Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.     

Amino acid residues that modulate the properties of tyrosine YZ and the manganese cluster in the water oxidizing complex of photosystem II

The catalytic site for photosynthetic water oxidation is embedded in a protein matrix consisting of nearly 30 different polypeptides. Residues from several of these polypeptides modulate the properties of the tetrameric Mn cluster and the redox-active tyrosine residue, Y_Z, that are located at the catalytic site. However, most or all of the residues that interact directly with Y_Z and the Mn cluster appear to be contributed by the D1 polypeptide. This review summarizes our knowledge of the environments of Y_Z and the Mn cluster as obtained from the introduction of site-directed, deletion, and other mutations into the photosystem II polypeptides of the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii.     

Blocking of electron donation by Mn(II) to Y(Z*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light

The donation of electrons by Mn(II) and Fe(II) to Y(Z*) through the high-affinity (HA(Z)) site in Mn-depleted photosystem II (PSII) membranes has been studied by flash-probe fluorescence yield measurements. Mn(II) and Fe(II) donate electrons to Y(Z*) with about the same efficiency, saturating this reaction at the same concentration (ca. 5 microM). However, following a short incubation of the membranes with 5 microM Fe(II), but not with Mn(II) in room light, added Mn(II) or Fe(II) can no longer be photooxidized by Y(Z)(*). This blocking effect is caused by specifically bound, photooxidized Fe [> or =Fe(III)] and is accompanied by a delay in the fluorescence yield decay kinetics attributed to the slowing down of the charge recombination rate between Q(a-) and Y(Z*). Exogenously added Fe(III), on the other hand, does not donate electrons to Y(Z*), does not block the donation of electrons by added Mn(II) and Fe(II), and does not change the kinetics of the decay of the fluorescence yield. These results demonstrate that the light-dependent oxidation of Fe(II) by Y(Z*) creates an Fe species that binds at the HA(Z) site and causes the blocking effect. The pH dependence of Mn(II) electron donation to Y(Z*) via the HA(Z) site and of the Fe-blocking effect is different. These results, together with sequence homologies between the C-terminal ends of the D1 and D2 polypeptides of the PSII reaction center and several diiron-oxo enzymes, suggest the involvement of two or perhaps more (to an upper limit of four to five) bound iron cations per reaction center of PSII in the blocking effect. Similarities in the interaction of Fe(II) and Mn(II) with the HA(Z) Mn site of PSII during the initial steps of the photoactivation process are discussed. The Fe-blocking effect was also used to investigate the relationship between the HA(Z) Mn site and the HA sites on PSII for diphenylcarbazide (DPC) and NH2OH oxidation. Blocking of the HA(Z) site with specifically bound Fe leads to the total inhibition of electron donation to Y(Z*) by DPC. Since DPC and Mn(II) donation to PSII is noncompetitive [Preston, C., and Seibert, M. (1991) Biochemistry 30, 9615-9624], the Fe bound to the HA(Z) site can also block the DPC donation site. On the other hand, electron donation by NH2OH to PSII still occurs in Fe-blocked membranes. Since hydroxylamine does not reduce the Fe [> or =Fe(III)] specifically bound to the HA(Z) site, NH2OH must donate to Y(Z*) through its own site or directly to P680+.     

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.     

Accessibility of tyrosine Y(.)(Z) to exogenous reductants and Mn(2+) in various Photosystem II preparations

The reduction of tyrosine Y(.)(Z) by benzidine and exogenous Mn(2+) was studied by kinetic EPR experiments in various Photosystem II (PSII) preparations. Using lanthanide treated PSII membranes it was demonstrated that neither the extrinsic polypeptides (17, 23 and 33 kDa) nor the Mn complex block the accessibility of Y(.)(Z) to exogenous reductants, such as benzidine. In addition, it was shown that in the presence of the native Mn complex exogenous Mn(2+) does not reduce Y(.)(Z).     

Orientation of the tetranuclear manganese cluster and tyrosine Z in the O(2)-evolving complex of photosystem II: An EPR study of the S(2)Y(Z)(*) state in oriented acetate-inhibited photosystem II membranes.

