided additional new information: the deactivation of the "O(2)-evolving centers," which cannot be measured by the O(2) method in the leaves, is in the 20- to 30-s range; and in the dark-adapted leaves, the secondary bound plastoquinone molecule (the so-called secondary electron acceptor Q(B)) is in equal concentration in its reduced and oxidized forms. The origin of thermoluminescence and delayed light emission, in terms of the recombination of charges on the O(2)-evolving and plastoquinone sides, is also discussed.
This chapter discusses the primary reactions of photosystems I and II of algae and higher plants. In photosynthetic organisms, the “primary reactions” fulfil the objective of converting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes—that is, milliseconds. In these reactions, a rather large fraction, approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major perspectives. Firstly, from a photochemical point of view: pigment molecules are excited to their lowest excited singlet state, which reacts in an electron transfer reaction, the first step of a process of charge separation. Secondly, from a biochemical point of view the reactions take place in highly organized complexes, the reaction centres, which are made up of several classes of molecules that cooperate in fulfilling complementary roles, such as: architectural support, light absorption, energy transfer and electron transfer. All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain photosystem I (PS I) and PS II reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity.
The oxidation‐reduction midpoint potential (E m) of the primary quinone (QA) of the acceptor quinone complex of bacterial photosynthetic reaction centers has been measured as a function of pH in the presence and absence of ubiquinone and o‐phenanthroline (o‐Phen). Reaction centers, isolated from Rhodopseudomonas sphaeroides, were incorporated into egg phosphatidylcholine vesicles. Contrary to earlier reports, the E m was found to exhibit a pH‐dependence very similar to that observed in chromatophores, with a slope of ‐ 60 mV/pH up to a p K for Q−A/Q−A(H+) at pH 9.5–10.0. In the presence of ubiquinone to reconstitute the secondary quinone (QB), the E m/pH curve of QA was shifted to lower potentials, indicating that the binding of Qn (actually QBH2) was suppressed by reduction of QA. o‐Phen, an inhibitor of electron transport from QA to QB, raised the pK of Q−A/Q−A(H+) and, at pH‐values below but not above this pK, reversed the effects of QB. In the absence of QB, o‐Phen lowered the E m of QA above the pK but had no effect below it. These results are discussed in terms of interactions between the binding sites for QA and QB (A‐ and B‐sites). It is suggested that ubiquinone and o‐Phen compete for the B‐site in a mutually exclusive fashion, and that their relative binding strengths are modulated by the redox and protonation state of QA. In preparations with low quinone content, o‐Phen inhibits photochemistry suggesting that it can also compete with ubiquinone at the A‐site. Competitive displacement of quinone from the B‐site by o‐Phen and other inhibitors is suggested as the primary mode of action of a broad class of herbicides active in Photosystem II of plants. The relative binding affinities of the various redox states of QB are also discussed and it is concluded that the order of binding strength is: Q−B → QB → QBH2. This accounts for the atypical stability of the semiquinone and the lower average E m for reduction to the quinol, compared to free ubiquinone in the quinone pool. It may also be significant in the functioning of quinones in communicating reducing equivalents from the reaction center to other electron transport complexes in the intact membrane.
A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S2Q−A state while that at 25°C is attributed to recombination of S2QAQ−B (and S3QAQ−B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S2QAQ−B and S3QAQ−B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to QA and thence to QB or Q−B) results in predictable changes in the extent of flash-induced thermoluminescence.
Patterns of O2 evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O2-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O2 evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O2 evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O2-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S3 was first order with a half-time of 105 s while that of S2 was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S2 phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S3 → S2 → S1, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S1 to S0 can be set by previously developed techniques, that S0 is formed mostly from activated S3 (S4), and that both S0 and S1 are stable in the dark.
