The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P(680+)Q(A-) limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P(680+) by the secondary electron donor Tyrosine Z cannot occur because Y(Z) is already oxidized. The 1.4 ms recombination is seen in Y(Z)-less mutants of the cyanobacterium Synechocystis. However, the rate of P(680+)Q(A-) recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P(680+) reduction by Y(Z) were obtained, and the rate of the competing P(680+)Q(A-) recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y(Z)(ox) slightly decreased the efficiency of excitation trapping but did not seem to accelerate P(680+)Q(A-) recombination. The two P(680+)Q(A-) lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

Characterization of carotenoid and chlorophyll photooxidation in photosystem II

Photosystem II (PSII) contains two accessory chlorophylls (Chl(Z), ligated to D1-His118, and Chl(D), ligated to D2-His117), carotenoid (Car), and heme (cytochrome b(559)) cofactors that function as alternate electron donors under conditions in which the primary electron-donation pathway from the O(2)-evolving complex to P680(+) is inhibited. The photooxidation of the redox-active accessory chlorophylls and Car has been characterized by near-infrared (near-IR) absorbance, shifted-excitation Raman difference spectroscopy (SERDS), and electron paramagnetic resonance (EPR) spectroscopy over a range of cryogenic temperatures from 6 to 120 K in both Synechocystis PSII core complexes and spinach PSII membranes. The following key observations were made: (1) only one Chl(+) near-IR band is observed at 814 nm in Synechocystis PSII core complexes, which is assigned to Chl(Z)(+) based on previous spectroscopic studies of the D1-H118Q and D2-H117Q mutants [Stewart, D. H., Cua, A., Chisholm, D. A., Diner, B. A., Bocian, D. F., and Brudvig, G. W. (1998) Biochemistry 37, 10040-10046]; (2) two Chl(+) near-IR bands are observed at 817 and 850 nm in spinach PSII membranes which are formed with variable relative yields depending on the illumination temperature and are assigned to Chl(Z)(+), and Chl(D)(+), respectively; (3) the Chl and Car cation radicals have significantly different stabilities at reduced temperatures with Car(+) decaying much faster; (4) in Synechocystis PSII core complexes, Car(+) decays by recombination with Q(A)(-) and not by Chl(Z)/Chl(D) oxidation, with multiphasic kinetics that are attributed to an ensemble of protein conformers that are trapped as the protein is frozen; and (5) in spinach PSII membranes, Car(+) decays mainly by recombination with Q(A)(-), but also partly by formation of the 850 nm Chl cation radical. The greater stability of Chl(Z)(+) at low temperatures enabled us to confirm that resonance Raman bands previously assigned to Chl(Z)(+) are correctly assigned. In addition, the formation and decay of these cations provide insight into the alternate electron-donation pathways to P680(+).     

Proton and hydrogen currents in photosynthetic water oxidation

The photosynthetic processes that lead to water oxidation involve an evolution in time from photon dynamics to photochemically-driven electron transfer to coupled electron/proton chemistry. The redox-active tyrosine, Y(Z), is the component at which the proton currents necessary for water oxidation are switched on. The thermodynamic and kinetic implications of this function for Y(Z) are discussed. These considerations also provide insight into the related roles of Y(Z) in preserving the high photochemical quantum efficiency in Photosystem II (PSII) and of conserving the highly oxidizing conditions generated by the photochemistry in the PSII reaction center. The oxidation of Y(Z) by P(680)(+) can be described well by a treatment that invokes proton coupling within the context of non-adiabatic electron transfer. The reduction of Y(.)(Z), however, appears to proceed by an adiabatic process that may have hydrogen-atom transfer character.     

Photosystem II of peas: effects of added divalent cations of Mn, Fe, Mg, and Ca on two kinetic components of P(+)(680) reduction in Mn-depleted core particles

