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This chapter compares two different techniques for monitoring photosynthetic O 2 production; the wide-spread Clark-type O 2 electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O 2 evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Green organisms evolve oxygen (O 2 ) via photosynthesis and consume it by respiration. Generally, net O 2 consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles display strong O 2 consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring (ES). By employing different electron transport chain inhibitors, we show that this unusual light-induced O 2 consumption occurs around photosystem (PS) I and correlates with higher abundance of flavodiiron (Flv) A protein in ES thylakoids. With P700 absorption changes, we demonstrate that electron scavenging from the acceptor-side of PSI via O 2 photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments., (© 2023. The Author(s).)

Photosynthetic water oxidation by Photosystem II (PSII) is a fascinating process because it sustains life on Earth and serves as a blue print for scalable synthetic catalysts required for renewable energy applications. The biophysical, computational, and structural description of this process, which started more than 50 years ago, has made tremendous progress over the past two decades, with its high-resolution crystal structures being available not only of the dark-stable state of PSII, but of all the semi-stable reaction intermediates and even some transient states. Here, we summarize the current knowledge on PSII with emphasis on the basic principles that govern the conversion of light energy to chemical energy in PSII, as well as on the illustration of the molecular structures that enable these reactions. The important remaining questions regarding the mechanism of biological water oxidation are highlighted, and one possible pathway for this fundamental reaction is described at a molecular level., (© 2023. The Author(s).)

The regulation of photosynthetic machinery with a nonoxidative approach is a powerful but challenging strategy for the selective inhibition of bloom-forming cyanobacteria. Acetylacetone (AA) was recently found to be a target-selective cyanocide for Microcystis aeruginosa , but the cause and effect in the studied system are still unclear. By recording of the chemical fingerprints of the cells at two treatment intervals (12 and 72 h with 0.1 mM AA) with omics assays, the molecular mechanism of AA in inactivating Microcystis aeruginosa was elucidated. The results clearly reveal the effect of AA on ferredoxin and the consequent effects on the physiological and biochemical processes of Microcystis aeruginosa . In addition to its role as an electron acceptor of photosystem I, ferredoxin plays pivotal roles in the assimilation of nitrogen in cyanobacterial cells. The effect of AA on ferredoxin and on nonheme iron of photosystem II first cut off the photosynthetic electron transfer flow and then interrupted the synthesis of adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which ultimately might affect carbon fixation and nitrogen assimilation metabolisms. The results here provide missing pieces in the current knowledge on the selective inhibition of cyanobacteria, which should shed light on the better control of harmful blooms.

Photosynthetic electron transfer comprises a series of light-induced redox reactions catalysed by multiprotein machinery in the thylakoid. These protein complexes possess cofactors susceptible to redox modifications by reactive small molecules. The gaseous radical nitric oxide (NO), a key signalling molecule in green algae and plants, has earlier been shown to bind to Photosystem (PS) II and obstruct electron transfer in plants. The effects of NO on cyanobacterial bioenergetics however, have long remained obscure. In this study, we exposed the model cyanobacterium Synechocystis sp. PCC 6803 to NO under anoxic conditions and followed changes in whole-cell fluorescence and oxidoreduction of P700 in vivo. Our results demonstrate that NO blocks photosynthetic electron transfer in cells by repressing PSII, PSI, and likely the NDH dehydrogenase-like complex 1 (NDH-1). We propose that iron‑sulfur clusters of NDH-1 complex may be affected by NO to such an extent that ferredoxin-derived electron injection to the plastoquinone pool, and thus cyclic electron transfer, may be inhibited. These findings reveal the profound effects of NO on Synechocystis cells and demonstrate the importance of controlled NO homeostasis in cyanobacteria., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Selective inhibition of photosynthesis is a fundamental strategy to solve the global challenge caused by harmful cyanobacterial blooms. However, there is a lack of specificity of the currently used cyanocides, because most of them act on cyanobacteria by generating nontargeted oxidative stress. Here, for the first time, we find that the simplest β-diketone, acetylacetone, is a promising specific cyanocide, which acts on Microcystis aeruginosa through targeted binding on bound iron species in the photosynthetic electron transport chain, rather than by oxidizing the components of the photosynthetic apparatus. The targeted binding approach outperforms the general oxidation mechanism in terms of specificity and eco-safety. Given the essential role of photosynthesis in both natural and artificial systems, this finding not only provides a unique solution for the selective control of cyanobacteria but also sheds new light on the ways to modulate photosynthesis.