Abstract— A quantitative analysis is made of a linear 4-step model for photosynthetic O2 evolution in which each photochemical trapping center or an associated enzyme cycles through 5 oxidation states (S0, S1, S2, S3, S4). The overall reaction is: S0→ S4, S4→ S0+ O2, where kd= rate of dark reaction. Based on data obtained with isolated chloroplasts, four aspects were considered: (1) The two perturbations which damp the oscillation of the O2 flash yield in a flash sequence given after a dark period-(a) a failure rate (α) of the trapping centers in the photochemical conversion (‘misses’) and (b) double effective excitations in a fraction (8) of the centers which are in the S0 and S1 states (‘double hits’). Best fit with the experimental data is obtained for α= 0.1, β=0.05. (2) The kinetics and the mechanism of deactivation—the loss of + charges (O2 precursors) in the dark. The momentary distribution of the four oxidation states (S0--S3) in a sample can be computed from the O2 yields of four consecutive flashes with corrections for a and β. The time course of the various states in the dark following specific preilluminations reveals that deactivation proceeds in single equivalent steps: S3→S2, S2,→S1. S1, itself stable in the dark, is the end product of deactivation. The ground state S0 cannot be formed by deactivation in the dark but is only formed during illumination. (3) The various ratios [S0]/[S1] which can be observed in a sample after deactivation following different preilluminations with flashes or continuous light. (4) The transients of the O2 evolution rate in weak continuous light as observed after deactivation with or without flash preillumination. In all instances satisfactory agreement is found between the observations and the predictions of the model.
In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S1 and S2 states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S2 state. Low-temperature illumination of samples prepared in the S2 state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH2OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl− with F− exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.
The oscillations of the ZV and A thermoluminescence bands were investigated in spinach chloroplasts which had been dark-adapted for various time periods and subjected to a series of flashes at +2°C before continuous illumination at various low temperatures. When excited with continuous light below −65°C, the ZV band exhibited period-4 oscillation, with maxima on preflashes 0, 4 and 8. Above −65°C, the oscillation pattern depended greatly on the dark-adaptation period of the chloroplasts. In preilluminated samples (15 s light followed by 3 min dark), when the QB pool is half oxidized, the oscillation of the thermoluminescence intensity measured at −50°C was similar to that observed below −65°C. However, after the thorough dark-adaptation of the chloroplasts (6 h), when the major fraction of the QB pool is assumed to be oxidized, a binary oscillation appeared in the oscillation pattern, with maxima at odd flash numbers. Below −65°C, period-2 oscillation of the ZV band could not be induced by the dark-adaptation of the chloroplasts, suggesting an inhibition of electron exchange between QA and QB. Upon excitation of the chloroplasts with continuous light at −30°C, the A band oscillated with a periodicity of 4 with maxima at preflash numbers 2 and 6. At pH 7.5, the period-4 oscillation was converted into a period-2 oscillation by thorough dark-adaptation of the chloroplasts (24 h). Model calculations of the oscillatory patterns suggest that the period-4 oscillations of the ZV and A bands are determined by the concentrations [S0] + [S1] and [S2] + [S3], respectively, which are present after the preflashes prior to the low-temperature continuous illumination. The period-2 oscillations in the amplitudes of the ZV and A bands reflect the changes occurring in the redox state of the QB pool in a sequence of flashes. The possible relationship between the characteristics of the ZV and A bands and the temperature-dependence of the S state transitions was investigated. Comparison of the amplitudal changes of the B (S2Q−B and S3Q−B recombination) and Q (S2Q−A recombination) thermoluminescence bands as a function of the excitation temperature suggests that the S2 → S3 and S3 → S4 transitions are blocked at about −65 and −40°C, respectively. It is also concluded that the thermoluminescence intensity emitted by the reaction center is about twice as high in the S3 state as in the S2 state.
We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S2 state gives rise to this multiline signal; we find that the S1 state can be fully advanced to the S2 state at temperatures as low as 160 K. The S2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.