The catalytic Mn cluster of the photosynthetic oxygen-evolving system is oxidized via a tyrosine, Y(Z), by a photooxidized chlorophyll a moiety, P(+)(680). The rapid reduction of P(+)(680) by Y(Z) in nanoseconds requires the intactness of an acid/base cluster around Y(Z) with an apparent functional pK of <5. The removal of Mn (together with bound Ca) shifts the pK of the acid/base cluster from the acid into the neutral pH range. At alkaline pH the electron transfer (ET) from Y(Z) to P(+)(680) is still rapid (<1 micros), whereas at acid pH the ET is much slower (10-100 micros) and steered by proton release. In the intermediate pH domain one observes a mix of these kinetic components (see R. Ahlbrink, M. Haumann, D. Cherepanov, O. B?gershausen, A. Mulkidjanian, W. Junge, Biochemistry 37 (1998)). The overall kinetics of P(680)(+) reduction by Y(Z) in Mn-depleted photosystem II (PS II) has been previously shown to be slowed down by divalent cations (added at >10 microM), namely: Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) (C.W. Hoganson, P.A. Casey, O. Hansson, Biochim. Biophys. Acta 1057 (1991)). Using Mn-depleted PS II core particles from pea as starting material, we re-investigated this phenomenon at nanosecond resolution, aiming at the effect of divalent cations on the particular kinetic components of P(+)(680) reduction. To our surprise we found only the slower, proton steered component retarded by some added cations (namely Co(2+)/Zn(2+)>Fe(2+)>Mn(2+)). Neither the fast component nor the apparent pK of the acid/base cluster around Y(Z) was affected. Apparently, the divalent cations acted (electrostatically) on the proton release channel that connects the oxygen-evolving complex with the bulk water, but not on the ET between Y(Z) and P(+)(680), proper. Contrastingly, Ca(2+) and Mg(2+), when added at >5 mM, accelerated the slow component of P(+)(680) reduction by Y(Z) and shifted the apparent pK of Y(Z) from 7.4 to 6.6 and 6.7, respectively. It was evident that the binding site(s) for added Ca(2+) and Mg(2+) were close to Y(Z) proper. The data obtained are discussed in relation to the nature of the metal-binding sites in photosystem II.     

A functional role for tyrosine-D in assembly of the inorganic core of the water oxidase complex of photosystem II and the kinetics of water oxidation.

The role of D2-Tyr160 (Y(D)), a photooxidizable residue in the D2 reaction center polypeptide of photosystem II (PSII), was investigated in both wild type and a mutant strain (D2-Tyr160Phe) in which phenylalanine replaces Y(D) in the cyanobacterium Synechocystis sp. (strain PCC 6803). Y(D) is the symmetry-related tyrosine that is homologous to the essential photoactive Tyr161(Y(Z)) of the D1 polypeptide of PSII. We compared the flash-induced yield of O(2) in intact, functional PSII centers from both wild-type and mutant PSII core complexes. The yield of O(2) in the intact holo-enzyme was found to be identical in the mutant and wild-type PSII cores using long (saturating) pulses or continuous illumination, but was observed to be appreciably reduced in the mutant using short (nonsaturating) light pulses (<50 ms). We also compared the rates of the first two kinetically resolved steps of photoactivation. Photoactivation is the assembly process for binding of the inorganic cofactors to the apo-water oxidation/PSII complex (apo-WOC-PSII) and their light-induced photooxidation to form the functional Mn(4)Ca(1)Cl(x)() core required for O(2) evolution. We show that the D2-Tyr160Phe mutant cores can assemble a functional WOC from the free inorganic cofactors, but at a much slower rate and with reduced quantum efficiency vs wild-type PSII cores. Both of these observations imply that the presence of Y(D)(*) leads to a more efficient photooxidation of the Mn cluster relative to deactivation (reductive processes). One possible explanation for this behavior is that the phenolic proton on Y(D) is retained within the reaction center following Y(D) oxidation. The positive charge, likely shared by D2-His189 and other residues, raises the reduction potential of P(680)(+)/P(680), thereby increasing the driving force for the oxidation of Mn(4)Y(Z). There is, therefore, a competitive advantage to organisms that retain the Y(D) residue, possibly explaining its retention in all sequences of psbD (encoding the D2 polypeptide) known to date. We also find that the sequence of metal binding steps during assembly of apo-WOC-PSII centers in cyanobacteria cores differs from that in higher plants. This is seen by a reduced calcium affinity at its effector site and reduced competition for binding to the Mn(II) site, resulting in acceleration of the initial lagtime by Ca(2+), in contrast to retardation in spinach. Ca(2+) binding to its effector site promotes the stability of the photointermediates (IM1 and above) by suppressing unproductive decay.     

