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Photosystem II (PSII) is a protein complex that performs water oxidation using light energy during photosynthesis. In PSII, electrons abstracted from water are eventually transferred to the secondary quinone electron acceptor, Q B , and upon double reduction, Q B is converted to quinol by binding two protons. Thus, excess electron transfer in PSII increases the pH of the stroma. In this study, to investigate the pH-dependent regulation of the electron flow in PSII, we have estimated the relaxation rate of the Q B - radical anion in the pH region between 5 and 8 by direct monitoring of its population using light-induced Fourier transform infrared difference spectroscopy. The decay of Q B - by charge recombination with the S 2 state of the water oxidation center in PSII membranes was shown to be accelerated at higher pH, whereas that of Q A - examined in the presence of a herbicide was virtually unaffected at pH ≤7.5 and slightly slowed at pH 8. These observations were consistent with the previous studies that included rather indirect monitoring of the Q B - and Q A - decays using fluorescence detection. The accelerated relaxation of Q B - was explained by the shift of a redox equilibrium between Q A - and Q B - to the Q A - side due to the decrease in the redox potential of Q B at higher pH, which is induced by deprotonation of a single amino acid residue near Q B . It is proposed that this pH-dependent Q B - relaxation is one of the mechanisms of electron flow regulation in PSII for its photoprotection.

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(•), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(•)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.

Can Trolox, a water-soluble analogue of α-tocopherol and a scavenger of singlet oxygen ((1)O(2)), provide photoprotection, under high irradiance, to the isolated photosystem II (PSII) reaction center (RC)? To answer the question, we studied the endogenous production of (1)O(2) in preparations of the five-chlorophyll PSII RC (RC5) containing only one β-carotene molecule. The temporal profile of (1)O(2) emission at 1270 nm photogenerated by RC5 in D(2)O followed the expected biexponential behavior, with a rise time, unaffected by Trolox, of 13 ± 1 μs and decay times of 54 ± 2 μs (without Trolox) and 38 ± 2 μs (in the presence of 25 μM Trolox). The ratio between the total (k(t)) and chemical (k(r)) bimolecular rate constants for the scavenging of (1)O(2) by Trolox in aqueous buffer was calculated to be ~1.3, with a k(t) of (2.4 ± 0.2) × 10(8) M(-1) s(-1) and a k(r) of (1.8 ± 0.2) × 10(8) M(-1) s(-1), indicating that most of the (1)O(2) photosensitized by methylene blue chemically reacts with Trolox in the assay buffer. The photoinduced oxygen consumption in the oxygen electrode, when RC5 and Trolox were mixed, revealed that Trolox was a better (1)O(2) scavenger than histidine and furfuryl alcohol at low concentrations (i.e., <1 mM). After its incorporation into detergent micelles in unbuffered solutions, Trolox was able to photoprotect the surface-exposed regions of the D1-D2 heterodimer, but not the RC5 pigments, which were oxidized, together with the membrane region of the protein matrix of the PSII RC, by (1)O(2). These results are discussed and compared with those of studies dealing with the physiological role of tocopherol molecules as a (1)O(2) scavenger in thylakoid membranes of photosynthetic organisms.

Psb27 is a highly conserved component of photosystem II. The three-dimensional structure has a well-defined helical core, composed of four helices arranged in a right-handed up-down-up-down fold, with a less ordered region of the structure located at the N-terminus. The position of conserved residues on the surface suggests conserved functional roles for distinct interconnected features encompassing a P-phi-P loop, a polar patch spanning helices alpha3 and alpha4, and the N-terminal sequence.

Psb27 is a membrane-extrinsic subunit of photosystem II (PSII) where it is involved in the assembly and maintenance of this large membrane protein complex that catalyzes one of the key reactions in the biosphere, the light-induced oxidation of water. Here, we report for the first time the structure of Psb27 that was not observed in the previous crystal structures of PSII due to its transient binding mode. The Psb27 structure shows that the core of the protein is a right-handed four-helix bundle with an up-down-up-down topology. The electrostatic potential of the surface generated by the amphipathic helices shows a dipolar distribution which fits perfectly to the major PsbO binding site on the PSII complex. Moreover, the presented docking model could explain the function of Psb27, which prevents the binding of PsbO to facilitate the assembly of the Mn(4)Ca cluster.

