Genetically introduced hydrogen bond interactions reveal an asymmetric charge distribution on the radical cation of the special-pair chlorophyll P680

The special-pair chlorophyll (Chl) P680 in photosystem II has an extremely high redox potential (E_m) to enable water oxidation in photosynthesis. Significant positive-charge localization on one of the Chl constituents, P_D1 or P_D2, in P680⁺ has been proposed to contribute to this high E_m. To identify the Chl molecule on which the charge is mainly localized, we genetically introduced a hydrogen bond to the 13¹-keto C=O group of P_D1 and P_D2 by changing the nearby D1-Val-157 and D2-Val-156 residues to His, respectively. Successful hydrogen bond formation at P_D1 and P_D2 in the obtained D1-V157H and D2-V156H mutants, respectively, was monitored by detecting 13¹-keto C=O vibrations in Fourier transfer infrared (FTIR) difference spectra upon oxidation of P680 and the symmetrically located redox-active tyrosines Y_Z and Y_D, and they were simulated by quantum-chemical calculations. Analysis of the P680⁺/P680 FTIR difference spectra of D1-V157H and D2-V156H showed that upon P680⁺ formation, the 13¹-keto C=O frequency upshifts by a much larger extent in P_D1 (23 cm^-1) than in P_D2 (<9 cm^-1). In addition, thermoluminescence measurements revealed that the D1-V157H mutation increased the E_m of P680 to a larger extent than did the D2-V156H mutation. These results, together with the previous results for the mutants of the His ligands of P_D1 and P_D2, lead to a definite conclusion that a charge is mainly localized to P_D1 in P680⁺.