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During photosynthetic water oxidation, the Mn4Ca cluster in Photosystem II progresses through five intermediate S-i (i = 0-4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S-3 state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S-3 state or in the subsequent S-3 to S-0 transition. Here we report conventional high-resolution data for the S-1, S-2, and S-3 states to a resolution of similar to 1.9 & Aring;, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, O-X (or O6), in the S-3 state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The O-X-O5 distance is similar to 2.1 & Aring;, supporting no strong interaction between them in the S-3 state, suggesting that if this is the O-O bond formation site, it is formed during the S-3 to S-0 transition initiated by the final oxidation of the cluster.

Spiro-OMeTAD is a widely used hole transport material in perovskite solar cells, contributing significantly to their high-power conversion efficiencies. In this study, Electron Paramagnetic Resonance (EPR) spectroscopy was employed to investigate F4TCNQ as a molecular dopant for Spiro-OMeTAD. The doping efficiency of F4TCNQ was examined by EPR spectroscopy by varying its concentration from 0.5 to 6 mol% in two different solvents: chloroform and chlorobenzene. Spiro-OMeTAD films prepared at these dopant concentrations in chloroform were additionally characterized with UV/VIS spectroscopic and ellipsometry measurements. EPR analysis of both solutions and films revealed that F4TCNQ doping is more effective in chloroform than in chlorobenzene, indicating a strong solvent influence on the doping efficiency of spiro-OMeTAD by F4TCNQ. Furthermore, an ambient air stability study was performed on F4TCNQ-doped spiro-OMeTAD films and compared with films containing conventional additives such as tBP, LiTFSI, and FK209. The results demonstrate that F4TCNQ serves as an efficient single dopant alternative to traditional additive mixtures. Results are discussed in the context of EPR spectroscopy as a powerful tool for identifying effective dopants for hole transport material thin films and elucidating the role of solvent-dopant interactions in determining doping efficiency.

Radical chemistry, once deemed too reactive for biological systems, is delicately controlled within metalloenzymes to catalyze challenging chemical transformations under physiological conditions. This review explores the diverse strategies employed by metalloenzymes to generate, stabilize, and utilize highly reactive radical intermediates. We discuss amino acid–based radicals (tyrosyl, tryptophan, cysteinyl, glycyl, and DOPA), radicals derived from molecular oxygen (in heme, manganese, copper, and nonheme iron enzymes), and cofactor-based radicals (specifically adenosylcobalamin and radical S-adenosylmethionine enzymes). We devote special attention to ribonucleotide reductases as a prime example of evolutionary convergence of radical mechanisms. The interplay among redox-active cofactors, metal ions, and protein scaffolds highlights nature's ingenuity in generating, controlling, and utilizing radicals. We also discuss emerging themes and open questions, emphasizing how advances in structural and spectroscopic techniques continue to deepen our understanding of these complex and vital enzymatic processes.

Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO₂ to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn₄CaO₅ cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated from Thermosynechococcus vestitus (T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn₄CaO₅ cluster during its S₂→S₃ and S₃→S₄→S₀ state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacterium Synechocystis sp. PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitus numbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn₄CaO₅ cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn₄CaO₅ cluster that includes the pentameric, near planar ‘water wheel’ of the O1 channel.

