Electron Transfer from Cyt b559 and Tyrosine-D to the S2 and S3 states of the water oxidizing complex in Photosystem II at Cryogenic Temperatures

The Mn4CaO5 cluster of photosystem II (PSII) catalyzes the oxidation of water to molecular oxygen through the light-driven redox S-cycle. The water oxidizing complex (WOC) forms a triad with Tyrosine(Z) and P-680, which mediates electrons from water towards the acceptor side of PSII. Under certain conditions two other redox-active components, Tyrosine(D) (Y-D) and Cytochrome b (559) (Cyt b (559)) can also interact with the S-states. In the present work we investigate the electron transfer from Cyt b (559) and Y-D to the S-2 and S-3 states at 195 K. First, Y-D (aEuro cent) and Cyt b (559) were chemically reduced. The S-2 and S-3 states were then achieved by application of one or two laser flashes, respectively, on samples stabilized in the S-1 state. EPR signals of the WOC (the S-2-state multiline signal, ML-S-2), Y-D (aEuro cent) and oxidized Cyt b (559) were simultaneously detected during a prolonged dark incubation at 195 K. During 163 days of incubation a large fraction of the S-2 population decayed to S-1 in the S-2 samples by following a single exponential decay. Differently, S-3 samples showed an initial increase in the ML-S-2 intensity (due to S-3 to S-2 conversion) and a subsequent slow decay due to S-2 to S-1 conversion. In both cases, only a minor oxidation of Y-D was observed. In contrast, the signal intensity of the oxidized Cyt b (559) showed a two-fold increase in both the S-2 and S-3 samples. The electron donation from Cyt b (559) was much more efficient to the S-2 state than to the S-3 state

Electron transfer from cytochrome b559 and tyrosineD to the S2 and S3 states of the water oxidizing complex in photosystem II

We have investigated the electron transfer from reduced tyrosine YD (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S2 state multiline EPR signal and the EPR signal from YD@? were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD@? signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD@?S1 centers, via the intermediate YD@?S2 state. The early increase of the S2 state multiline signal involves electron transfer from YDred to the S3 state resulting in formation of YD@?S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the YDredS2->YD@?S1 and YDredS3->YD@?S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed

pH dependence of the four individual transitions in the catalytic S-cycle during photosynthetic oxygen evolution.

We have investigated the pH dependence for each individual redox transition in the S-cycle of the oxygen evolving complex (OEC) of photosystem II by electron paramagnetic resonance (EPR) spectroscopy. In the experiments, OEC is advanced to the appropriate S-state at normal pH. Then, the pH is rapidly changed, and a new flash is given. The ability to advance to the next S-state in the cycle at different pHs is determined by measurements of the decrease or increase of characteristic EPR signals from the OEC in different S-states. In some cases the measured EPR signals are very small (this holds especially for the S0 ML signal at pH >7.5 and pH S2 transition is independent of pH between 4.1 and 8.4. All other S-transitions are blocked at low pH. In the acidic region, the pK's for the inhibition of the S2 --> S3, the S3 --> [S4] --> S0, and the S0 --> S1 transitions are about 4.0, 4.5, and 4.7, respectively. The similarity of these pK values indicates that the inhibition of the steady-state oxygen evolution in the acidic range, which occurs with pK approximately 4.8, is a consequence of similar pH blocks in three of the redox steps involved in the oxygen evolution. In the alkaline region, we report a clear pH block in the S3 --> [S4] --> S0 transition with a pK of about 8.0. Our study also indicates the existence of a pH block at very high pH (pK approximately 9.4) in the S2 --> S3 transition. The S0 --> S1 transition is not affected, at least up to pH 9.0. This suggests that the inhibition of the steady-state oxygen evolution, which occurs with a pK of 8.0, is dominated by the inhibition of the S3 --> [S4] --> S0 transition. Our results are obtained in the presence of 5% methanol (v/v). However, it is unlikely that the determined pK's are affected by the presence of methanol since our results also show that the pH dependence of the steady-state oxygen evolution is not affected by methanol. The results in the alkaline region are in good agreement with a model, which suggests that the redox potential of Y(Z*)/Y(Z) is directly affected by high pH. At high pH the Y(Z*)/Y(Z) potential becomes lower than that of S2/S1 and S3/S2. The acidic block, with a pK of 4-5 in three S-transitions, implies that the inhibition mechanism is similar, and we suggest that it reflects protonation of a carboxylic side chain in the proton relay that expels protons from the OEC

