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

Abstract

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<sup/>.

Keywords: FTIR; chlorophyll; cyanobacteria; hydrogen bond; photosynthesis; photosystem II; quantum chemical calculation; site-directed mutagenesis; special pair; thermoluminescence.

Figure 1.

Cofactors attached to the heterodimeric D1/D2 subunits in PSII with an expanded view of P680 that consists of P_D1 and P_D2. Red arrows indicate the 13¹-keto C=O groups of P_D1 and P_D2. In the D1-V157H and D2-V156H mutants, a Val residue located near P_D1 (D1-V157) and P_D2 (D2-V156), respectively, was replaced with His to introduce a hydrogen bond to the 13¹-keto C=O group.

Figure 2.

TL glow curves of wild-type and mutant cells in the presence of DCMU due to S₂Q_A⁻ recombination. a, D1-WT (black) and D1-V157H (red). b, D2-WT (black) and D2-V156H (blue).

Figure 3.

P680⁺/P680 FTIR difference spectra of the PSII core complexes in the 1800–1100-cm⁻¹ region (A) and the expanded view of the 13¹-keto C=O stretching region (B). a, D1-WT (black) and D1-V157H (red). b, D2-WT (black) and D2-V156H (blue). The spectra were normalized by the intensities of the 1312–1311/1286 cm⁻¹ peaks.

Figure 4.

Y_Z^•/Y_Z FTIR difference spectra of the PSII core complexes in the 1800–1200-cm⁻¹ region (A) and the expanded view of the 13¹-keto C=O stretching region of P680 (B). a, D1-WT (black) and D1-V157H (red). b, D2-WT (black) and D2-V156H (blue). The spectra were normalized by the intensity of the CO stretching band of the Y_Z^• radical at 1512 cm⁻¹.

Figure 5.

Y_D^•/Y_D FTIR difference spectra of the PSII core complexes in the 1800–1200-cm⁻¹ region (A) and the expanded view of the 13¹-keto C=O stretching region of P680 (B). a, D1-WT (black) and D1-V157H (red). b, D2-WT (black) and D2-V156H (blue). The spectra were normalized by the intensity of the CO stretching band of the Y_D^• radical at 1503 cm⁻¹.

Figure 6.

QM and MM regions of P680 model (WT) used for QM/MM calculations (A, top view; B, side view). Atoms in the QM region (the headgroups of P_D1 and P_D2 and the side chains of D1-His-198, D2-His-197, D1-Val-157, and D2-Val-156) are expressed as balls, and cofactors, amino acid residues, and water molecules in the MM region are shown as tubes (cofactors) and wire frame (others) images. The phytol chains of P_D1 and P_D2 are colored green, and the monomeric Chls (Chl_D1 and Chl_D2) and Pheos (Pheo_D1 and Pheo_D2) are colored blue and magenta, respectively.

Figure 7.

Optimized geometries of the P680 models of WT (A), D1-V157H (B), and D2-V156H (C) obtained by QM/MM calculations. Magnesium, green; carbon, gray; oxygen, red; nitrogen, blue; and hydrogen, cyan.