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...

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