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 investiga ted after chemical modification of histidyl and carboxylic acid residu es in the presence or absence of the native manganese cluster. K-M val ues for Mn2+ were determined from steady-state electron transfer betwe en Mn2+ and 2,6-dichlorophenolindophenol, the dissociation constant fo r Mn2+ was measured by observing the effect of added Mn2+ on the reduc tion of the primary donor P680(+) after a saturating flash, and single -turnover electron donation from Mn2+ was followed by monitoring the d ecay kinetics of the EPR signal from the flash-induced tyrosine Z(OX) radical. K-M values for Mn2+ were found to be highly pH-dependent in b oth modified and unmodified photosystem II membranes. Treatment with h istidine modifiers after removal of the manganese complex increased th e KM values between 2.5 and 10 times and increased the dissociation co nstant for Mn2+ 8-fold, compared to membranes that were modified in th e presence of the manganese cluster. Modification of carboxylic acid r esidues after removal of the manganese cluster increased the K-M about 5-fold compared to membranes that were modified in the presence of th e manganese cluster. The reduction rate of tyrosine Z(OX) by Mn2+ was diminished after modification of either histidine or carboxylic acid r esidues. 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 modifica tion 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 mang anese, and to 0.18 x 10(6) M(-1) s(-1) after carboxylic acid modificat ion in the absence of manganese. Our results indicate the existence of two different manganese binding sites containing histidine, and at le ast two manganese sites with carboxylic acid residues, which are diffe rently shielded against modifying agents by the native manganese clust er.