A Mn-containing enzyme complex is involved in the oxidation of H/sub 2/O to O/sub 2/ in algae and higher plants. X-ray absorption spectroscopy is well suited for studying the structure and function of Mn in this enzyme complex. Results of X-ray K-edge and extended X-ray absorption fine structure (EXAFS) studies of Mn in the S/sub 1/ and S/sub 2/ states of the photosynthetic O/sub 2/-evolving complex in photosystem II preparations from spinach are presented in this paper. The S/sub 2/ state was prepared by illumination at 190 K or by illumination at 277 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); these are protocols that limit the photosystem II reaction center to one turnover. Both methods produce an S/sub 2/ state characterized by a multiline electron paramagnetic resonance (EPR) signal. An additional protocol, illumination at 140 K, produces a state characterized by the g = 4.1 EPR signal. The authors have previously observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark-adapted S/sub 1/ state to the S/sub 2/ state produced by illumination at 190 K. The Mn K-edge spectrum of the 277 K illuminated sample is similar to that produced atmore » 190 K, indicating that the S/sub 2/ state is similar when produced at 190 or 277 K. A similar edge shape and an edge shift of the same magnitude are seen for the 140 K illuminated sample. These results indicate that the g = 4.1 signal arises from oxidation of the Mn complex and that the structural differences between the species responsible for the g = 4.1 signal and the multiline EPR signal are subtle. They conclude from the edge and EXAFS studies that the light-induced S/sub 1/ to S/sub 2/ transition at 190 K or at 277 K involves a change in the oxidation state of Mn with no EXAFS-detectable change in the coordination of Mn in the O/sub 2/-evolving complex.« less
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
The thermoluminescence band observed in chloroplasts after flash excitation at ambient temperatures has recently been identified as being due to recombination of the electron on the semiquinone form of the secondary plastoquinone acceptor, QB, with positive charges on the oxygen-evolving enzyme, S2 and S3 (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). Further investigation of this thermoluminescence confirms this assignment and provides information on the function of PS II. The following data are reported: (1) Washing of chloroplasts with ferricyanide lowers the concentration of QB− in the dark and predictable changes in the extent of the thermoluminescence band are observed. (2) The thermoluminescence intensity arising from S2QB− is approximately one half of that arising from S3QB−. (3) Preflash treatment followed by dark adaptation results in changes in the intensity of the thermoluminescence band recorded after a series of flashes. These changes can be explained according to the above assignments for the origin of the thermoluminescence and if QB− provides an important source of deactivating electrons for the S states. Computer simulations of the preflash data are reported using the above assumptions. Previously unexplained data already in the literature (Läufer, A. and Inoue, Y. (1980) Photobiochem. Photobiophys. 1, 339–346) can be satisfactorily explained and are simulated using the above assumptions. (4) Lowering the pH to pH 5.5 results in a shift of the S2QB− thermoluminescence band to higher temperatures while that arising from S3QB− does not shift. This effect is interpreted as indicating that QB− is protonated and the S2 to S3 reaction involves deprotonation while the S1 to S2 reaction does not.
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.
The spin-lattice relaxation time of Signal II, which arises from two plastoquinol cation radicals, D+ and Z+, has been measured with electron spin-echo spectroscopy in Photosystem II preparations with inactivated and with intact oxygen-evolving complex. In Tris- and subsequently EDTA-washed Photosystem II preparations the spin-lattice relaxation times of D+ and Z+ are equal and remain unchanged if the pH is increased from 6.0 to 8.3. In preparations in which the oxygen-evolving complex is not inactivated by the Tris washing, the spin-lattice relaxation times of D+ and Z+ are affected by the redox state of the oxygen-evolving complex. At pH 6.0 the spin-lattice relaxation time decreases with higher redox state of the manganese cluster. The relaxation behavior at pH 8.3 indicates that at this pH the stability of the S1 state is decreased such that in dark-adapted samples nearly 100% of the systems is in the S0 state. This was confirmed by optical experiments where the period-four oscillation in the absorption at 350 nm was monitored as a function of the flash number.
The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S0 → S1, S1 → S2 and S2 → S3 transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z+ and the three Mn(IV) ions.
Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y4) is lower compared to Y3 than at lower flash frequency. At 4 Hz, the calculated S0 concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent ratio increases to about 0.2. This is explained by a relatively fast donation () of one electron by an electron donor to S2 and S3 in 15–25% of the Photosystem II reaction chains. The one-electron donor to S2 and S3 appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal IIs. (ii) The probability for the fast one-electron donation to S2 and S3 has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S2 decay becomes 10-fold faster () in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S2 decay was extremely slow. The S3 decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S2 is reduced mainly by Q−A, whereas S3 is not. (iii) In the absence of CO2/HCO−A and in the presence of formate, the fast one-electron donation to S2 and S3 does not occur. Addition of HCO−3 restores the fast decay of part of S2 and S3 to almost the same extent as in control thylakoids. The slow phase of S2 and S3 decay is not influenced significantly by CO2/HCO−3. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q−A oxidation without interference of QB, were 2.3-fold slower in the absence of CO2/HCO−3 than in its presence. (iv) An almost 3-fold decrease in decay rate of S2 is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q−A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q−B is responsible for this shift of equilibrium Q−A concentration, these data would suggest that the pKa value for Q−B protonation is somewhat higher than 7.6, assuming that the protonated form of Q−B cannot reduce QA.
