Analysis of flash-induced FTIR difference spectra of the S-state cycle in the photosynthetic water-oxidizing complex by uniform 15N and 13C isotope labeling

Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform N-15 and C-13 isotopic labeling of photosystem II (PS II). The difference spectra upon first- to fourth-flash illumination were obtained with hydrated (for the 1800-1200 cm(-1) region) or deuterated (for the 3500-3100 cm(-1) region) films of unlabeled, N-15-labeled, and C-13-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon N-15 and C-13 labeling provided the assignments of major peaks in the regions of 3450-3250 and 1700-1630 cm(-1) to the NH stretches and amide I modes of polypeptide backbones, respectively, and the assignments of some of the peaks in the 1600-1500 cm(-1) region to the amide II modes of backbones. Other prominent peaks in the latter region and most of the peaks in the 1450-1300 cm(-1) region exhibited large downshifts upon C-13 labeling but were unchanged by N-15 labeling, and hence assigned to the asymmetric and symmetric COO- stretching vibrations, respectively, of carboxylate groups in Glu, Asp, or the C-terminus. Peak positions corresponded well with each other among the first- to fourth-flash spectra, and most of the bands in the first- and/or second-flash spectra appeared with opposite signs of intensity in the third- and/or fourth-flash spectra. This observation indicates that the protein movements in the S-1-->S-2 and/or S-2-->S-3 transitions are mostly reversed in the S-3-->S-0 and/or S-0-S-1 transitions, representing a catalytic role of the protein moieties of the water-oxidizing complex. Drastic structural changes in carboxylate groups over the S-state cycle suggest that the Asp and/or Glu side chains play important roles in the reaction mechanism of photosynthetic water oxidation.