ENDOR AND SPECIAL TRIPLE-RESONANCE STUDIES OF CHLOROPHYLL CATION RADICALS IN PHOTOSYSTEM-2

Electron nuclear double resonance (ENDOR) and special triple (ST) reso nance spectroscopies have been used to study the cation radicals of th e primary donor, P680, and two secondary donor chlorophylls (Chi) in p hotosystem 2 (PS2). Two different preparations were employed, Tris-was hed PS2 membranes and PS2 reaction centers (D1-D2-I-Cytb(559) complex) . One secondary donor Chl a cation radical, Chl1(.+), was generated in the Tris-washed preparation, while the P680(.+) radical cation and a further Chl a cation radical, Chl2(.+), were produced in the reaction center preparation. The ENDOR spectrum of the primary donor radical ca tion of photosystem 1 (P700(.+)) is also presented for comparison. Hyp erfine coupling constants for methyl groups have been measured for all three PS2 radical species and assigned by comparison with previously published spectra of Chi a radicals in vitro. Electron spin densities were calculated from these hyperfine couplings. Comparison of ENDOR sp ectral features with those of Chla(.+) in vitro indicates similar valu es for Chl1(.+) and Chl2(.+) radicals but an apparent reduction in unp aired electron spin density for P680(.+). It has been proposed from th e more detailed studies of purple bacterial reaction centers that such a reduction in spin density can be interpreted as a delocalization ov er two Chl a molecules. Our calculations therefore suggest that P680(. +) is a weakly coupled chlorophyll pair with 82% of the unpaired elect ron spin located on one chlorophyll of the pair at 15 K. Environmental or geometrical changes to the chlorin ring structure to give a novel monomeric primary donor are also possible. The secondary donor Chl a's are both monomeric, the species in the reaction center preparation po ssibly lacking amino acid coordination at the magnesium ion. These dat a are discussed in the light of previous studies of P680 and oxidized chlorophyll species in PS2 using other techniques.