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

Abstract

Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform ¹⁵N and ¹³C 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, ¹⁵N-labeled, and ¹³C-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon ¹⁵N and ¹³C 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 ¹³C labeling but were unchanged by ¹⁵N 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 S₁→S₂ and/or S₂→S₃ transitions are mostly reversed in the S₃→S₀ and/or S₀→S₁ 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.