The photosynthetic electron flux from photosystem I (PSI) is mainly directed to NADP+ and CO2 fixation, but a fraction is always shared between alternative and cyclic electron transport. Although the electron transfer from P700 to ferredoxin, via phylloquinone and the FeSX, FeSB and FeSA clusters, is well characterized, the regulatory role of these redox intermediates in the delivery of electrons from PSI to NADP+, alternative and cyclic electron transport under environmental stress remains elusive. Here we provide evidence for sequential damage to PSI FeS clusters under high light and subsequent slow recovery under low light in Arabidopsis thaliana. Wild-type plants showed 10–35% photodamage to their FeSA/B clusters with increasing high-light duration, without much effect on P700 oxidation capacity, FeSX function or CO2 fixation rate, and without additional oxygen consumption (O2 photoreduction). Parallel FeSA/B cluster damage in the pgr5 mutant was more pronounced at 50–85%, probably due to weak photosynthetic control and low non-photochemical quenching. Such severe electron pressure on PSI was also shown to damage the FeSX clusters, with a concomitant decrease in P700 oxidation capacity and a decrease in thylakoid-bound ferredoxin in the pgr5 mutant. The results from wild-type and pgr5 plants reveal controlled damage of PSI FeS clusters under high light. In wild-type plants, this favours electron transport to linear over alternative pathways by intact PSI centres, thereby preventing reactive oxygen species production and probably promoting harmless charge recombination between P700+ and FeSX− as long as the majority of FeSA/B clusters remain functional.

The production of molecular hydrogen (H2) by microalgae holds great promise, and immobilization techniques offer potential for further advancement in this field. The current study focuses on investigating the positive impact of immobilization on maintaining the stability and activity of photosystem II (PSII) over incubation time, with the aim of enhancing H2 production potential in green microalgae Chlamydomonas reinhardtii. For this purpose, immobilized cells within alginate beads were treated with small concentrations of 2,5-dibromo-3methyl-6-isopropyl-p-benzoquinone (DBMIB) inhibitor to induce the partial inhibition of inter-photosystem electron transport, recently reported as a novel approach for sustaining microalgal H2 production. A comparative analysis of fluorescence decay kinetic changes and EPR spectroscopy of the cell beads revealed the superior capacity of immobilization for sustaining PSII stability and activity in batch culture over time. Treatment of the cell beads with 3.5 mu M DBMIB led to sustained H2 production yielding over 200 mu mol H2/mg Chl within 3 weeks, with an average H2 production rate of approximately 10 mu mol/mg Chl per day, both of which were roughly twice as high as those observed in free cells treated with DBMIB. Our findings underscore the significance of integrating immobilization with a proven and effective method for H2 production, thereby enhancing its sustainability and productivity.

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.

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.

Periodic surges of H₂ evolution were observed in the wild-type strain CC-5325 of the green unicellular alga Chlamydomonas reinhardtii in the presence of the electron transport inhibitors dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB, 3.5 μM) and 2,4-dinitrophenylether and iodonitrothymol (DNP-INT, 0.6 μM). Addition of DBMIB partly inhibited the electron transfer from Cytochrome b₆f complex to Photosystem I, over-reduced the plastoquinone pool, gradually inhibited photosystem II and created anoxic conditions in cells. During 30 days of anaerobic incubation, continues H₂ photoproduction with a minimum rate of 1 ml/L of culture per day was accompanied with additional outbursts of H₂ evolution. The first noticeable peak of H₂ evolution was observed on day 6 of incubation, with maximum rate of 23 ml of H₂ per L of culture per day. It was repeated on day 9 and day 22 with the 2 and 4 times lower rates respectively. Addition of DNP-NT showed similar effect by inducing the H₂ photoproduction for 15 days, albeit at much lower rates. Contribution of the direct and indirect pathways to the H₂ production is shown by fluorescence, thermoluminescence and electron paramagnetic resonance spectroscopy. It is proposed that photosynthetic electron transport in combination with photorespiration, chlororespiration and starch accumulation can switch on and off between photosynthetic, H₂ producing and survival modes of cell metabolism. Controlled switching between these modes could potentially maintain the long lasting photosynthetic H₂ production in the wild-type of Chlamydomonas.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn₄CaO₅ cluster first stores four oxidizing equivalents, the S₀ to S₄ intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry^1,2,3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S₃→[S₄]→S₀ transition where O₂ is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn₄CaO₅ cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O_x, which was introduced as a bridging ligand between Ca and Mn1 during the S₂→S₃ transition^4,5,6, disappears or relocates in parallel with Y_z reduction starting at approximately 700 μs after the third flash. The onset of O₂ evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.