We report the first parallel polarization EPR signal from the Mn(III) ion formed by photooxidation of Mn(II) bound at the high affinity Mn-binding site of photosystem II (PSII). This species corresponds to the first photoactivation intermediate formed on the pathway to assembly of the water-splitting Mn cluster. The parallel mode EPR spectrum of the photooxidation product of 1.2/1 stoichiometry Mn(II)/Mn-depleted wildtype Synechocystis sp. PCC 6803 PSII particles consists of six well-resolved transitions split by a relatively small 55 Mn hyperfine coupling (44 G). This spectral signature is absent in photooxidized Mn apoPSII complexes prepared from D1-Asp170Glu and D1-Asp170His mutants, providing direct spectral evidence for a role for this specific D1-Asp170 residue in the initial photoactivation chemistry. Temperature-dependence measurements and spectral simulations performed on the Mn(III) parallel mode EPR signal of the wild-type sample give an axial zero-field splitting value of D ≈ -2.5 cm -1 and a rhombic zero-field splitting value of |E| ≈ 0.269 cm -1 . The negative D value for this d 4 ion is indicative of either a 5 B 1g symmetry ground state of an octahedral Mn(III) geometry or a 5 B 1 symmetry ground state of a five-coordinate square-pyramidal Mn(III) geometry. The parallel mode Mn(III) EPR spectrum obtained from the wild-type photooxidized Mn apoPSII complex is contrasted with that obtained from the five-coordinate Mn(III) form of native Mn superoxide dismutase, which has a trigonal-bipyramidal geometry and a 5 A 1 symmetry ground state giving rise to a positive D value and a much larger 55 Mn hyperfine coupling of 100 G. The D1-Asp170His mutant displays a parallel mode EPR spectrum similar to that observed in a Mn(III) model complex. The D1-Asp170Glu mutant shows no parallel mode spectrum, but in perpendicular mode it shows a broad feature near g ) 5 which has spectral characteristics of an S ) 3 / 2 Mn(IV) ion. This suggests that this mutant provides a binding site with a less positive Mn(III)/ Mn(IV) reduction potential.Photosystem II of oxygenic photosynthesis utilizes a tetranuclear Mn cluster and a redox active tyrosine residue (Y Z ) to couple the reduction of the photooxidized Chl species P 680 + to the enzymatic oxidation of water to dioxygen. 1 The same photochemistry is utilized to assemble the Mn cluster through a process termed "photoactivation", 2-6 where bioavailable Mn(II) ions are oxidized to the higher Mn(III)/Mn(IV) valencies of the catalytic cluster. The first photooxidation event occurs at a unique high-affinity Mn(II) binding site, where Mn(II) is oxidized to Mn(III) by the neutral Y Z • radical formed upon the coupled deprotonation and oxidation of Y Z by P 680 + . 3 It is likely, but not yet rigorously proven, that this first photooxidation site remains a Mn ligation site for the intact Mn cluster. Mutagenesis studies have implicated the D1 protein residue aspartate-170 in high affinity binding of this photooxidizable Mn(II). 4 There have been relatively few EPR investigatio...

The redox-active tyrosines, Y(Z) and Y(D), of Photosystem II are oxidized by P680+ to the neutral tyrosyl radical. This oxidation thus involves the transfer of the phenolic proton as well as an electron. It has recently been proposed that tyrosine Y(Z) might replace the lost proton by abstraction of a hydrogen atom or a proton from a water molecule bound to the manganese cluster, thereby increasing the driving force for water oxidation. To compare and contrast with the intact system, we examine here, in a simplified Mn-depleted PSII core complex, isolated from a site-directed mutant of Synechocystis PCC 6803 lacking Y(D), the role of proton transfer in the oxidation and reduction of Y(Z). We show how the oxidation and reduction rates for Y(Z), the deuterium isotope effect on these rates, and the Y(Z)* - Y(Z) difference spectra all depend on pH (from 5.5 to 9.5). This simplified system allows examination of electron-transfer processes over a broader range of pH than is possible with the intact system and with more tractable rates. The kinetic isotope effect for the oxidation of P680+ by Y(Z) is maximal at pH 7.0 (3.64). It decreases to lower pH as charge recombination, which shows no deuterium isotope, starts to become competitive with Y(Z) oxidation. To higher pH, Y(Z) becomes increasingly deprotonated to form the tyrosinate, the oxidation of which at pH 9.5 becomes extremely rapid (1260 ms(-1)) and no longer limited by proton transfer. These observations point to a mechanism for the oxidation of Y(Z) in which the tyrosinate is the species from which the electron occurs even at lower pH. The kinetics of oxidation of Y(Z) show elements of rate limitation by both proton and electron transfer, with the former dominating at low pH and the latter at high pH. The proton-transfer limitation of Y(Z) oxidation at low pH is best explained by a gated mechanism in which Y(Z) and the acceptor of the phenolic proton need to form an electron/proton-transfer competent complex in competition with other hydrogen-bonding interactions that each have with neighboring residues. In contrast, the reduction of Y(Z)* appears not to be limited by proton transfer between pH 5.5 and 9.5. We also compare, in Mn-depleted Synechocystis PSII core complexes, Y(Z) and Y(D) with respect to solvent accessibility by detection of the deuterium isotope effect for Y(Z) oxidation and by 2H ESEEM measurement of hydrogen-bond exchange. Upon incubation of H2O-prepared PSII core complexes in D2O, the phenolic proton of Y(Z) is exchanged for a deuterium in less than 2 min as opposed to a t(1/2) of about 9 h for Y(D). In addition, we show that Y(D)* is coordinated by two hydrogen bonds. Y(Z)* shows more disordered hydrogen bonding, reflecting inhomogeneity at the site. With 2H ESEEM modulation comparable to that of Y(D)*, Y(Z)* would appear to be coordinated by two hydrogen bonds in a significant fraction of the centers.