Redox control and hydrogen bonding networks: proton-coupled electron transfer reactions and tyrosine Z in the photosynthetic oxygen-evolving complex.

In photosynthetic oxygen evolution, redox active tyrosine Z (YZ) plays an essential role in proton-coupled electron transfer (PCET) reactions. Four sequential photooxidation reactions are necessary to produce oxygen at a Mn(4)CaO(5) cluster. The sequentially oxidized states of this oxygen-evolving cluster (OEC) are called the S(n) states, where n refers to the number of oxidizing equivalents stored. The neutral radical, YZ•, is generated and then acts as an electron transfer intermediate during each S state transition. In the X-ray structure, YZ, Tyr161 of the D1 subunit, is involved in an extensive hydrogen bonding network, which includes calcium-bound water. In electron paramagnetic resonance experiments, we measured the YZ• recombination rate, in the presence of an intact Mn(4)CaO(5) cluster. We compared the S(0) and S(2) states, which differ in Mn oxidation state, and found a significant difference in the YZ• decay rate (t(1/2) = 3.3 ± 0.3 s in S(0); t(1/2) = 2.1 ± 0.3 s in S(2)) and in the solvent isotope effect (SIE) on the reaction (1.3 ± 0.3 in S(0); 2.1 ± 0.3 in S(2)). Although the YZ site is known to be solvent accessible, the recombination rate and SIE were pH independent in both S states. To define the origin of these effects, we measured the YZ• recombination rate in the presence of ammonia, which inhibits oxygen evolution and disrupts the hydrogen bond network. We report that ammonia dramatically slowed the YZ• recombination rate in the S(2) state but had a smaller effect in the S(0) state. In contrast, ammonia had no significant effect on YD•, the stable tyrosyl radical. Therefore, the alterations in YZ• decay, observed with S state advancement, are attributed to alterations in OEC hydrogen bonding and consequent differences in the YZ midpoint potential/pK(a). These changes may be caused by activation of metal-bound water molecules, which hydrogen bond to YZ. These observations document the importance of redox control in proton-coupled electron transfer reactions.

Proton coupled electron transfer and redox-active tyrosine Z in the photosynthetic oxygen-evolving complex.

Proton coupled electron transfer (PCET) reactions play an essential role in many enzymatic processes. In PCET, redox-active tyrosines may be involved as intermediates when the oxidized phenolic side chain deprotonates. Photosystem II (PSII) is an excellent framework for studying PCET reactions, because it contains two redox-active tyrosines, YD and YZ, with different roles in catalysis. One of the redox-active tyrosines, YZ, is essential for oxygen evolution and is rapidly reduced by the manganese-catalytic site. In this report, we investigate the mechanism of YZ PCET in oxygen-evolving PSII. To isolate YZ(•) reactions, but retain the manganese-calcium cluster, low temperatures were used to block the oxidation of the metal cluster, high microwave powers were used to saturate the YD(•) EPR signal, and YZ(•) decay kinetics were measured with EPR spectroscopy. Analysis of the pH and solvent isotope dependence was performed. The rate of YZ(•) decay exhibited a significant solvent isotope effect, and the rate of recombination and the solvent isotope effect were pH independent from pH 5.0 to 7.5. These results are consistent with a rate-limiting, coupled proton electron transfer (CPET) reaction and are contrasted to results obtained for YD(•) decay kinetics at low pH. This effect may be mediated by an extensive hydrogen-bond network around YZ. These experiments imply that PCET reactions distinguish the two PSII redox-active tyrosines.

Proton-coupled electron transfer in photosystem II: proton inventory of a redox active tyrosine.

Photosystem II (PSII) catalyzes the light driven oxidation of water and the reduction of plastoquinone. PSII is a multisubunit membrane protein; the D1 and D2 polypeptides form the heterodimeric core of the PSII complex. Water oxidation occurs at a manganese-containing oxygen evolving complex (OEC). PSII contains two redox active tyrosines, Y(Z) and Y(D), which form the neutral tyrosyl radicals, Y(z)(*) and Y(D)(*). Y(D) has been assigned as tyrosine 160 in the D2 polypeptide through isotopic labeling and site-directed mutagenesis. Whereas Y(D) is not directly involved in the oxidation of water, it has been implicated in the formation and stabilization of the OEC. PSII structures have shown Y(D) to be within hydrogen-bonding distance of histidine 189 in the D2 polypeptide. Spectroscopic studies have suggested that a proton is transferred between Y(D) and histidine 189 when Y(D) is oxidized and reduced. In our previous work, we used (2)H(2)O solvent exchange to demonstrate that the mechanism of Y(D) proton-coupled electron transfer (PCET) differs at high and low pH. In this article, we utilize the proton inventory technique to obtain more information concerning PCET mechanism at high pH. The hypercurvature of the proton inventory data provides evidence for the existence of multiple, proton-donation pathways to Y(D)(*). In addition, at least one of these pathways must involve the transfer of more than one proton.

