INCREASES IN PEROXIDE FORMATION BY THE PHOTOSYSTEM-II OXYGEN-EVOLVINGREACTIONS UPON REMOVAL OF THE EXTRINSIC 16, 22 AND 33 KDA PROTEINS ARE REVERSED BY CACL2 ADDITION

This communication introduces a new spectrophotometric assay for the d etection of peroxide generated by Photosystem II (PS II) under steady state illumination in the presence of an electron acceptor. The assay is based on the formation of an indamine dye in a horseradish peroxida se coupled reaction between 3-(dimethylamino)benzoic acid and 3-methyl -2-benzothiazolinone hydrazone. Using this assay, we found that as the O-2 evolution activity of PS II-enriched membrane fragments is decrea sed by treatments which cause the dissociation of the 33 and/or 23 and 16 kDa extrinsic proteins (i.e., CaCl2-washing, NaCl-washing, lauroyl choline-treatment and ethylene glycol-treatment), light-induced peroxi de formation increases. Both the losses of O-2 evolution and increases in peroxide formation seen under these conditions are reversed by CaC l2 addition, indicating that the two activities originate from the wat er-splitting site. However, the increased rates of peroxide formation do not quantitatively match the losses in O-2 evolution activity. We s uggest that a rapid consumption of the peroxide takes place via a cata lase/peroxidase activity at the water-splitting site which competes wi th both the O-2 evolution and peroxide formation reactions. The observ ed peroxide formation is interpreted as arising from enhanced water ac cessibility to the catalytic site upon perturbation of the extrinsic p roteins which then leads to alternate water oxidation side reactions.