Electron donation to Photosystem II in reaction center preparations

The room-temperature EPR characteristics of Photosystem II reaction center preparations from spinach, pokeweed and Chlamydomonas reinhardii have been investigated. In all preparations a light-induced increase in EPR Signal II, which arises from the oxidized form of a donor to P-680⁺, is observed. Spin quantitation, with potassium nitrosodisulfonate as a spin standard, demonstrates that the Signal II species, Z^?, is present in approx. 60% of the reaction centers. In response to a flash, the increase in Signal II spin concentration is complete within the 98 μs response time of our instrument. The decay of Z^? is dependent on the composition of the particle suspension medium and is accelerated by addition of either reducing agents or lipophilic anions in a process which is first order in these reagents. Comparison of these results with optical data reported previously (Diner, B.A. and Bowes, J.M. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoyunoglou, G., ed.), Vol. 3, pp. 875–883, Balaban, Philadelphia), supports the identification of Z with the P-680⁺ donor, D₁. From the polypeptide composition of the particles used in this study, we conclude that Z is an integral component of the reaction center and use this conclusion to construct a model for the organization of Photosystem II.     

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.     

Effects of pH on the S(3) state of the oxygen evolving complex in photosystem II probed by EPR split signal induction

The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (～5 K) Y(Z) can be oxidized to its neutral radical, Y(Z)(?). This can be followed by the induction of a split EPR signal from Y(Z)(?) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(?). Here we report on the pH dependence for the induction of S(2)'Y(Z)(?) between pH 4.0 and pH 8.7. At acidic pH the split S(3) EPR signal decreases with the apparent pK(a) (pK(app)) ～ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y(Z)-His(Z) motif. At alkaline pH, the split S(3) EPR signal decreases with the pK(app) ～ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK(app) ～ 8.3) promoted by a change in the redox potential of Y(Z). Our results allow dissection of the proton-coupled electron transfer reactions in the S(3) state and provide further evidence that the radical involved in the split EPR signals is indeed Y(Z)(?).     

Electrogenic reactions and dielectric properties of photosystem II

This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P(680) and primary quinone acceptor Q(A); (ii) P(680)(+) reduction by the redox-active tyrosine Y(Z) of polypeptide D1; (iii) oxidation of Mn cluster by Y(Z)(ox) followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor Q(B). The electrogenicity related to (i) proton-coupled electron transfer between Q(A)(-) and preoxidized non-heme iron (Fe(3+)) in native and (ii) electron transfer between protein-water boundary and Y(Z)(ox) in the presence of redox-dye(s) in Mn-depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.     

Electrostatics and proton transfer in photosynthetic water oxidation

Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), S(3) --> S(4) --> S(0)). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H(+)/e(-) under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y(Z)), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogen-bonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y(Z) acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y(Z) and the primary donor of PSII P(+)(680) from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y(Z), is probably further remote from Y(Z) than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y(Z) to P(+)(680) (in nanoseconds) and from the Mn cluster to Y(ox)(Z).     

Water, water everywhere, and its remarkable chemistry

Photosystem II (PSII), the multisubunit pigment-protein complex localised in the thylakoid membranes of oxygenic photosynthetic organisms, uses light energy to drive a series of remarkable reactions leading to the oxidation of water. The products of this oxidation are dioxygen, which is released to the atmosphere, and reducing equivalents destined to reduce carbon dioxide to organic molecules. The water oxidation occurs at catalytic sites composed of four manganese atoms (Mn(4)-cluster) and powered by the redox potential of an oxidised chlorophyll a molecule (P680(*+)). Gerald T (Jerry) Babcock and colleagues showed that electron/proton transfer processes from substrate water to P680(*+) involved a tyrosine residue (Y(Z)) and proposed an attractive reaction mechanism for the direct involvement of Y(Z) in the chemistry of water oxidation. The 'hydrogen-atom abstract/metalloradical' mechanism he formulated is an expression of his genius and a highlight of his many other outstanding contributions to photosynthesis research. A structural basis for Jerry's model is now being revealed by X-ray crystallography.     

Interaction of a phenolic inhibitor with photosystem II particles

Photosystem II particles have been prepared from spinach and Chlamydomonas reinhardii CW 15 thylakoids. Photosynthetic electron transport in these particles is inhibited by phenolic compounds like dinoseb, but not by atrazine and diuron. The labeling patterns obtained by photoaffinity labels derived from either atrazine (azido-atrazine) or the phenolic herbicide dinoseb (azido-dinoseb) were compared in photosystem II particles and thylakoids. Whereas azido-atrazine in thylakoids of spinach as well as of Chlamydomonas labels a 32-kilodalton peptide, this label does not react in photosystem II particle preparations. Azido-dinoseb, however, labels both the thylakoid membranes and the particles, predominantly polypeptides in the 40-53 kilodalton molecular weight region. Since the latter polypeptides are probably part of the reaction center of photosystem II, it is suggested that phenolic compounds have their inhibition site within the reaction center complex. This indicates that the atrazine-binding 32-kilodalton peptide is either absent or functionally inactive in photosystem II particles, whereas the phenol inhibitor-binding peptides are not.     