The light-induced EPR multiline signal is studied in O2-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S2 state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q−AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S2. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S2 signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.
The role of chloride on the S-state transition in spinach Photosystem II (PS II) particles was investigated by EPR spectroscopy at low temperature and the following results were obtained. (1) After excitation by continuous light at 200 K, chloride-depleted particles did not show the EPR multiline signal associated with the S2 state, but only showed the broad signal at g = 4.1. The S2 multiline signal was completely restored upon chloride repletion. (2) In the absence of chloride the S2 multiline signal was not induced by a single flash excitation at 0°C. However, upon addition of chloride after the flash the signal was developed in darkness. (3) The amplitude of the multiline S2 signal thus developed upon chloride addition after flash illumination did not show oscillations dependent upon flash number. These results indicate that the O2-evolving complex in chloride-depleted PS II membranes is able to store at least one oxidizing equivalent, a modified S2 state, which does not give rise to the multiline signal. Addition of chloride converts this oxidizing equivalent to the normal S2 state which gives rise to the multiline signal. The modified S2 state is more stable than the normal S2 state, showing decay kinetics about 20-times slower than those of the normal S2 state, and the formation of higher S states is blocked.
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).
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.
In this report we show that, under flashing light, NaCl-washed, Photosystem-II particles which do not evolve oxygen, are unable to go beyond the S3Z+ state. Addition of Ca2+ alone restores the S3Z+ → S0 transition and oxygen evolution. These conclusions were reached by analysis of the oxygen and luminescence oscillations.
Flash-induced enhancements of the proton NMR relaxation rate R1 of the solvent in suspensions of PS II particles have been recorded for sequences of 1-5 saturating flashes. The one-flash relaxation transient is a positive R1 enhancement of 0.0080 s-1, which relaxes to the preflash baseline with a half-time of 25 s. The two-flash response is a positive relaxation transient of nearly identical amplitude to the one-flash response, but with a slower decay (t1/2 [approximate] 40 s). The appearance of a strongly relaxing paramagnetic center after one flash is consistent with the expected properties of an Mn(III) --> Mn(IV) oxidation. In contrast, the absence of a further R1 enhancement after the second flash shows that a strongly relaxing paramagnetic center is not formed by S2 --> S3 as is expected for manganese redox chemistry involving oxidation of Mn(III); the NMR experiment gives no indication that manganese redox chemistry occurs on the S2 --> S3 transition. The three-flash R1 response is a positive R1 transient of 0.0066 s-1, which decays with a half-time of approx. 50 s to a stable value of 0.002 s-1 above the preflash baseline. Positive R1 enhancements have been observed in the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP), an agent known to accelerate the decay of S2 and S3, at concentrations which very effectively suppress the one-flash and two-flash decays. These enhancements indicate that the formation of the S0 state from S1 involves the production of a strongly relaxing center, and the sign of the R1 response is consistent with the expected properties of an Mn(III) --> Mn(II) reduction. The kinetic behavior of the three-flash R1 transient is indicative of slow redox equilibria involving the strongly relaxing center of the S0 state. Flash transients across a five-flash cycle exhibit oscillatory behavior with a local minimum on the fourth flash. Simulations of the flash profile indicate the presence of an R1 contribution from a minor fraction of centers which are capable of transition to S2 and S3, but are inhibited at S3 --> S0. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/26015/1/0000082.pdf
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)
A polynuclear manganese complex functions in Photosystem II both to accumulate oxidizing equivalents and to bind water and catalyze its four-electron oxidation. Recent electron paramagnetic resonance (EPR) spectroscopic studies of the manganese complex show that four manganese ions are required to account for its magnetic properties. The exchange couplings between manganese ions in the S2 state are characteristic of a Mn4O4 "cubane"-like structure. Based on this structure for the manganese complex in the S2 state, as well as a consideration of the known properties of the manganese complex in Photosystem II and the coordination chemistry of manganese, structures are proposed for the five intermediate oxidation states of the manganese complex. A molecular mechanism for the formation of an O-O bond and the displacement of O2 from the S4 state is suggested.
The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q− to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center: 1.(1) In dark-adapted chloroplasts (i.e. in States S0+S1 according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.2.(2) When chloroplasts are placed in the S2+S3 states by a two-flash preillumination at room temperature, the reoxidation of Q− after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.3.(3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O2 molecule.4.(4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.