Site-directed mutations at D1-His198 and D2-His 97 of photosystem II in synechocystis PCC 6803: Sites of primary charge separation and cation and triplet stabilization

Site-directed mutations were introduced to replace D1-His198 and D2-His197 of the Dl and D2 polypeptides, respectively, of the photosystem II (PSII) reaction center of Synechocystis PCC 6803. These residues coordinate chlorophylls P-A and P-B which are homologous to the special pair Bchlorophylls of the bacterial reaction centers that are coordinated respectively by histidines L-173 and M-200 (202). P-A and P-B together serve as the primary electron donor, P, in purple bacterial reaction centers. In PS II, the site-directed mutations at D1 His198 affect the P+-P-absorbance difference spectrum. The bleaching maximum in the Soret region (in WT at 433 nm) is blue-shifted by as much as 3 nm. In the D1 His 198Gln mutant, a similar displacement to the blue is observed for the bleaching maximum in the Q(y) region (672.5 nm in WT at 80 K), whereas features attributed to a band shift centered at 681 nm are not altered. In the Y-z. -Y-z-difference spectrum, the band shift of a reaction center chlorophyll centered in WT at 433-434 nm is shifted by 2-3 nm to the blue in the D1-His198Gln mutant. The D1-His198Gln mutation has little effect on the optical difference spectrum, P-3-P-1, of the reaction center triplet formed by P(+)Pheo(-) charge recombination (bleaching at 681-684 nm), measured at 5-80 K, but becomes visible as a pronounced shoulder at 669 nm at temperatures greater than or equal to 150 K. Measurements of the kinetics of oxidized donor-Q(A)(-) charge recombination and of the reduction of P+ by redox active tyrosine, Y-z, indicate that the reduction potential of the redox couple P+/P can be appreciably modulated both positively and negatively by ligand replacement at D1-198 but somewhat less so at D2-197. On the basis of these observations and others in the literature, we propose that the monomeric accessory chlorophyll, B-A, is a long-wavelength trap located at 684 nm at 5 K. B-A* initiates primary charge separation at low temperature, a function that is increasingly shared with P-A* in an activated process as the temperature rises. Charge separation from B-A* would be potentially very fast and form PA+BA- and/or B(A)(+)Pheo(-) as observed in bacterial reaction centers upon direct excitation of B-A (van Brederode, M. E., et al. (1999) Proc. Nad. Acad Sci. 96, 2054-2059). The cation, generated upon primary charge separation in PSII, is stabilized at all temperatures primarily on P-A, the absorbance spectrum of which is displaced to the blue by the mutations. In WT, the cation is proposed to be shared to a minor extent (similar to 20%) with P-B, the contribution of which can be modulated up or down by mutation. The band shift at 681 nm, observed in the P+-P difference spectrum, is attributed to an electrochromic effect of P-A(+) on neighboring B-A. Because of its low-energy singlet and therefore triplet state, the reaction center triplet state is stabilized on B-A at less than or equal to 80 K but can be shared with P-A at > 80 K in a thermally activated process.