Site-Directed Mutations at D1-His198 and D2-His197 of Photosystem II in Synechocystis PCC 6803:  Sites of Primary Charge Separation and Cation and Triplet Stabilization

Abstract

Site-directed mutations were introduced to replace D1-His198 and D2-His197 of the D1 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 His198Gln 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, 3 P-1 P, 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 g150 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 P A + B A -and/or B A + Pheo -as observed in bacterial reaction centers upon direct excitation of B A ) Proc. Natl. 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 (∼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 e80 K but can be shared with P A at >80 K in a thermally activated process.

Initial electron donor and acceptor in isolated Photosystem II reaction centers identified with femtosecond mid-IR spectroscopy

“…An alternative assignment for the Chl D1 keto stretch, however, is at 1,670 cm Ϫ1 , meaning that it is completely masked in our RP1 spectrum by the large positive (amide CAO) band at this frequency: The keto frequency of the tripletcarrying Chl at low temperature [either Chl D1 or Chl D2 (28,40,41)] has been reported to be at 1,670 cm Ϫ1 (28,31). Because the low temperature triplet-singlet spectrum in the visible coincides with the band in the P ϩ ͞P spectrum assigned to Chl D1 (9), Diner and coworkers (9) proposed Chl D1 as the triplet carrying Chl.…”

“…This analysis seems a likely interpretation because the cation state is localized mainly on P D1 (9), and the 1,704 cm Ϫ1 band has the largest amplitude in the P ϩ ͞P spectrum. The additional bands at 1,681, 1,670, and 1,653 cm Ϫ1 may be either due to electrochromic responses of nearby Chl, H, and amide CAO molecules or reflect the partial localization of the cation on pigments other than P D1, and in PC-P*͞P reflect the delocalization of the excited state over several pigments.…”

“…Excitation of the PSII RC with a short laser flash leads to ultrafast charge separation followed by the formation of the radical pair P D1 ϩ H D1 Ϫ , consisting of the H molecule in the D 1 branch, H D1 , and one of the Chl forming the special pair, P D1 (9). Electron transport stops at the H molecule because the final quinone acceptors are missing in these preparations.…”

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

“…An anodic current originating from HTPP oxidation appears only in the presence of the mutant foldon or in a solution of free 5-HTPP with E Pa ϭ 400 mV and E Pa ϭ 450 mV [vs. saturated calomel electrode (SCE)], respectively, indicating the presence of 5-HTPP in the mutant foldon. The slight decrease in the oxidation potential for the mutant protein probably results from differential stabilization of the oxidized and reduced forms of 5-HTPP in aqueous solution vs. the hydrophobic protein core (33,34). No current was observed on attempts to oxidize the WT foldon (data not shown).…”

Spectroelectrochemical determination of the redox potential of pheophytina, the primary electron acceptor in photosystem II

“…A similar value of Ϫ165 meV was reported from time-resolved photovoltage measurements on unstacked PS II membrane fragments and data analysis in terms of the exciton ion-pair equilibrium model (50). ⌬G S is generally referred to as a static Coulombic interaction calculated as Ϫq 2 /(4 r), where is the permittivity, and r is the ionic center-to-center distance: The interaction energy between P680 and Phe a ranges from Ϫ45 to Ϫ225 mV for r (relative permittivity) of 20 to 4, respectively, by assuming that the cation is located predominantly on the D 1 side of the special pair (51). Considering an entropic equilibration at an excited state between P680 and antenna Chls (AnChl) and charge separation driven by the equilibrated [AnChl P680]*; however, ⌬G S should be within the difference between the equilibrated state and the charge- (12).…”