Redox chemistry of the calcium-manganese cluster of Photosystem II as probed by chemical reducing agents

The reactions between the chemical reducing agents hydroquinone (H 2Q), N,N,N'N'-tetramethyl-p-phenylenediamine (TMPD), hydroxylamine (NH2OH), N-methyl hydroxylamine (NMHA), N,N-dimethyl hydroxylamine (DMHA) and the Ca-Mn4-Cl cluster of Photosystem II (PSII) were investigated in order to probe the redox properties of this active site metal center. The reactions between all of the reductants with PSII preparations depleted of the 17 and 23 kDa extrinsic polypeptides were studied; the reaction between intact PSII and NH2OH was also investigated. Of these reductants, NH2OH alone was found to display substrate-like characteristics in its reactions with PSII; it reacted more quickly with the Mn4 cluster in the presence of Cl-, an essential activator of PSII. The stoichiometries of the reactions between H2Q, NH2OH, NMHA, DMHA and the PSII Mn were obtained by measuring the loss of activity and Mn2+ from PSII centers treated with these reductants. Modeling of the data obtained suggested that PSII centers can be reduced to an S -5 state or below, providing the first example of chemical evidence that the oxidation states of the Mn4 cluster in its dark stable state are 2 Mn3+/2 Mn4+. The models used to fit the data obtained also indicate that an S-1 state formed by chemical reduction is stabilized and active in intact, but not in polypeptide depleted PSII centers. Further, it was found that the reactions between higher potential reducing agents and the Mn4 cluster are possible in the absence, but not in the presence of Ca2+. Because these reactions appear to be screened by Ca2+ on the basis of the reduction potential but not the size of the reducing agent employed, it is likely that Ca2+ is located close to a subset of higher potential Mn within the metal cluster. An analysis of the time dependent formation of the radical formed (TMPD•+) in the reactions between TMPD and polypeptide depleted PSII under various conditions allowed for a proposed chemical mechanism for the reaction between TMPD and PSII. The proposed mechanism provided further chemical evidence, in addition to the existing biophysical evidence, that the Mn oxidation states in S1 are 2 Mn3+/2 Mn 4+. The proposed mechanism also supports the view that Ca2+ is located within the Mn4 cluster in close proximity to a distinct group of high potential Mn.