Dual-mode EPR detects the initial intermediate in photoassembly of the photosystem II mn cluster: The influence of amino acid residue 170 of the D1 polypeptide on Mn coordination

We report the first parallel polarization EPR signal from the Mn(III) ion f ormed by photooxidation of Mn(II) bound at the high affinity Mn-binding sit e of photosystem II (PSII). This species corresponds to the first photoacti vation intermediate formed on the pathway to assembly of the water-splittin g Mn cluster. The parallel mode EPR spectrum of the photooxidation product of 1.2/1 stoichiometry Mn(II)/Mn-depleted wildtype Synechocystis sp. PCC 68 03 PSII particles consists of six well-resolved transitions split by a rela tively small Mn-55 hyperfine coupling (44 G). This spectral signature is ab sent in photooxidized Mn apoPSII complexes prepared from D1-Asp170Glu and D 1-Asp170His mutants, providing direct spectral evidence for a role for this specific D1-Asp170 residue in the initial photoactivation chemistry. Tempe rature-dependence measurements and spectral simulations performed on the Mn (III) parallel mode EPR signal of the wild-type sample give an axial zero-f ield splitting value of D approximate to -2.5 cm(-1) and a rhombic zero-fie ld splitting value of \E\ approximate to 0.269 cm(-1). The negative D value for this d(4) ion is indicative of either a B-5(1g) symmetry ground state of an octahedral cm Mn(III) geometry or a B-5(1) symmetry ground state of a five-coordinate square-pyramidal Mn(III) geometry. The parallel mode Mn(II I) EPR spectrum obtained from the wild-type photooxidized Mn apoPSII comple x 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 55Mn hyperfine coupling of 100 G. The D1-Asp170His mutant disp lays a parallel mode EPR spectrum similar to that observed in a Mn(III) mod el 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 pr ovides a binding site with a less positive Mn(III)/MnOV) reduction potentia l.