Deprotonation of the 33-kDa, extrinsic, manganese-stabilizing subunit accompanies photooxidation of manganese in photosystem II.

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.

EPR kinetic studies of oxygen release in thylakoids and PSII membranes: a kinetic intermediate in the S3 to S0 transition.

Time-resolved EPR oximetry has been used to determine the oxygen release kinetics in spinach thylakoids and PSII membranes. We observe release kinetics with half-times of approximately 0.85 and approximately 1.45 ms for thylakoids and PSII membranes, respectively, which are in close agreement with the EPR determined Yz decay kinetics for the S3 --> --> S0 transition in these systems. The results show conclusively that water-oxygen chemistry is not a rate-limiting step in the donor side of PSII under normal turnover conditions. By analyzing the oxygen release kinetics in thylakoids under nonphysiological, but still functionally competent conditions (low pH or high salt), we observed an initial delay in the O2 release of up to 200 microseconds following flash turnover from the S3 state. This is the first direct indication of a probable quasi-stable intermediate in the S3 --> --> S0 turnover of PSII, possibly representing the putative S4 state. Under conditions more closely approaching physiological, no such delay was resolved, indicating that the S4 --> O2 transition occurs within 50 microseconds under such circumstances. Two possible reaction sequences for O2 formation consistent with these and other data are discussed. It is suggested that the more probable form of "S4" is in fact the S3 + Yz* combination, which must undergo some molecular rearrangement on the tens to hundreds of microseconds time scale before O2 formation chemistry occurs.

Yz. reduction kinetics in the absence of the manganese-stabilizing protein of photosystem II.

Decay of Signal IIvf of photosystem II (PSII), under repetitive flash conditions, was examined in whole cells of wild-type Synechocystis sp. PCC6803 and in cells of an engineered strain, delta psbO, which lacks the extrinsic 33 kDa manganese-stabilizing protein (MSP). Previous polarographic analysis had shown that O2 release during the S3-->[S4]-->S0 transition of the catalytic cycle is significantly retarded in the delta psbO strain relative to the wild-type [Burnap et al. (1992) Biochemistry 31, 7404-7410]. The present experiments provide evidence that a parallel retardation in the rate of reduction of photooxidized Yz by the H2O oxidation complex is due to the absence of MSP. The half-time of the Signal IIvf component, corresponding to Yz. reduction during the S3-->[S4]-->S0 transition, was estimated to be 1.2 and 6.0 ms in the wild-type and delta psbO cells, respectively.

A time-resolved FTIR difference study of the plastoquinone QA and redox-active tyrosine YZ interactions in photosystem II.

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].

Electron paramagnetic resonance kinetic studies of the S states in spinach thylakoids.

The Tyrz+ decay kinetics have been analyzed by using time-resolved EPR to determine the half-time of each Si-->S(i + 1) transition in the O2-evolving complex of spinach thylakoids under physiological conditions. Using dark-adapted thylakoids and appropriate single-turnover flash sequences, we were able to detect the signal IIvf kinetics of the Tyrz+ S0-->Tyrz S1, Tyrz+ S1-->Tyrz S2, Tyrz+ S2-->Tyrz S3, and Tyrz+ S3-->(S4)-->Tyrz S0 transitions. To correct for damping of the S state synchronization during the flash sequence, the Kok parameters were estimated by measuring the oxygen flash pattern in situ using nitroxide-based EPR oximetry. Following deconvolution of the individual S state contributions, the signal IIvf decay kinetics yield the following half-times for the S state transitions: S0-->S1 in 40-60 microseconds, S1-->S2 in 85 microseconds, S2-->S3 in 140 microseconds, and S3-->(S4)-->S0 in 750 microseconds. Preliminary results with detergent-solubilized PSII membranes suggest that the S3-->S0 transition at least is slowed by a factor of approximately 2 in this system. Ramifications of these half-times in terms of electron transfer events on the donor site of PSII are discussed.