pH Dependence of the Donor Side Reactions in Ca2+-Depleted Photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca2+ ion cofactor. For these samples, it was found that the EPR signal from the YZ radical decays faster at low pH than at high pH. At 20 C, YZ decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of ~0.8 s, while the slow phase has a lifetime of ~22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of ~0.3 s. The kinetic changes occurred with an approximate pKa of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S2YZ state that is induced in Ca2+-depleted PSII membranes because of an inability of YZ to oxidize the S2 state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pKa of 4.5. In the same samples, the stable multiline signal from the S2 state, which is modified by the removal of Ca2+, was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S2YZ state at lower pH was not due to inability to oxidize the modified S2 state induced by the Ca2+ depletion. Instead, we propose that the low pH makes YZ able to oxidize the S2 state, making the S2 S3 transition available in Ca2+-depleted PSII. Implications of these results for the catalytic role of Ca2+ and the role of proton transfer between the Mn cluster and YZ during oxygen evolution is discussed

pH-dependent oxidation of Cytochrome b559 is different in the different S-states

We have studied the state of Cyt b559 and the Mn cluster by EPR and optical spectroscopy in the different S-states between pH 4.0-10.0. In these experiments, the pH was changed after the OEC turnover to the appropriate S-state (at pH 6.0) by 0-3 saturating flashes. Furthermore, the state of Cyt b559 after a subsequent flash given after the pH modification was also investigated. The results show that low ( pH 9.0) resulted in an increase in the oxidized form of Cyt b559 in the S2 and S3 states. The effect was more pronounced at low than high pH. There was no further oxidation after the subsequent flash. Different to this, the pH induced oxidized Cyt b559 population in the S0 and S1 states was significantly smaller. In the S1 state, the pH dependent oxidation of Cyt b559 was very weak, with a slight increase from pH 10.0 to 4.0. A subsequent flash in S0 or S1 resulted in a significant Cyt b559 oxidation at both low and high pH’s. The ratio of the high and low potential forms of Cyt b559 at different pH’s was also different in different S-states. At low and normal pH’s both oxidized LP and HP forms were observed, while at high pH’s, only the LP form was oxidized. The possible involvement of Cyt b559 in electron donation to the Mn cluster during S-cycle at extreme pH is discussed

Spin conversion of cytochrome b559 in photosystem II induced by exogenous high potential quinone

The spin-state of cytochrome b559 (Cyt b559) was studied in photosystem II (PSII) membrane fragments by low-temperature EPR spectroscopy. Treatment of the membranes with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) converts the native low-spin (LS) form of Cyt b559 to the high-spin (HS) form characterized with the g= 6.19 and g= 5.95 split signal. The HS Cyt b559 was pH dependent with the amplitude increasing toward more acidic pH values (pH 5.5-8.5). The HS state was not photochemically active upon 77 and 200 K continuous illumination under our conditions and was characterized by a low reduction potential (=<0 V). It was also demonstrated that DDQ treatment damages the oxygen evolving complex, leading to inhibition of oxygen evolution, decrease of the S2-state EPR multiline signal and release of Mn2+. In parallel, studies of model systems containing iron(III) protoporphyrin IX chloride (FeIIIPor), which is a good model compound for the Cyt b559 prosthetic group, were performed by using optical and EPR spectroscopy. The interaction of FeIIIPor with imidazole (Im) in weakly polar solvent results in formation of bis-imidazole coordinated heme iron (FeIIIPor Im2) which mimic the bis-histidine axial ligation of Cyt b559. The reaction of DDQ with the LS FeIIIPor Im2 complex leads to its transformation into the HS state (g@?=5.95, g@?=2.00). It was shown that the spin conversion occurs due to the donor-acceptor interaction of coordinated imidazole with this high-potential quinone causing the displacement of imidazole from the axial position. The similar mechanism of DDQ-induced spin change is assumed to be valid for the native membrane Cyt b559 in PSII centers

Stepwise charge separation from a ruthenium-tyrosine complex to a nanocrystalline TiO2 film

A supramolecular complex composed of Ru(II) tris-bipyridine, tyrosine, and dipicolylamine was synthesized and characterized. This complex was attached to TiO2 nanocrystalline films via ester groups at the Ru(II) chromophore, and photoinduced multistep electron transfer was investigated by laser flash photolysis and electron paramagnetic resonance techniques. Following ultrafast electron injection from the metal-ligand charge transfer excited states of Ru(II) to the conduction band of TiO2, fast intramolecular electron transfer from the tyrosine moiety to the photogenerated Ru(III) was observed, characterized by a rate constant of similar to2 x 10(6) s(-1). By comparison of recovery kinetics at the isosbestic point with that of the reference compound lacking the tyrosine, it was found that the intramolecular electron-transfer efficiency is 90%. A hydrogen-bond-promoted electron-transfer mechanism is proposed