Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.

The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.

Changes in the chemical structure of ␣-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [13 C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S 2 /S 1 FTIR difference spectrum were affected by selective 13 C labeling of the ␣-carboxylate of Ala with L-[1-13 C]alanine, whereas most of the isotopic effects failed to be induced in a sitedirected mutant in which Ala-344 was replaced with Gly. Labeling of the ␣-methyl of Ala with L-[3-13 C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH 3 deformation coupled with the ␣-carboxylate. The isotopic bands for the ␣-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S 1 -to-S 2 transition and to a lesser extent upon the S 2 -to-S 3 transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S 3 -to-S 0 transition. No obvious isotopic band appeared upon the S 0 -to-S 1 transition. These results indicate that the ␣-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S 1 -to-S 2 transition and reduced reversely upon the S 3 -to-S 0 transition but is not associated with manganese ion(s) oxidized during the S 0 -to-S 1 (and S 2 -to-S 3 ) transition(s). Consistently, L-[1-13 C]alanine labeling also induced spectral changes in the low frequency (670 -350 cm ؊1 ) S 2 /S 1 FTIR difference spectrum.Photosynthetic water oxidation takes place in an oxygenevolving complex (OEC) 1 in which the catalytic metal cluster located on the lumenal side of the D1 protein is composed of four manganese ions and one Ca 2ϩ ion. Most of the potential ligands of the manganese/Ca 2ϩ cluster appear to be located on the D1 protein based on site-directed mutagenesis studies using the cyanobacterium Synechocystis sp. PCC 6803 (reviewed in Refs. 1-3). These include , which are arranged in close proximity to the cluster according to the x-ray structural model of S 1 state OEC (10 -13).The D1 protein is synthesized and assembled into the PS II complex with a short C-terminal extension except for the protein in Euglena (14). Light-dependent assembly of the manganese/Ca 2ϩ cluster requires a free ␣-carboxylate of the C-terminal Ala-344, which occurs via cleavage of the C-terminal extension by the D1 C-terminal processing protease (CtpA) (15). None of the C-terminal truncated Synechocystis mutants in which Asn-335, Asp-342, Leu-343, and Ala-344 were replaced with a stop codon grew photoautotrophically and evolved oxygen (5). Site-directed replacement of D1-Ala-344 with Gly, Met, Ser, Val, Glu, or Gln in the D1-Ala-344-stop strain did not eliminate the capability for photoautotrophic growth and o...