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 ( Em ) to enable water oxidation in photosynthesis. Significant positive-charge localization on one of the Chl constituents, PD1 or PD2 , in P680+ has been proposed to contribute to this high Em 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 PD1 and PD2 by changing the nearby D1-Val-157 and D2-Val-156 residues to His, respectively. Successful hydrogen bond formation at PD1 and PD2 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 YZ and YD , 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 PD1 (23 cm-1 ) than in PD2 (<9 cm-1 ). In addition, thermoluminescence measurements revealed that the D1-V157H mutation increased the Em 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 PD1 and PD2 , lead to a definite conclusion that a charge is mainly localized to PD1 in P680<sup/>.