Time-resolved quasielastic neutron scattering with laser excitation is a promising novel pump-probe approach, which opens up new perspectives for the study of protein-membrane dynamics in specific functional states of even complex systems. This is demonstrated here for the case of photosystem II membrane fragments with inhibited electron transfer. In contrast to the case of the model system bacteriorhodopsin, a transient reduction of the dynamics is observed approximately 160 micros after the actinic laser flash. This effect is the first observation of a modulated structural dynamics in photosystem II membrane fragments.

The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ* and the Mn4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0 degrees C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ* and the Mn4Ca cluster.

The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.

We have employed absorption, circular dichroism (CD), and persistent spectral hole-burning measurements at 1.7 K to study the photoconversion properties and exciton coupling of low-energy chlorophylls (Chls) in the CP43 proximal antenna light-harvesting subunit of photosystem II (PSII) isolated from spinach. These approximately 683 nm states act as traps for excitation energy in isolated CP43. They "bleach" at 683 nm upon illumination and photoconvert to a form absorbing in the range approximately 660-680 nm. We present new data that show the changes in the CD spectrum due to the photoconversion process. These changes occur in parallel with those in absorption, providing evidence that the feature undergoing the apparent bleach is a component of a weakly exciton-coupled system. From our photoconversion difference spectra, we assign four states in the Chl long-wavelength region of CP43, two of which are the known trap states and are both highly localized on single Chls. The other two states are associated with weak exciton coupling (maximally approximately 50 cm(-)(1)) to one of these traps. We propose a mechanism for photoconversion that involves Chl-protein hydrogen bonding. New hole-burning data are presented that indicate this mechanism is distinct to that for narrow-band spectral hole burning in CP43. We discuss the photophysical behavior of the Chl trap states in isolated CP43 compared to their behavior in intact PSII preparations. The latter represent a more intact, physiological complex, and we find no clear evidence that they exhibit the photoconversion process reported here.

The photosystem-II-associated 33-kDa extrinsic manganese-stabilizing protein is found in all oxygen-evolving organisms. In this paper, we show that this protein undergoes pH-induced conformational changes in the physiological pH range. At a neutral pH of 7.2, the hydrophobic amino acid residues that are most likely located inside the beta barrel are "closed" and the protein binds neither Mn2+ nor Ca2+ ions. When the protein is transferred to a solution with a slightly acidic pH of 5.7, hydrophobic amino acid residues become exposed to the surrounding medium, enabling them to bind the fluorescent probe 8,1-ANS. At this pH-induced open state, Mn2+ and Ca2+ bind to the manganese-stabilizing protein. The pH values used in this study, 7.2 and 5.7, are typical of the pH found in the thylakoid lumen in the dark and light, respectively. A model is presented in which the manganese-stabilizing protein undergoes a pH-dependent conformational change that in turn influences its capacity to bind calcium and manganese. In this model, the proton-dependent conformational changes of the tertiary structure of the manganese-stabilizing protein are of functional relevance for the regulation of substrate (water) delivery to and product (proton) release from the water-oxidizing complex by forming a proton-sensing proton-transport pathway.

Under strong light, photosystem II (PSII) of oxygenic photosynthetic organisms is inactivated, and this phenomenon is called photoinhibition. In a widely accepted model, photoinhibition is induced by excess light energy, which is absorbed by chlorophyll but not utilized in photosynthesis. Using monochromatic light from the Okazaki Large Spectrograph and thylakoid membranes from Thermosynechococcus elongatus, we observed that UV and blue light inactivated the oxygen-evolving complex much faster than the photochemical reaction center of PSII. These observations suggested that the light-induced damage was associated with a UV- and blue light-absorbing center in the oxygen-evolving complex of PSII. The action spectrum of the primary event in photodamage to PSII revealed the strong effects of UV and blue light and differed considerably from the absorption spectra of chlorophyll and thylakoid membranes. By contrast to the photoinduced inactivation of the oxygen-evolving complex in untreated thylakoid membranes, red light efficiently induced inactivation of the PSII reaction center in Tris-treated thylakoid membranes, and the action spectrum resembled the absorption spectrum of chlorophyll. Our observations suggest that photodamage to PSII occurs in two steps. Step 1 is the light-induced inactivation of the oxygen-evolving complex. Step 2, occurring after step 1 is complete, is the inactivation of the PSII reaction center by light absorbed by chlorophyll. We confirmed our model by illumination of untreated thylakoid membranes with blue and UV light, which inactivated the oxygen-evolving complex, and then with red light, which inactivated the photochemical reaction center.