Inhibitory treatment by acetate, followed by illumination and rapid freezing, is known to trap the S(2)Y(Z)(*) state of the O(2)-evolving complex (OEC) in photosystem II (PS II). An EPR spectrum of this state exhibits broad split signals due to the interaction of the tyrosyl radical, Y(Z)(*), with the S = 1/2 S(2) state of the Mn(4) cluster. We present a novel approach to analyze S(2)Y(Z)(*) spectra of one-dimensionally (1-D) oriented acetate-inhibited PS II membranes to determine the magnitude and relative orientation of the S(2)Y(Z)(*) dipolar vector within the membrane. Although there exists a vast body of EPR data on isolated spins in oriented membrane sheets, the present study is the first of its kind on dipolar-coupled electron spin pairs in such systems. We demonstrate the feasibility of the technique and establish a rigorous treatment to account for the disorder present in partially oriented 1-D membrane preparations. We find that (i) the point-dipole distance between Y(Z)(*) and the Mn(4) cluster is 7.9 +/- 0.2 A, (ii) the angle between the interspin vector and the thylakoid membrane normal is 75 degrees, (iii) the g(z)()-axis of the Mn(4) cluster is 70 degrees away from the membrane normal and 35 degrees away from the interspin vector, and (iv) the exchange interaction between the two spins is -275 x 10(-)(4) cm(-)(1), which is antiferromagnetic. Due to the sensitivity of EPR line shapes of oriented spin-coupled pairs to the interspin distance, the present study imposes a tighter constraint on the Y(Z)-Mn(4) point-dipole distance than obtained from randomly oriented samples. The geometric constraints obtained from the 1-D oriented sample are combined with published models of the structure of Mn-depleted PS II to propose a location of the Mn(4) cluster. A structure in which Y(Z) is hydrogen bonded to a manganese-bound hydroxide ligand is consistent with available data and favors maximal orbital overlap between the two redox center that would facilitate direct electron- and proton-transfer steps.     

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).     

Amino acid residues involved in the coordination and assembly of the manganese cluster of photosystem II. Proton-coupled electron transport of the redox-active tyrosines and its relationship to water oxidation

The combination of site-directed mutagenesis, isotopic labeling, new magnetic resonance techniques and optical spectroscopic methods have provided new insights into cofactor coordination and into the mechanism of electron transport and proton-coupled electron transport in photosystem II. Site-directed mutations in the D1 polypeptide of this photosystem have implicated a number of histidine and carboxylate residues in the coordination and assembly of the manganese cluster, responsible for photosynthetic water oxidation. Many of these are located in the carboxy-terminal region of this polypeptide close to the processing site involved in its maturation. This maturation is a required precondition for cluster assembly. Recent proposals for the mechanism of water oxidation have directly implicated redox-active tyrosine Y(Z) in this mechanism and have emphasized the importance of the coupling of proton and electron transfer in the reduction of Y(Z)(radical) by the Mn cluster. The interaction of both homologous redox-active tyrosines Y(Z) and Y(D) with their respective homologous proton acceptors is discussed in an effort to better understand the significance of such coupling.     

Electron,proton and hydrogen-atom transfers in photosynthetic water oxidation

When photosynthetic organisms developed so that they could use water as an electron source to reduce carbon dioxide, the stage was set for efficient proliferation. Algae and plants spread globally and provided the foundation for our atmosphere and for O(2)-based chemistry in biological systems. Light-driven water oxidation is catalysed by photosystem II, the active site of which contains a redox-active tyrosine denoted Y(Z), a tetramanganese cluster, calcium and chloride. In 1995, Gerald Babcock and co-workers presented the hypothesis that photosynthetic water oxidation occurs as a metallo-radical catalysed process. In this model, the oxidized tyrosine radical is generated by coupled proton/electron transfer and re-reduced by abstracting hydrogen atoms from substrate water or hydroxide-ligated to the manganese cluster. The proposed function of Y(Z) requires proton transfer from the tyrosine site upon oxidation. The oxidation mechanism of Y(Z) in an inhibited and O(2)-evolving photosystem II is discussed. Domino-deprotonation from Y(Z) to the bulk solution is shown to be consistent with a variety of data obtained on metal-depleted samples. Experimental data that suggest that the oxidation of Y(Z) in O(2)-evolving samples is coupled to proton transfer in a hydrogen-bonding network are described. Finally, a dielectric-dependent model for the proton release that is associated with the catalytic cycle of photosystem II is discussed.     

