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Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of Photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn₄CaO₅ cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S₁) and the first illuminated state (S₂) of PS II. Our simultaneous XRD/XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn₄CaO₅ cluster, opening new directions for future dynamics studies.

Over 40 years ago, Joliot et al. (Photochem Photobiol 10:309-329, 1969) designed and employed an elegant and highly sensitive electrochemical technique capable of measuring O2 evolved by photosystem II (PSII) in response to trains of single turn-over light flashes. The measurement and analysis of flash-induced oxygen evolution patterns (FIOPs) has since proven to be a powerful method for probing the turnover efficiency of PSII. Stemler et al. (Proc Natl Acad Sci USA 71(12):4679-4683, 1974), in Govindjee's lab, were the first to study the effect of "bicarbonate" on FIOPs by adding the competitive inhibitor acetate. Here, we extend this earlier work by performing FIOPs experiments at various, strictly controlled inorganic carbon (Ci) levels without addition of any inhibitors. For this, we placed a Joliot-type bare platinum electrode inside a N2-filled glove-box (containing 10-20 ppm CO2) and reduced the Ci concentration simply by washing the samples in Ci-depleted media. FIOPs of spinach thylakoids were recorded either at 20-times reduced levels of Ci or at ambient Ci conditions (390 ppm CO2). Numerical analysis of the FIOPs within an extended Kok model reveals that under Ci-depleted conditions the miss probability is discernibly larger (by 2-3 %) than at ambient conditions, and that the addition of 5 mM HCO3 (-) to the Ci-depleted thylakoids largely restores the original miss parameter. Since a "mild" Ci-depletion procedure was employed, we discuss our data with respect to a possible function of free or weakly bound HCO3 (-) at the water-splitting side of PSII.

Cyanobacteria, algae and plants oxidize water to the O₂ we breathe, and consume CO₂ during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO₂/HCO₃⁻ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO₃⁻ acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water-oxidation out into the chloroplast's lumen, resulting in a light-driven production of O₂ and CO₂. Depletion of HCO₃⁻ from the media leads, in the absence of added buffers, to a reversible down-regulation of O₂ production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis, and conclude the more than 50-y-long quest for the function of CO₂/ HCO₃⁻ in photosynthetic water oxidation.

Oxygen evolution catalysed by calcium manganese and manganese-only oxides was studied in (18) O-enriched water. Using membrane-inlet mass spectrometry, we monitored the formation of the different O(2) isotopologues (16) O(2) , (16) O(18) O and (18) O(2) in such reactions simultaneously with good time resolution. From the analysis of the data, we conclude that entirely different pathways of dioxygen formation catalysis exist for reactions involving hydrogen peroxide (H(2) O(2) ), hydrogen persulfate (HSO(5) (-) ) or single-electron oxidants such as Ce(IV) and [Ru(III) (bipy)(3) ](3+) . Like the studied oxide catalysts, the active sites of manganese catalase and the oxygen-evolving complex (OEC) of photosystem II (PSII) consist of μ-oxido manganese or μ-oxido calcium manganese sites. The studied processes show very similar (18) O-labelling behaviour to the natural enzymes and are therefore interesting model systems for in vivo oxygen formation by manganese metalloenzymes such as PSII.

Co-oxides are promising water oxidation catalysts for artificial photosynthesis devices. Their catalytic mechanism was studied previously both experimentally and theoretically, but there is presently no agreement whether the O-O bond formation occurs via nucleophilic attack or by direct coupling, and whether or not bridging oxygens participate as substrate during O-O bondformation. Here we present time-resolved ¹⁸O-labelling isotope-ratio membrane-inlet massspectrometry experiments employing the previously introduced Co/methylenediphosphonate (Co/M2P) system in combination with [Ru(bpy)₃]³⁺ (bpy = 2,2’-bipyridine) as chemical oxidant. Our data demonstrate that for Co/M2P-oxide O-O bond formation occurs between two pre-bound, fast exchanging oxygen species, i.e. likely via direct coupling between two terminal water-derived oxygen ligands. Detailed modeling of the dependence of the O₂-isotope ratios on the [Ru(bpy)₃]³⁺ concentration revealed that in the Co/M2P-oxidenanoparticles almost all Co ions are catalytically active (2.35 Co per catalytic site) and that, starting from a ‘resting state‘, 3.5 electrons need to be removed from each catalytic site for thefirst O₂ formation. Since it was previously demonstrated that in the resting state most Co ions are in the oxidation state Co(III), we conclude that the coupling mechanism involves at least one Co(IV)-O^• radical.