The S1 to S2 and S2 to S3 state transitions in plant photosystem II: relevance to the functional and structural heterogeneity of the water oxidizing complex.

In Photosystem II, light-induced water splitting occurs via the S state cycle of the CaMn4O5-cluster. To understand the role of various possible conformations of the CaMn4O5-cluster in this process, the temperature dependence of the S1 → S2 and S2 → S3 state transitions, induced by saturating laser flashes, was studied in spinach photosystem II membrane preparations under different conditions. The S1 → S2 transition temperature dependence was shown to be much dependent on the type of the cryoprotectant and presence of 3.5% methanol, resulting in the variation of transition half-inhibition temperature by 50 K. No similar effect was observed for the S2 → S3 state transition, for which we also show that both the low spin g = 2.0 multiline and high spin g = 4.1 EPR configurations of the S2 state advance with similar efficiency to the S3 state, both showing a transition half-inhibition temperature of 240 K. This was further confirmed by following the appearance of the Split S3 EPR signal. The results are discussed in relevance to the functional and structural heterogeneity of the water oxidizing complex intermediates in photosystem II.

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center.

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77-295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of Cytochrome b559 and ChlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730-750 nm) spectral regions. Temperature dependence of the S2 state signal induction after single flash at 730-750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the ChlD1 molecule.

Molecular basis for turnover inefficiencies (misses) during water oxidation in photosystem II.

Photosynthesis stores solar light as chemical energy and efficiency of this process is highly important. The electrons required for CO2 reduction are extracted from water in a reaction driven by light-induced charge separations in the Photosystem II reaction center and catalyzed by the CaMn4O5-cluster. This cyclic process involves five redox intermediates known as the S0-S4 states. In this study, we quantify the flash-induced turnover efficiency of each S state by electron paramagnetic resonance spectroscopy. Measurements were performed in photosystem II membrane preparations from spinach in the presence of an exogenous electron acceptor at selected temperatures between -10 °C and +20 °C and at flash frequencies of 1.25, 5 and 10 Hz. The results show that at optimal conditions the turnover efficiencies are limited by reactions occurring in the water oxidizing complex, allowing the extraction of their S state dependence and correlating low efficiencies to structural changes and chemical events during the reaction cycle. At temperatures 10 °C and below, the highest efficiency (i.e. lowest miss parameter) was found for the S1 → S2 transition, while the S2 → S3 transition was least efficient (highest miss parameter) over the whole temperature range. These electron paramagnetic resonance results were confirmed by measurements of flash-induced oxygen release patterns in thylakoid membranes and are explained on the basis of S state dependent structural changes at the CaMn4O5-cluster that were determined recently by femtosecond X-ray crystallography. Thereby, possible "molecular errors" connected to the e - transfer, H+ transfer, H2O binding and O2 release are identified.

Luminescence and reactivity of a charge-transfer excited iron complex with nanosecond lifetime.

Iron's abundance and rich coordination chemistry are potentially appealing features for photochemical applications. However, the photoexcitable charge-transfer states of most iron complexes are limited by picosecond or subpicosecond deactivation through low-lying metal-centered states, resulting in inefficient electron-transfer reactivity and complete lack of photoluminescence. In this study, we show that octahedral coordination of iron(III) by two mono-anionic facial tris-carbene ligands can markedly suppress such deactivation. The resulting complex [Fe(phtmeimb)2]+, where phtmeimb is {phenyl[tris(3-methylimidazol-1-ylidene)]borate}-, exhibits strong, visible, room temperature photoluminescence with a 2.0-nanosecond lifetime and 2% quantum yield via spin-allowed transition from a doublet ligand-to-metal charge-transfer (2LMCT) state to the doublet ground state. Reductive and oxidative electron-transfer reactions were observed for the 2LMCT state of [Fe(phtmeimb)2]+ in bimolecular quenching studies with methylviologen and diphenylamine.

Kα X-ray Emission Spectroscopy on the Photosynthetic Oxygen-Evolving Complex Supports Manganese Oxidation and Water Binding in the S3 State.