Proton-coupled electron transfer and tyrosine D of photosystem II.

Photosystem II (PSII) is a photosynthetic reaction center that oxidizes water and reduces bound plastoquinone. PSII electron transfer is mediated by two redox-active tyrosine residues. One of these residues, tyrosine D (YD), has been assigned as Tyr160 of the D2 polypeptide by site-directed mutagenesis and isotopic labeling. Previous spectroscopic evidence has established that His189 in the D2 subunit forms a hydrogen bond with YD* and donates a proton to YD* when the radical is reduced. However, the mechanism of this reaction has not been elucidated. In this report, EPR spectroscopy and 2H2O solvent exchange were used to investigate the pL dependence of the YD* reduction rate. The kinetic isotope effect (KIE), induced by solvent exchange, was also measured as a function of pL. Under the conditions employed, the reduction of YD* is attributed to recombination with the QA- plastoquinone acceptor of PSII. The kinetic data were fit with a biexponential function. The majority, slow phase exhibited a pL-dependent rate constant, with a minimum at pL 7.5. Solvent exchange gave significant KIE at values between pL 5.5 and 8.0. In particular, at high pL (> or =7.5), the values of the KIE were determined to be 2.1 +/- 0.6 and 2.4 +/- 0.5. These values are consistent with a coupled electron and proton reaction, which occurs with a single kinetic step at pL values > or =7.5. The lower KIE values and the rate acceleration observed at low pL may be consistent with a change of mechanism in which the protonation of YD* occurs first, followed by rate-limiting electron transfer. The more modest acceleration in rate at high pL values is attributed to a small, pL-induced change in the distance between YD* and QA-.

Calcium exchange and structural changes during the photosynthetic oxygen evolving cycle.

PSII catalyzes the oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. PSII contains an oxygen-evolving complex, which is located on the lumenal side of the PSII reaction center and which contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen. This process produces five Sn-states, where n refers to the number of oxidizing equivalents stored. Calcium is required for oxygen production. Strontium is the only divalent cation that replaces calcium and maintains activity. In our previous FT-IR work, we assessed the effect of strontium substitution on substrate-limited PSII preparations, which were inhibited at the S3 to S0 transition. In this work, we report reaction-induced FT-IR studies of hydrated PSII preparations, which undergo the full S-state cycle. The observed difference FT-IR spectra reflect long-lived photoinduced conformational changes in the oxygen-evolving complex; strontium exchange identifies vibrational bands sensitive to substitutions at the calcium site. During the S1' to S2' transition, the data are consistent with an electrostatic or structural perturbation of the calcium site. During the S3' to S0' and S0' to S1' transitions, the data are consistent with a perturbation of a hydrogen bonding network, which contains calcium, water, and peptide carbonyl groups. To explain our data, persistent shifts in divalent cation coordination must occur when strontium is substituted for calcium. A modified S-state model is proposed to explain these results and results in the literature.

Calcium ligation in photosystem II under inhibiting conditions.

In oxygenic photosynthesis, PSII carries out the oxidation of water and reduction of plastoquinone. The product of water oxidation is molecular oxygen. The water splitting complex is located on the lumenal side of the PSII reaction center and contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen from water; the five sequentially oxidized forms of the water splitting complex are known as the Sn states, where n refers to the number of oxidizing equivalents stored. Calcium plays a role in water oxidation; removal of calcium is associated with an inhibition of the S state cycle. Although calcium can be replaced by other cations in vitro, only strontium maintains activity, and the steady-state rate of oxygen evolution is decreased in strontium-reconstituted PSII. In this article, we study the role of calcium in PSII that is limited in water content. We report that strontium substitution or 18OH2 exchange causes conformational changes in the calcium ligation shell. The conformational change is detected because of a perturbation to calcium ligation during the S1 to S2 and S2 to S3 transition under water-limited conditions.