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 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 wild-type Synechocystis sp. PCC 6803 PSII particles consists of six well-resolved transitions split by a relatively small ⁵⁵Mn 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 d⁴ ion is indicative of either a ⁵B(1g) symmetry ground state of an octahedral Mn(III) geometry or a ⁵B₁ 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 ⁵A₁ symmetry ground state giving rise to a positive D value and a much larger ⁵⁵Mn 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.