The unique manganese-calcium catalyst in photosystem II (PSII) is the natural paragon for efficient light-driven water oxidation to yield O2. The oxygen-evolving complex (OEC) in the dark-stable state (S1) comprises a Mn4CaO4 core with five metal-bound water species. Binding and modification of the water molecules that are substrates of the water-oxidation reaction is mechanistically crucial but controversially debated. Two recent crystal structures of the OEC in its highest oxidation state (S3) show either a vacant Mn coordination site or a bound peroxide species. For purified PSII at room temperature, we collected Mn Kα X-ray emission spectra of the S0, S1, S2, and S3 intermediates in the OEC cycle, which were analyzed by comparison to synthetic Mn compounds, spectral simulations, and OEC models from density functional theory. Our results contrast both crystallographic structures. They indicate Mn oxidation in three S-transitions and suggest additional water binding at a previously open Mn coordination site. These findings exclude Mn reduction and render peroxide formation in S3 unlikely.

The wavelength of the incident light determines the primary charge separation pathway in Photosystem II.

Charge separation is a key component of the reactions cascade of photosynthesis, by which solar energy is converted to chemical energy. From this photochemical reaction, two radicals of opposite charge are formed, a highly reducing anion and a highly oxidising cation. We have previously proposed that the cation after far-red light excitation is located on a component different from PD1, which is the location of the primary electron hole after visible light excitation. Here, we attempt to provide further insight into the location of the primary charge separation upon far-red light excitation of PS II, using the EPR signal of the spin polarized 3P680 as a probe. We demonstrate that, under far-red light illumination, the spin polarized 3P680 is not formed, despite the primary charge separation still occurring at these conditions. We propose that this is because under far-red light excitation, the primary electron hole is localized on ChlD1, rather than on PD1. The fact that identical samples have demonstrated charge separation upon both far-red and visible light excitation supports our hypothesis that two pathways for primary charge separation exist in parallel in PS II reaction centres. These pathways are excited and activated dependent of the wavelength applied.

A low-spin Fe(iii) complex with 100-ps ligand-to-metal charge transfer photoluminescence.

Transition-metal complexes are used as photosensitizers, in light-emitting diodes, for biosensing and in photocatalysis. A key feature in these applications is excitation from the ground state to a charge-transfer state; the long charge-transfer-state lifetimes typical for complexes of ruthenium and other precious metals are often essential to ensure high performance. There is much interest in replacing these scarce elements with Earth-abundant metals, with iron and copper being particularly attractive owing to their low cost and non-toxicity. But despite the exploration of innovative molecular designs, it remains a formidable scientific challenge to access Earth-abundant transition-metal complexes with long-lived charge-transfer excited states. No known iron complexes are considered photoluminescent at room temperature, and their rapid excited-state deactivation precludes their use as photosensitizers. Here we present the iron complex [Fe(btz)3]3+ (where btz is 3,3'-dimethyl-1,1'-bis(p-tolyl)-4,4'-bis(1,2,3-triazol-5-ylidene)), and show that the superior σ-donor and π-acceptor electron properties of the ligand stabilize the excited state sufficiently to realize a long charge-transfer lifetime of 100 picoseconds (ps) and room-temperature photoluminescence. This species is a low-spin Fe(iii) d5 complex, and emission occurs from a long-lived doublet ligand-to-metal charge-transfer (2LMCT) state that is rarely seen for transition-metal complexes. The absence of intersystem crossing, which often gives rise to large excited-state energy losses in transition-metal complexes, enables the observation of spin-allowed emission directly to the ground state and could be exploited as an increased driving force in photochemical reactions on surfaces. These findings suggest that appropriate design strategies can deliver new iron-based materials for use as light emitters and photosensitizers.

Tyrozine D oxidation and redox equilibrium in photosystem II.