Role of D1-His190 in the proton-coupled oxidation of tyrosine YZ in manganese-depleted photosystem II.

To further characterize the role of D1-His190 in the oxidation of tyrosine Y(Z) in photosystem II, the pH dependence of P(680)(*)()(+) reduction was measured in H190A and Mn-depleted wild-type PSII particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803. Measurements were conducted in the presence and absence of imidazole and other small organic bases. In H190A PSII particles, rapid reduction of P(680)(*)()(+) attributed to electron transfer from Y(Z) increased dramatically above pH 9, with an apparent pK(A) of approximately 10.3. In the presence of ethanolamine and imidazole, this dramatic increase occurred at lower pH values, with the efficiency of Y(Z) oxidation correlating with the solution pK(A) value of the added base. We conclude that the pK(A) of Y(Z) is approximately 10.3 in D1-H190A PSII particles. In Mn-depleted wild-type PSII particles, P(680)(*)()(+) reduction was accelerated by all exogenous bases examined (substituted imidazoles, histidine, Tris, and 1,4-diazabicyclo[2.2.2]octane). We conclude that Y(Z) is solvent accessible in Mn-depleted wild-type PSII particles and that its pK(A) is near that of tyrosine in solution. In Mn-depleted wild-type PSII particles, over 80% of the kinetics of P(680)(*)()(+) reduction after a flash could be described by three kinetic components. The individual rate constants of these components varied slightly with pH, but their relative proportions varied dramatically with pH, showing apparent pK(A) values of 7.5 and 6.25 (6.9 and 5.8 in the presence of Ca(2+) and Mg(2+) ions). An additional pK(A) value (pK(A) < 4.5) may also be present. To describe these data, we propose (1) the pK(A) of His190 is 6.9-7.5, depending on buffer ions, (2) the deprotonation of Y(Z) is facilitated by the transient formation of a either a hydrogen bond or a hydrogen-bonded water bridge between Y(Z) and D1-His190, and (3) when protonated, D1-His190 interacts with nearby residues having pK(A) values near 6 and 4. Because Y(Z) and D1-His190 are located near the Mn cluster, these residues may interact with the Mn cluster in the intact system.     

Effects of Ca2+ and EGTA on P680*+ reduction kinetics and O2 evolution of Photosystem II

We report for the first time significant changes in the P680*+ reduction kinetics of Photosystem II (PS II) in which the 17 and 23 kDa extrinsic polypeptides are intact, in the presence of Ca(2+) or ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) which were added to vary the Ca(2+) concentration from 5 microM to 30 mM. The decrease in the extent of normal P680*+ reduction decay with lifetimes of 40-370 ns and a corresponding increase in the extent of kinetics with lifetimes of 20-220 micros was interpreted as being due to electron transfer from Y(Z) to P680*+ being replaced by slow forward conduction and by processes including P680*+/Q(A)(-) recombination. The question of whether changes in P680*+ reduction kinetics were caused by loss of Ca(2+) from PS II or by direct interaction of EGTA with PS II was addressed by lowering the free-Ca(2+) concentration of suspensions of PS II core complexes by serial dilution in the absence of EGTA. Despite a significant decrease in the rate of O(2) evolution after this treatment, only small changes in the P680*+ reduction kinetics were observed. Loss of Ca(2+) did not affect P680*+ reduction associated with electron transfer from Y(Z). Since much larger changes in the P680*+ reduction kinetics of intact PS II occurred at comparable free-Ca(2+) concentrations in the presence of EGTA, we conclude that EGTA influenced the P680*+ reduction kinetics by directly interacting with PS II rather than by lowering the free Ca(2+) concentration of the surrounding media. Notwithstanding these effects, we show that useful information about Ca(2+) binding to PS II can be obtained when direct interaction of EGTA is taken into account.     

