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 ( ) to enable water oxidation in photosynthesis. Significant positive-charge localization on one of the Chl constituents, P or P, in P680 has been proposed to contribute to this high To identify the Chl molecule on which the charge is mainly localized, we genetically introduced a hydrogen bond to the 13-keto C=O group of P and P by changing the nearby D1-Val-157 and D2-Val-156 residues to His, respectively. Successful hydrogen bond formation at P and P in the obtained D1-V157H and D2-V156H mutants, respectively, was monitored by detecting 13-keto C=O vibrations in Fourier transfer infrared (FTIR) difference spectra upon oxidation of P680 and the symmetrically located redox-active tyrosines Y and Y, 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 13-keto C=O frequency upshifts by a much larger extent in P (23 cm) than in P (<9 cm). In addition, thermoluminescence measurements revealed that the D1-V157H mutation increased the 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 and P, lead to a definite conclusion that a charge is mainly localized to P in P680^.