Different manganese binding sites in photosystem II probed by selective chemical modification of histidyl and carboxylic acid residues.

The binding of Mn2+ to manganese-depleted photosystem II was investigated after chemical modification of histidyl and carboxylic acid residues in the presence or absence of the native manganese cluster. K(M) values for Mn2+ were determined from steady-state electron transfer between Mn2+ and 2,6-dichlorophenolindophenol, the dissociation constant for Mn2+ was measured by observing the effect of added Mn2+ on the reduction of the primary donor P680+ after a saturating flash, and single-turnover electron donation from Mn2+ was followed by monitoring the decay kinetics of the EPR signal from the flash-induced tyrosine Zox radical. K(M) values for Mn2+ were found to be highly pH-dependent in both modified and unmodified photosystem II membranes. Treatment with histidine modifiers after removal of the manganese complex increased the K(M) values between 2.5 and 10 times and increased the dissociation constant for Mn2+ 8-fold, compared to membranes that were modified in the presence of the manganese cluster. Modification of carboxylic acid residues after removal of the manganese cluster increased the K(M) about 5-fold compared to membranes that were modified in the presence of the manganese cluster. The reduction rate of tyrosine Zox by Mn2+ was diminished after modification of either histidine or carboxylic acid residues. The apparent second-order rate constant decreased from 2.6 x 10(6) M(-1) s(-1) to 0.05 x 10(6) M(-1) s(-1) after histidine modification in the presence or absence of manganese, to 0.77 x 10(6) M(-1) s(-1) after carboxylic acid residue modification in the presence of manganese, and to 0.18 x 10(6) M(-1) s(-1) after carboxylic acid modification in the absence of manganese. Our results indicate the existence of two different manganese binding sites containing histidine, and at least two manganese sites with carboxylic acid residues, which are differently shielded against modifying agents by the native manganese cluster.