Trapping of metalloradical intermediates of the S-states at liquid helium temperatures. Overview of the phenomenology and mechanistic implications

The oxygen-evolving complex (OEC) of photosystem II (PSII) consists of a Mn cluster (believed to be tetranuclear) and a tyrosine (Tyr Z or Y(Z)). During the sequential absorption of four photons by PSII, the OEC undergoes four oxidative transitions, S(0) to S(1), ..., S(3) to (S(4))S(0). Oxygen evolves during the S(3) to S(0) transition (S(4) being a transient state). Trapping of intermediates of the S-state transitions, particularly those involving the tyrosyl radical, has been a goal of ultimate importance, as that can test critically models employing a role of Tyr Z in proton (in addition to electron) transfer, and also provide important clues about the mechanism of water oxidation. Until very recently, however, critical experimental information was lacking. We review and evaluate recent observations on the trapping of metalloradical intermediates of the S-state transitions, at liquid helium temperatures. These transients are assigned to Tyr Z(*) magnetically interacting with the Mn cluster. Besides the importance of trapping intermediates of this unique catalytic mechanism, liquid helium temperatures offer the additional advantage that proton motions (unlike electron transfer) are blocked except perhaps across strong hydrogen bonds. This paper summarizes the recent observations and discusses the constraints that the phenomenology imposes.     

The basic properties of the electronic structure of the oxygen-evolving complex of photosystem II are not perturbed by Ca2+ removal

Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair.     

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.     

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

The active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)CaO(5) cluster and Tyr(Z), a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7? away from the Mn(4)CaO(5) cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I(-) for Cl(-) in the cyanobacterium Thermosynechococcus elongatus with either Ca(2+) or Sr(2+) biosynthetically assembled into the Mn(4) cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV-visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl(-)/I(-) exchange considerably slowed down the two S(3)Tyr(Z)(?)→(S(3)Tyr(Z)(?))'→S(0) reactions in which the fast phase, S(3)Tyr(Z)(?)→(S(3)Tyr(Z)(?))', reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on Tyr(Z)(?) and the slow phase corresponds to the S(0) and O(2) formations and to a second proton release. The t(1/2) for S(0) formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the Tyr(Z)(?) reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca(2+) binding site and the Cl(-) binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl(-) and Ca(2+) binding sites by which proton release may be channelled.     

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)(?).     

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.     

Water oxidation chemistry of photosystem II

The O(2)-evolving complex of photosystem II catalyses the light-driven four-electron oxidation of water to dioxygen in photosynthesis. In this article, the steps leading to photosynthetic O(2) evolution are discussed. Emphasis is given to the proton-coupled electron-transfer steps involved in oxidation of the manganese cluster by oxidized tyrosine Z (Y(*)(Z)), the function of Ca(2+) and the mechanism by which water is activated for formation of an O-O bond. Based on a consideration of the biophysical studies of photosystem II and inorganic manganese model chemistry, a mechanism for photosynthetic O(2) evolution is presented in which the O-O bond-forming step occurs via nucleophilic attack on an electron-deficient Mn(V)=O species by a calcium-bound water molecule. The proposed mechanism includes specific roles for the tetranuclear manganese cluster, calcium, chloride, Y(Z) and His190 of the D1 polypeptide. Recent studies of the ion selectivity of the calcium site in the O(2)-evolving complex and of a functional inorganic manganese model system that test key aspects of this mechanism are also discussed.     

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.     

