Structural Changes of D1 C-terminal α-Carboxylate during S-state Cycling in Photosynthetic Oxygen Evolution

Abstract

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...

No Evidence from FTIR Difference Spectroscopy That Glutamate-189 of the D1 Polypeptide Ligates a Mn Ion That Undergoes Oxidation during the S0 to S1, S1 to S2, or S2 to S3 Transitions in Photosystem II

“…If D1-Glu189 was indeed a ligand of a Mn ion and sensed the oxidation of the Mn 4 cluster during one or more of the S state transitions, then replacing Glu with Arg or Gln should eliminate 1 − 2 specific carboxylate modes from one or more of the S n+1 -minus-S n FTIR difference spectra, replacing them with a combination of CNH, NH, and/or CO modes. The elimination of the carboxylate modes should occur in both mutants and should be as obvious as the [1-13 C]alanine-induced shift of the ν sym (COO − ) mode of the α-COO − group of D1-Ala344 in prior studies (31,34,35). Yet, no such elimination is observed ( Figures 1A -1D, traces b and d).…”

“…PCC 6803 PSII particles (28,(31)(32)(33). Nevertheless, the WT(D1a) and WT (D1b) spectra show subtle differences between 1700 and 1300 cm −1 ( Figures 1A -1D, traces a).…”

“…The identification of these modes will complement X-ray crystallography by providing information about the dynamic structural changes that accompany water oxidation. Few of these modes have yet been identified (31,(33)(34)(35). We recently employed isotopic labeling to identify the symmetric carboxylate stretching mode of the α-COO − group of D1-Ala344 in the S 2 -minus-S 1 FTIR difference spectrum of wild-type PSII particles containing Ca or Sr (34,35) [also see (31)].…”

“…Few of these modes have yet been identified (31,(33)(34)(35). We recently employed isotopic labeling to identify the symmetric carboxylate stretching mode of the α-COO − group of D1-Ala344 in the S 2 -minus-S 1 FTIR difference spectrum of wild-type PSII particles containing Ca or Sr (34,35) [also see (31)]. The frequency of this mode in the S state and its ∼ 17 cm −1 or ∼ 36 cm −1 downshift in the S 2 state imply that this group is a unidentate ligand of a Mn ion whose charge increases during the S 1 → S 2 transition (31,34,35).…”

“…We recently employed isotopic labeling to identify the symmetric carboxylate stretching mode of the α-COO − group of D1-Ala344 in the S 2 -minus-S 1 FTIR difference spectrum of wild-type PSII particles containing Ca or Sr (34,35) [also see (31)]. The frequency of this mode in the S state and its ∼ 17 cm −1 or ∼ 36 cm −1 downshift in the S 2 state imply that this group is a unidentate ligand of a Mn ion whose charge increases during the S 1 → S 2 transition (31,34,35). A similar identification of one of the carboxylate stretching modes of D1-Glu189 in any of the S n → S n+1 transitions could provide unequivocal spectroscopic evidence for the ligation of the Mn 4 cluster by D1-Glu189 and could provide information about the type of carboxylate ligation and the environment of the carboxylate group.…”