Tyrosine D (TyrD) is an auxiliary redox active tyrosine residue in photosystem II (PSII). The mechanism of TyrD oxidation was investigated by EPR spectroscopy, flash-induced fluorescence decay and thermoluminescence measurements in PSII enriched membranes from spinach. PSII membranes were chemically treated with 3mM ascorbate and 1mM diaminodurene and subsequent washing, leading to the complete reduction of TyrD. TyrD oxidation kinetics and competing recombination reactions were measured after a single saturating flash in the absence and presence of DCMU (inhibitor of the QB-site) in the pH range of 4.7-8.5. Two kinetic phases of TyrD oxidation were observed by the time resolved EPR spectroscopy - the fast phase (msec-sec time range) and the pH dependent slow phase (tens of seconds time range). In the presence of DCMU, TyrD oxidation kinetics was monophasic in the entire pH range, i.e. only the fast kinetics was observed. The results obtained from the fluorescence and thermoluminescence analysis show that when forward electron transport is blocked in the presence of DCMU, the S2QA- recombination outcompetes the slow phase of TyrD oxidation by the S2 state. Modelling of the whole complex of these electron transfer events associated with TyrD oxidation fitted very well with our experimental data. Based on these data, structural information and theoretical considerations we confirm our assignment of the fast and slow oxidation kinetics to two populations of PSII centers with different water positions (proximal and distal) in the TyrD vicinity.

The protonation state around TyrD/TyrD• in photosystem II is reflected in its biphasic oxidation kinetics.

The tyrosine residue D2-Tyr160 (TyrD) in photosystem II (PSII) can be oxidized through charge equilibrium with the oxygen evolving complex in PSII. The kinetics of the electron transfer from TyrD has been followed using time-resolved EPR spectroscopy after triggering the oxidation of pre-reduced TyrD by a short laser flash. After its oxidation TyrD is observed as a neutral radical (TyrD•) indicating that the oxidation is coupled to a deprotonation event. The redox state of TyrD was reported to be determined by the two water positions identified in the crystal structure of PSII [Saito et al. (2013) Proc. Natl. Acad. Sci. USA 110, 7690]. To assess the mechanism of the proton coupled electron transfer of TyrD the oxidation kinetics has been followed in the presence of deuterated buffers, thereby resolving the kinetic isotope effect (KIE) of TyrD oxidation at different H/D concentrations. Two kinetic phases of TyrD oxidation - the fast phase (msec-sec time range) and the slow phase (tens of seconds time range) were resolved as was previously reported [Vass and Styring (1991) Biochemistry 30, 830]. In the presence of deuterated buffers the kinetics was significantly slower compared to normal buffers. Furthermore, although the kinetics were faster at both high pH and pD values the observed KIE was found to be similar (~2.4) over the whole pL range investigated. We assign the fast and slow oxidation phases to two populations of PSII centers with different water positions, proximal and distal respectively, and discuss possible deprotonation events in the vicinity of TyrD.

Homogeneous Cobalt/Vanadium Complexes as Precursors for Functionalized Mixed Oxides in Visible-Light-Driven Water Oxidation.

The heterometallic complexes (NH4 )2 [Co(H2 O)6 ]2 [V10 O28 ]⋅4 H2 O (1) and (NH4 )2 [Co(H2 O)5 (ß-HAla)]2 [V10 O28 ]⋅4 H2 O (2) have been synthesized and used for the preparation of mixed oxides as catalysts for water oxidation. Thermal decomposition of 1 and 2 at relatively low temperatures (<500 °C) leads to the formation of the solid mixed oxides CoV2 O6 /V2 O5 (3) and Co2 V2 O7 /V2 O5 (4). The complexes (1, 2) and heterogeneous materials (3, 4) act as catalysts for photoinduced water oxidation. A modification of the thermal decomposition procedure allowed the deposition of mixed metal oxides (MMO) on a mesoporous TiO2 film. The electrodes containing Co/V MMOs in TiO2 films were used for electrocatalytic water oxidation and showed good stability and sustained anodic currents of about 5 mA cm-2 at 1.72 V versus relative hydrogen electrode (RHE). This method of functionalizing TiO2 films with MMOs at relatively low temperatures (<500 °C) can be used to produce other oxides with different functionality for applications in, for example, artificial photosynthesis.

Room-Temperature Energy-Sampling Kß X-ray Emission Spectroscopy of the Mn4Ca Complex of Photosynthesis Reveals Three Manganese-Centered Oxidation Steps and Suggests a Coordination Change Prior to O2 Formation.