Heat sensitivity of chloroplasts and leaves: Leakage of protons from thylakoids and reversible activation of cyclic electron transport

In illuminated intact spinach chloroplasts, warming to and beyond 40 °C increased the proton permeability of thylakoids before linear electron transport through Photosystem II was inhibited. Simultaneously, antimycin A-sensitive cyclic electron transport around Photosystem II was activated with oxygen or CO2, but not with nitrite as electron acceptors. Between 40 to 42 °C, activation of cyclic electron transport balanced the loss of protons so that a sizeable transthylakoid proton gradient was maintained. When the temperature of darkened spinach leaves was slowly increased to 40°C, reduction of the quinone acceptor of Photosystem II, Q_A, increased particularly when respiratory CO2 production and autoxidation of plastoquinones was inhibited by decreasing the oxygen content of the atmosphere from 21 to 1%. Simultaneously, Photosystem II activity was partially lost. The enhanced dark Q_A reduction disappeared after the leaf temperature was decreased to 20 °C. No membrane energization was detected by light-scattering measurements during heating the leaf in the dark. In illuminated spinach leaves, light scattering and nonphotochemical quenching of chlorophyll fluorescence increased during warming to about 40 °C while Photosystem II activity was lost, suggesting extra energization of thylakoid membranes that is unrelated to Photosystem II functioning. After P700 was oxidized by far-red light, its reduction in the dark was biphasic. It was accelerated by factors of up to 10 (fast component) or even 25 (slow component) after short heat exposure of the leaves. Similar acceleration was observed at 20 °C when anaerobiosis or KCN were used to inhibit respiratory oxidation of reductants. Methyl viologen, which accepts electrons from reducing side of Photosystem II, completely abolished heat-induced acceleration of P700+ reduction after far-red light. The data show that increasing the temperature of isolated chloroplasts or intact spinach leaves to about 40 °C not only inhibits linear electron flow through Photosystem II but also activates Photosystem I-driven cyclic electron transport pathways capable of contributing to the transthylakoid proton gradient. Heterogeneity of the kinetics of P700+ reduction after far-red oxidation is discussed in terms of Photosystem I-dependent cyclic electron transport in stroma lamellae and grana margins.     

The temperature dependence of P680(+) reduction in oxygen-evolving photosystem II

The temperature dependence for the reduction of the oxidized primary electron donor P680(+) by the redox active tyrosine Y(Z) has been studied in oxygen-evolving photosystem II preparations from spinach. The observed temperature dependence is found to vary markedly with the S-state of the manganese cluster. In the higher oxidation states, S(2) and S(3), sub-microsecond P680(+) reduction exhibits activation energies of about 260 meV. In contrast, there is only a small temperature dependence for the sub-microsecond reaction in the S(0) and S(1) states (an activation energy of approximately 50 meV). Slower microsecond components of P680(+) reduction show an activation energy of about 250 meV which, within experimental error, is independent of the oxidation state of the Mn cluster. By combining these values with measurements of DeltaG for electron transfer, the reorganization energies for each component of P680(+) reduction have been calculated. High activation and reorganization energies are found for sub-microsecond P680(+) reduction in S(2) and S(3), demonstrating that these electron transfers are coupled to significant reorganization events which do not occur in the presence of the lower S-states. One interpretation of these results is that there is an increase in the net charge on the manganese cluster on the S(1) to S(2) transition which acts as a barrier to electron transfer in the higher S-states. This argues against the electroneutrality requirement for some models of the function of the manganese cluster and hence against a role for Y(Z) as a hydrogen abstractor on all S-state transitions. An alternative or additional possibility is that there are proton (or other ion) motions in the sub-microsecond phases in S(2) and S(3) which contribute to the large reorganization energies observed, these motions being absent in the S(0) and S(1) states. Indeed charge accumulation may directly cause the increased reorganization energy.     

Secondary stabilization reactions and proton-coupled electron transport in photosystem II investigated by electroluminescence and fluorescence spectroscopy

The oxidized primary electron donor in photosystem II, P(680)(+), is reduced in several phases, extending over 4 orders of magnitude in time. Especially the slower phases may reflect the back-pressure exerted by water oxidation and provide information on the reactions involved. The kinetics of secondary electron-transfer reactions in the microseconds time range after charge separation were investigated in oxygen-evolving thylakoids suspended in H2O or D2O. Flash-induced changes of chlorophyll fluorescence yield and electric field-induced recombination luminescence were decomposed into contributions from oxidation states S(0), S(1), S(2), and S(3) of the oxygen-evolving complex and interpreted in terms of stabilization kinetics of the initial charge-separated state S(j)Y(Z)P(680)(+)Q(A)(-)Q(B). In approximately 10% of the centers, only charge recombination took place. Otherwise, no static heterogeneity was involved in the microsecond reduction of P(680)(+) by Y(Z) (stabilization) or Q(A)(-) (recombination). The recombination component in active centers occurs mainly upon charge separation in S(3), and, in the presence of D2O, in S(2) as well and is tentatively attributed to the presence of Y(Z)(ox)S(j-1) in equilibrium with Y(Z)S(j). A 20-30 micros stabilization occurs in all S-states, but to different extents. Possible mechanisms for this component are discussed. D2O was found to decrease: (i) the rate of the reaction Y(Z)(ox)S(1) to Y(Z)S(2), (ii) the equilibrium constant between P680(+)Y(Z)S(2) and P(680)Y(Z)(ox)S(2), (iii) the rate of the slow phase of P(680)(+) reduction for the S(3) --> S(0) transition, and (iv) the rate of electron transfer from Q(A)(-) to Q(B) /Q(B)(-). The increased 'miss probability' in D2O is due to (iii).     

