In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the YZoxQA-/YZQA vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), with a spectral resolution of 4 cm-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of YZox and QA- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the YZox decay, but does substantially slow down the QA- decay. By making use of the difference in the decay kinetics using DCMU, the QA-/QA signals could be separated from the YZox/YZ signals and a pure QA-/QA difference spectrum obtained. By comparison of the YZoxQA-/YZQA difference spectrum with the pure QA-/QA difference spectrum, a large differential band at 1706/1699 cm-1 could be identified and associated with YZ oxidation. In contrast, an intense band at 1478 cm-1, whose DCMU-sensitive decay follows the QA- decay based on the chlorophyll a fluorescence measurements, was present in all of the time-resolved spectra. Since no significant reversible Chl+ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion QA- [Berthomieu, C., Nabedryk, E., Mäntele, W., & Breton, J. (1990) FEBS Lett. 269, 363-367].

Plastoquinone (PQ-9) is active as an electron/proton transfer component in photosynthetic membranes. For example, in the photosynthetic complex, photosystem II (PSII), PQ-9 acts as Q A , a one-electron acceptor, and as Q B , a two electron, two proton accepting species. Light-minus-dark difference Fourier transform infrared (FT-IR) spectroscopy is a technique with which mechanistic information can be obtained concerning PSII. Here, we present combined experimental and computational studies designed to identify the vibrational contributions of the electron acceptor, Q A , in its oxidized and one-electron reduced states to the difference FT-IR spectrum. Infrared spectra of decyl-PQ and PQ-9 were obtained; the difference infrared spectra associated with the formation of the corresponding anion radicals were also generated in ethanol solutions. Vibrational mode assignments were made based on hybrid Hartree-Fock/density functional (HF/DF) B3LYP calculations with a 6-31G(d) basis set. Calculations were performed for hydrogen bonded models of PQ-1 and its radical anion. In addition, a methionine-tolerant strain of the cyanobacterium, Synechocystis sp. PCC 6803, was used to deuterate PQ-9 in PSII. The macrocycle and phytol tail of chlorophyll were not labeled by this procedure. Mass spectral data may be consistent with partial 13 3 methoxy labeling of chlorophyll. Lack of phytol labeling implies that carotenoids were unlabeled. Difference FT-IR spectra were then obtained by illumination at 80 K, resulting in the one-electron reduction of Q A . When spectra were obtained of PSII preparations, in which 39% of PQ was 2 H 3 labeled and 48% was 2 H 6 labeled, isotope-induced shifts were observed. Comparison of these data to vibrational spectra obtained in vitro and to mode frequencies and intensities from B3LYP/ 6-31G(d) calculations provides the basis for vibrational mode assignments.Photosystem II (PSII), a membrane-associated pigmentprotein complex, carries out the oxidation of water and reduction of PQ-9 (plastoquinone-9) in all oxygen-evolving plants, algae, and cyanobacteria. Photoexcitation of the primary electron donor, P 680 , results in electron transfer to a bound PQ-9, called Q A , via a pheophytin molecule. Reduced Q A is reoxidized by an exchangeable PQ-9, named Q B . Q A functions as a oneelectron acceptor, and the reduced form, Q A -, is an unprotonated semiquinone anion radical. Q B , on the other hand, is a twoelectron, two-proton acceptor {reviewed in ref 1}. Electron transfer events on the acceptor side of PSII resemble reactions occurring on the acceptor side of the photosynthetic bacterial reaction center. 2 This enzyme, for which high-resolution structural information is available, uses UQ (ubiquinone) or menaquinone, instead of PQ-9, as acceptor molecules {reviewed in ref 3}.On the donor side of PSII, P 680 + is reduced by a redox-active tyrosine, Z. 4-8 The tyrosine radical, Z • , is reduced by a multinuclear manganese cluster on the microsecond to millisecond time range {see ref 9 and references ther...

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Photosystem II catalyzes photosynthetic water oxidation. The oxidation of water to molecular oxygen requires four sequential oxidations; the sequentially oxidized forms of the catalytic site are called the S states. An extrinsic subunit, the manganese-stabilizing protein (MSP), promotes the efficient turnover of the S states. MSP can be removed and rebound to the reaction center; removal and reconstitution is associated with a decrease in and then a restoration of enzymatic activity. We have isotopically edited MSP by uniform 13 C labeling of the Escherichia coli-expressed protein and have obtained the Fourier transform infrared spectrum associated with the S 1 to S 2 transition in the presence either of reconstituted 12 C or 13 C MSP. 13 C labeling of MSP is shown to cause 30 -60 cm ؊1 shifts in a subset of vibrational lines. The derived, isotope-edited vibrational spectrum is consistent with a deprotonation of glutamic/ aspartic acid residues on MSP during the S 1 to S 2 transition; the base, which accepts this proton(s), is not located on MSP. This finding suggests that this subunit plays a role as a stabilizer of a charged transition state and, perhaps, as a general acid/base catalyst of oxygen evolution. These results provide a molecular explanation for known MSP effects on oxygen evolution.In oxygenic photosynthesis, the multi-subunit protein complex, photosystem II (PSII), 1 uses light energy to oxidize water and to form molecular oxygen. Hydrophobic subunits, such as the D1 and D2 proteins, bind most of the prosthetic groups involved in charge separation. Water oxidation occurs at a catalytic site containing four manganese atoms. The catalytic site accumulates the four oxidizing equivalents required for water oxidation. The five sequentially oxidized states of the catalytic site are called the S states. Each oxidation of the catalytic site is associated with the reduction of quinone acceptor molecules, Q A , a single electron acceptor, and then Q B , a two-electron, two-proton acceptor (reviewed in Ref. 1).The 33-kDa, manganese-stabilizing protein (MSP) of PSII plays an important role in water oxidation (reviewed in Ref. 2). As an extrinsic subunit, MSP can be removed from the plant reaction center by several different types of biochemical treatments (3-6). MSP has also been removed by mutagenesis in the cyanobacterium, Synechocystis sp. PCC 6803 (7), and in a green algae, Chlamydomonas reinhardtii (8). In the presence of low concentrations of calcium and chloride, plant MSP was found to be required for photosynthetic oxygen evolution and for maintaining the stability of the manganese cluster (9, 10). In the presence of high concentrations of calcium and chloride, oxygen evolution occurs, but the steady state rate of enzymatic activity is impaired upon removal of MSP (7,9,(11)(12)(13)(14). In addition, removal of MSP and replacement with calcium and chloride result in kinetic inhibition of the S state transitions (12,(15)(16)(17)(18)(19). MSP can be rebound to PSII (20), and this reconstitution reverses ...