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kß1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kß1,3 line energies suggest a high-valence configuration of the Mn4Ca complex with Mn(III)3Mn(IV) in S0, Mn(III)2Mn(IV)2 in S1, Mn(III)Mn(IV)3 in S2, and Mn(IV)4 in S3 and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S4 state unlikely. The difference spectra of both manganese Kß1,3 emission and K-edge X-ray absorption display different shapes for Mn(III) oxidation in the S2 → S3 transition when compared to Mn(III) oxidation in the S1 → S2 transition. Comparison to spectra of manganese compounds with known structures and oxidation states and varying metal coordination environments suggests a change in the manganese ligand environment in the S2 → S3 transition, which could be oxidation of five-coordinated Mn(III) to six-coordinated Mn(IV). Conceivable options for the rearrangement of (substrate) water species and metal-ligand bonding patterns at the Mn4Ca complex in the S2 → S3 transition are discussed.

Challenges facing an understanding of the nature of low-energy excited states in photosynthesis.

While the majority of the photochemical states and pathways related to the biological capture of solar energy are now well understood and provide paradigms for artificial device design, additional low-energy states have been discovered in many systems with obscure origins and significance. However, as low-energy states are naively expected to be critical to function, these observations pose important challenges. A review of known properties of low energy states covering eight photochemical systems, and options for their interpretation, are presented. A concerted experimental and theoretical research strategy is suggested and outlined, this being aimed at providing a fully comprehensive understanding.

Photodamage of iron-sulphur clusters in photosystem I induces non-photochemical energy dissipation.

Photosystem I (PSI) uses light energy and electrons supplied by photosystem II (PSII) to reduce NADP(+) to NADPH. PSI is very tolerant of excess light but extremely sensitive to excess electrons from PSII. It has been assumed that PSI is protected from photoinhibition by strict control of the intersystem electron transfer chain (ETC). Here we demonstrate that the iron-sulphur (FeS) clusters of PSI are more sensitive to high light stress than previously anticipated, but PSI with damaged FeS clusters still functions as a non-photochemical photoprotective energy quencher (PSI-NPQ). Upon photoinhibition of PSI, the highly reduced ETC further triggers thylakoid phosphorylation-based mechanisms that increase energy flow towards PSI. It is concluded that the sensitivity of FeS clusters provides an additional photoprotective mechanism that is able to downregulate PSII, based on PSI quenching and protein phosphorylation.

Iron sensitizer converts light to electrons with 92% yield.

Solar energy conversion in photovoltaics or photocatalysis involves light harvesting, or sensitization, of a semiconductor or catalyst as a first step. Rare elements are frequently used for this purpose, but they are obviously not ideal for large-scale implementation. Great efforts have been made to replace the widely used ruthenium with more abundant analogues like iron, but without much success due to the very short-lived excited states of the resulting iron complexes. Here, we describe the development of an iron-nitrogen-heterocyclic-carbene sensitizer with an excited-state lifetime that is nearly a thousand-fold longer than that of traditional iron polypyridyl complexes. By the use of electron paramagnetic resonance, transient absorption spectroscopy, transient terahertz spectroscopy and quantum chemical calculations, we show that the iron complex generates photoelectrons in the conduction band of titanium dioxide with a quantum yield of 92% from the (3)MLCT (metal-to-ligand charge transfer) state. These results open up possibilities to develop solar energy-converting materials based on abundant elements.

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis.

Spectroscopic evidence for a redox-controlled proton gate at tyrosine D in Photosystem II.

Tyrosine D (TyrD) is one of two well-studied redox active tyrosines in Photosystem II. TyrD shows redox kinetics much slower than that of its homologue, TyrZ, and is normally present as a stable deprotonated radical (TyrD(•)). We have used time-resolved continuous wave electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy to show that deuterium exchangeable protons can access TyrD on a time scale that is much faster (50-100 times) than that previously observed. The time of H/D exchange is strongly dependent on the redox state of TyrD. This finding can be related to a change in position of a water molecule close to TyrD.

Defining the far-red limit of photosystem I: the primary charge separation is functional to 840 nm.

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P700, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P700 (+) formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391-2401). The same far-red limit for the P700 (+) formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers FA and FB by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P700 similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.

The photochemistry in Photosystem II at 5 K is different in visible and far-red light.