Neurophilin-1 is a downstream target of transcription factor Ets-1 in human umbilical vein endothelial cells

Photosystem II complex (PSII) of thylakoid membranes uses light energy to oxidise extremely stable water and produce oxygen (2H(2)O-->O(2)+4H(+)+4e(-)). PSII is compared with cytochrome c oxidase that catalyses the opposite reaction coupled to proton translocation. Cytochrome c oxidase has proton and water channels, and a tentative oxygen channel. I propose that functional PSII complexes also need a specific oxygen channel to direct O(2) from the water molecules bound to specific Mn atoms of the Mn cluster within PSII out to the membrane surface. The function of this channel will be to prevent oxygen being accessible to the radical pair P680(+)Pheo(-), thereby preventing singlet oxygen generation from the triplet P680 state in functional PSII. The important role of singlet oxygen in structurally perturbed non-functional photosystem II is also discussed.     

Similarity of EPR Signal IIf rise and P-680+ decay kinetics in Tris-washed chloroplast Photosystem II preparations as a function of pH

The rise time, of Signal II_f and the decay time of P-680⁺ have been measured kinetically as a function of pH by using EPR. The Photosystem II-enriched preparations which were used as samples were derived from spinach chloroplasts, and they evolved oxygen before Tris washing. The onset kinetics of Signal II_f are in agreement, within experimental error, with the fast component of the decay of an EPR signal attributable to P-680⁺. The signal II_f rise kinetics also show good agreement with published values of the pH dependence of the decay of P-680⁺ measured optically (Conjeaud, H. and Mathis, P. (1980) Biochim. Biophys. Acta 590, 353–359). These results are consistent with a model where the species Z (or D₁) responsible for Signal II_f is the immediate electron donor to P-680⁺ in tris-washed Photosystem II fragments.     

Site-directed mutations at D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803 modify the spectroscopic properties of the accessory chlorophyll in the D1-branch of the reaction center

D1-Thr179, which overlies the reaction center chlorophyll Chl D1 of Photosystem II was replaced with His and Glu through site-directed mutation in Synechocystis sp. PCC 6803. Spectroscopic characterization of the mutants indicates that, compared to wild type, the main bleaching in the triplet-minus-singlet absorbance difference spectrum and the electrochromic band shift in the (P680 (+)Q A (-)-P680Q A) absorbance difference spectrum are displaced to the red by approximately 2 nm in the D1-Thr179His mutant and to the blue by approximately 1 nm in the D1-Thr179Glu mutant. These difference spectra are compared with the absorbance difference spectra, measured on the same states in the D1-His198Gln mutant in which the axial ligand D1-His198 of the special pair chlorophyll, P D1, was replaced by glutamine. Together, these results give direct evidence that (a) the reaction center triplet state, produced upon charge recombination from (3)[P (+)Pheo (-)], is primarily localized on Chl D1; (b) the cation of the oxidized donor P (+) is predominantly localized on chlorophyll P D1 of the special pair; and (c) the Q Y band of the accessory chlorophyll Chl D1 is electrochromically shifted in response to charges on P (+) and Q A (-). Light-induced absorbance difference spectra (between 650 and 710 nm), associated with the oxidation of secondary donors and the reduction of Q A, exhibit a bleaching attributed to the oxidation of a Chl Z and strong electrochromic band shifts. On the basis of mutation-induced spectroscopic changes and of structure-based calculations, we conclude that the experimental spectra are best explained by a blue-shift of the Q Y band of the accessory chlorophyll Chl D1, arising from charges on Car D2 (+) and Chl ZD2 (+) and on reduced Q A.