FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700+ and localization of the triplet character in 3P700

The effect of global N-15 or H-2 labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-1) and at 1697 and 1637 cm(-1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-1), which does not shift upon global labeling or H-1-H-2 exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P-1 and P-2) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of tripler formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P1). Thus, while the P-2 carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P-1 are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P-1 and P-2 appear to be five-coordinated. No localization of the triplet on the Pt half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P-1 and P-2 upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P-2) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P-1, indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.