We have earlier shown that all electron transfer reactions in Photosystem II are operational up to 800 nm at room temperature [Thapper, A., et al. (2009) Plant Cell 21, 2391-2401]. This led us to suggest an alternative charge separation pathway for far-red excitation. Here we extend these studies to a very low temperature (5 K). Illumination of Photosystem II (PS II) with visible light at 5 K is known to result in oxidation of almost similar amounts of YZ and the Cyt b559/ChlZ/CarD2 pathway. This is reproduced here using laser flashes at 532 nm, and we find the partition ratio between the two pathways to be 1:0.8 at 5 K [the partition ratio is here defined as (yield of YZ/CaMn4 oxidation):(yield of Cyt b559/ChlZ/CarD2 oxidation)]. The result using far-red laser flashes is very different. We find partition ratios of 1.8 at 730 nm, 2.7 at 740 nm, and >2.7 at 750 nm. No photochemistry involving these pathways is observed above 750 nm at this temperature. Thus, far-red illumination preferentially oxidizes YZ, while the Cyt b559/ChlZ/CarD2 pathway is hardly touched. We propose that the difference in the partition ratio between visible and far-red light at 5 K reflects the formation of a different first stable charge pair. In visible light, the first stable charge pair is considered to be PD1+QA-. In contrast, we propose that the electron hole is residing on the ChlD1 molecule after illumination by far-red light at 5 K, resulting in the first stable charge pair being ChlD1+QA-. ChlD1 is much closer to YZ (11.3 Å) than to any component in the Cyt b559/ChlZ/CarD2 pathway (shortest ChlD1-CarD2 distance of 28.8 Å). This would then explain that far-red illumination preferentially drives efficient electron transfer from YZ. We also discuss mechanisms for accounting for the absorption of the far-red light and the existence of hitherto unobserved charge transfer states. The involvement of two or more of the porphyrin molecules in the core of the Photosystem II reaction center is proposed.

A tandem mass spectrometric method for singlet oxygen measurement.

Singlet oxygen, a harmful reactive oxygen species, can be quantified with the substance 2,2,6,6-tetramethylpiperidine (TEMP) that reacts with singlet oxygen, forming a stable nitroxyl radical (TEMPO). TEMPO has earlier been quantified with electron paramagnetic resonance (EPR) spectroscopy. In this study, we designed an ultra-high-performance liquid chromatographic-tandem mass spectrometric (UHPLC-ESI-MS/MS) quantification method for TEMPO and showed that the method based on multiple reaction monitoring (MRM) can be used for the measurements of singlet oxygen from both nonbiological and biological samples. Results obtained with both UHPLC-ESI-MS/MS and EPR methods suggest that plant thylakoid membranes produce 3.7 × 10(-7) molecules of singlet oxygen per chlorophyll molecule in a second when illuminated with the photosynthetic photon flux density of 2000 µmol m(-2 ) s(-1).

Water oxidation by manganese oxides formed from tetranuclear precursor complexes: the influence of phosphate on structure and activity.

Two types of manganese oxides have been prepared by hydrolysis of tetranuclear Mn(iii) complexes in the presence or absence of phosphate ions. The oxides have been characterized structurally using X-ray absorption spectroscopy and functionally by O2 evolution measurements. The structures of the oxides prepared in the absence of phosphate are dominated by di-µ-oxo bridged manganese ions that form layers with limited long-range order, consisting of edge-sharing MnO6 octahedra. The average manganese oxidation state is +3.5. The structure of these oxides is closely related to other manganese oxides reported as water oxidation catalysts. They show high oxygen evolution activity in a light-driven system containing [Ru(bpy)3](2+) and S2O8(2-) at pH 7. In contrast, the oxides formed by hydrolysis in the presence of phosphate ions contain almost no di-µ-oxo bridged manganese ions. Instead the phosphate groups are acting as bridges between the manganese ions. The average oxidation state of manganese ions is +3. This type of oxide has much lower water oxidation activity in the light-driven system. Correlations between different structural motifs and the function as a water oxidation catalyst are discussed and the lower activity in the phosphate containing oxide is linked to the absence of protonable di-µ-oxo bridges.