The collapse of the tyrosine Z*-Mn spin-spin interaction above approximately 100 K reveals the spectrum of tyrosine Z*. An application of rapid-scan EPR to the study of intermediates of the water splitting mechanism of photosystem II

Tyr Z of photosystem II mediates electron transfer from the water splitting site, a Mn4Ca cluster, to the specialized chlorophyll assembly P680. Due to its proton-limited redox properties and the proximity to the Mn cluster, it is thought to play a critical role in the proton-coupled electron transfer reactions that constitute the four-step oxidation mechanism (so-called S-state transitions) of water to molecular oxygen. Spectroscopic evidence for the Tyr Z radical has been scarce in intact preparations (it is difficult to probe it optically, and too short-lived for EPR characterization) until recently. Advances in recent years have allowed the trapping at liquid helium temperatures and EPR characterization of metalloradical intermediates, attributed to tyrosyl Z* magnetically interacting with the Mn cluster. We have extended these studies and examined the evolution of the spectra of five intermediates: S0YZ*, S0YZ* (with 5% MeOH), S1YZ*, S2YZ*, and S2YZ* (with 5% MeOH) in the temperature range of 11-230 K. A rapid-scan EPR method has been applied at elevated temperatures. The tyrosyl radical decouples progressively from Mn, as the Mn relaxation rate increases with an increase in temperature. Above approximately 100 K, the spectra collapse to the unperturbed spectrum of Tyr Z*, which is found to be somewhat broader than that of the stable Tyr D* radical. This study provides a simple means for recording the spectrum of Tyr Z* and extends earlier observations that link the photochemistry at liquid helium temperatures to the photochemistry at temperatures that support S-state transitions.     

pH dependence of the donor side reactions in Ca2+-depleted photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.     

Characteristic changes of the S2/S1 difference FTIR spectrum induced by Ca2+ depletion and metal cation substitution in the photosynthetic oxygen-evolving complex

Effects of Ca2+ depletion and substitution with other metal cations on the structure of the protein matrices of the oxygen-evolving complex (OEC) and their corresponding changes upon the S1 to S2 transition were examined using Fourier transform infrared (FTIR) spectroscopy. Ca2+ depletion and further supplementation with Li+, Na+, Mg2+, Ca2+, or Sr2+ did not significantly affect the typical vibrational features in the double difference S2/S1 spectrum, including the symmetric [1365(+)/1404(-) cm(-1)] and the asymmetric [1587(+)/1566(-) cm(-1)] stretching modes of the carboxylate ligand and the amide I and II modes of the backbone polypeptides. On the other hand, supplementation with K+, Rb+, Cs+, or Ba2+ significantly modified the S2/S1 spectrum, in which the carboxylate modes disappeared and the amide I and II modes were modified. Results indicate that the binding of metal cations that have ionic radii larger than that of Ca2+ to the Ca2+ site induces perturbations in the protein matrices in the vicinity of the Mn cluster to interrupt the characteristic structural and/or conformational changes upon the oxidation of the Mn cluster accompanied with the S1 to S2 transition. The spectrum was also altered by the supplementation of Cd2+, which has an ionic radius comparable to that of Ca2+. A single-pulse-induced S2/S1 difference spectrum revealed that bands that have been assigned to the vibrational modes for the Y(Z) tyrosine and the histidine ligand for the Mn cluster were not induced in the K+-supplemented membranes, although the histidine band is likely to be preserved in the Ca2+-depleted membranes. The Y(Z) band was considerably small in the double difference S2/S1 spectrum in the Ca2+-depleted and the cation-substituted membranes but distinctively present in the Sr2+- or Ca2+-replenished membranes. Furthermore, cation supplementation induced several new bands that disappeared following the Ca2+ replenishment. These results suggest that the proper organization of the hydrogen bond network within OEC for the water oxidation chemistry requires the Ca2+ ion and indicate that the role of Ca2+ is not purely structurally defined by the physical properties of the ion, such as valence and ionic radius. On the basis of these and other findings, we propose that Ca2+ is necessary for the formation of the hydrogen bond network that is involved in the reaction step of water oxidation.