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H 2 production. H 2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H 2 ase), the key enzyme responsible for H 2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H 2 ase by O 2 coevolved in photosynthesis. Recently, we achieved sustainable H 2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H 2 ase. Employing membrane-inlet mass spectrometry and [Formula: see text], we now present clear evidence that efficient H 2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H 2 ase downstream of photosystem I. This occurs exclusively in the absence of CO 2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H 2 photoproduction ceases and instead a slow overall H 2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H 2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H 2 yield., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The effect of bicarbonate (HCO 3 - ) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO 3 - enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO 2 and H 2 O. The other is for the electron-acceptor side, in which (i) reduction of the Q A quinone leads to the release of HCO 3 - from its binding site on the non-heme iron and (ii) the E m potential of the Q A /Q A •- couple increases when HCO 3 - dissociates. This suggested a protective/regulatory role of HCO 3 - as it is known that increasing the E m of Q A decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with 13 C isotope labeling of HCO 3 - allows donor- and acceptor-side formation of CO 2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.

Knowledge of the manganese oxidation states of the oxygen-evolving Mn 4 CaO 5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S 2 state of this cluster. This signal implies an overall oxidation state of either Mn(III) 3 Mn(IV) or Mn(III)Mn(IV) 3 for the S 2 state. These 2 competing assignments are commonly known as "low oxidation (LO)" and "high oxidation (HO)" models of the Mn 4 CaO 5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn 4 CaO 5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn 4 CaO 5 cluster's oxidation states., Competing Interests: The authors declare no competing interest.

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water-oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane-inlet mass spectrometry and O 2 -polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these 'PSII birth defects' in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de-etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O 2 -polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, Q B -inhibitor binding, and thermoluminescence studies indicate that the decline of the high-light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability Q A -  → Q B during de-etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer-range energy transfer., (© 2019 Scandinavian Plant Physiology Society.)

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle 1,2 . The model comprises four (meta)stable intermediates (S 0 , S 1 , S 2 and S 3 ) and one transient S 4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn 4 CaO 5 ) cluster in the oxygen-evolving complex 3-7 . This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q B at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S 2 →S 3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S 3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O 2 release. Thus, our results exclude peroxo-bond formation in the S 3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

This chapter compares two different techniques for monitoring photosynthetic O 2 production: the widespread Clark-type O 2 electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell, and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O 2 evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Layers of amorphous manganese oxides were directly formed on the surfaces of different carbon materials by exposing the carbon to aqueous solutions of permanganate (MnO 4 - ) followed by sintering at 100-400 °C. During electrochemical measurements in neutral aqueous buffer, nearly all of the MnO x /C electrodes show significant oxidation currents at potentials relevant for the oxygen evolution reaction (OER). However, by combining electrolysis with product detection by using mass spectrometry, it was found that these currents were only strictly linked to water oxidation if MnO x was deposited on graphitic carbon materials (faradaic O 2 yields >90 %). On the contrary, supports containing sp 3 -C were found to be unsuitable as the OER is accompanied by carbon corrosion to CO 2 . Thus, choosing the "right" carbon material is crucial for the preparation of stable and efficient MnO x /C anodes for water oxidation catalysis. For MnO x on graphitic substrates, current densities of >1 mA cm -2 at η=540 mV could be maintained for at least 16 h of continuous operation at pH 7 (very good values for electrodes containing only abundant elements such as C, O, and Mn) and post-operando measurements proved the integrity of both the catalyst coating and the underlying carbon at OER conditions., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The concept of the Z-scheme of oxygenic photosynthesis is in all the textbooks. However, its evolution is not. We focus here mainly on some of the history of its biophysical aspects. We have arbitrarily divided here the 1941-2016 period into three sub-periods: (a) Origin of the concept of two light reactions: first hinted at, in 1941, by James Franck and Karl Herzfeld; described and explained, in 1945, by Eugene Rabinowitch; and a clear hypothesis, given in 1956 by Rabinowitch, of the then available cytochrome experiments: one light oxidizing it and another reducing it; (b) Experimental discovery of the two light reactions and two pigment systems and the Z-scheme of photosynthesis: Robert Emerson's discovery, in 1957, of enhancement in photosynthesis when two light beams (one in the far-red region, and the other of shorter wavelengths) are given together than when given separately; and the 1960 scheme of Robin Hill & Fay Bendall; and, ((c) Evolution of the many versions of the Z-Scheme: Louis Duysens and Jan Amesz's 1961 experiments on oxidation and reduction of cytochrome f by two different wavelengths of light, followed by the work of many others for more than 50 years.)

Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, i.e. another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn 4 CaO 5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S 0 to S 4 ), in which S 1 is the dark-stable state and S 3 is the last semi-stable state before O-O bond formation and O 2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S 1 ), two-flash illuminated (2F; S 3 -enriched), and ammonia-bound two-flash illuminated (2F-NH 3 ; S 3 -enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S 1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn 4 CaO 5 cluster in the S 2 and S 3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de-etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll-binding protein complexes and development of water splitting via O2 production in plastids (etiochloroplasts) isolated during de-etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light-harvesting antenna [PSII-light-harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de-etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de-etiolation, etiochloroplasts revealed the same water-splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de-etiolation precedes assembly of the PSII-LHCII supercomplexes. Taken together, data show a rapid establishment of water-splitting activity during etioplast-to-chloroplast transition and emphasize that assembly of the functional water-splitting site of PSII is not the rate-limiting step in the formation of photoactive thylakoid membranes., (© 2016 John Wiley & Sons Ltd.)

The two-helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b-binding motif; however, binding of chlorophyll has not been demonstrated. We find that Lil3.2 from Arabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25±7.8 nM) is favored relative to homodimerization (431±59 nM). Interaction of Lil3.2 with chlorophyll a (231±49 nM) suggests that heterodimerization precedes binding of chlorophyll in Arabidopsis thaliana., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 (-) increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3 (-) from CO2. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3 (-) on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C-terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live-cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull-downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high-light exposure. These include not only photosynthetic and stress-related proteins but also RNA-processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress-induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions., (© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

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/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that 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 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/ in photosynthetic water oxidation.

Monitoring isotopic compositions of gaseous products (e.g., H2, O2, and CO2) by time-resolved isotope-ratio membrane-inlet mass spectrometry (TR-IR-MIMS) is widely used for kinetic and functional analyses in photosynthesis research. In particular, in combination with isotopic labeling, TR-MIMS became an essential and powerful research tool for the study of the mechanism of photosynthetic water-oxidation to molecular oxygen catalyzed by the water-oxidizing complex of photosystem II. Moreover, recently, the TR-MIMS and (18)O-labeling approach was successfully applied for testing newly developed catalysts for artificial water-splitting and provided important insight about the mechanism and pathways of O2 formation. In this mini-review we summarize these results and provide a brief introduction into key aspects of the TR-MIMS technique and its perspectives for future studies of the enigmatic water-splitting chemistry.

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.

In photosynthesis, cyanobacteria, algae and plants fix carbon dioxide (CO(2)) into carbohydrates; this is necessary to support life on Earth. Over 50 years ago, Otto Heinrich Warburg discovered a unique stimulatory role of CO(2) in the Hill reaction (i.e., O(2) evolution accompanied by reduction of an artificial electron acceptor), which, obviously, does not include any carbon fixation pathway; Warburg used this discovery to support his idea that O(2) in photosynthesis originates in CO(2). During the 1960s, a large number of researchers attempted to decipher this unique phenomenon, with limited success. In the 1970s, Alan Stemler, in Govindjee's lab, perfected methods to get highly reproducible results, and observed, among other things, that the turnover of Photosystem II (PSII) was stimulated by bicarbonate ions (hydrogen carbonate): the effect would be on the donor or the acceptor, or both sides of PSII. In 1975, Thomas Wydrzynski, also in Govindjee's lab, discovered that there was a definite bicarbonate effect on the electron acceptor (the plastoquinone) side of PSII. The most recent 1.9Å crystal structure of PSII, unequivocally shows HCO(3)(-) bound to the non-heme iron that sits in-between the bound primary quinone electron acceptor, Q(A), and the secondary quinone electron acceptor Q(B). In this review, we focus on the historical development of our understanding of this unique bicarbonate effect on the electron acceptor side of PSII, and its mechanism as obtained by biochemical, biophysical and molecular biological approaches in many laboratories around the World. We suggest an atomic level model in which HCO(3)(-)/CO(3)(2-) plays a key role in the protonation of the reduced Q(B). In addition, we make comments on the role of bicarbonate on the donor side of PSII, as has been extensively studied in the labs of Alan Stemler (USA) and Vyacheslav Klimov (Russia). We end this review by discussing the uniqueness of bicarbonate's role in oxygenic photosynthesis and its role in the evolutionary development of O(2)-evolving PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study we employ isotope ratio membrane-inlet mass spectrometry to probe the turnover efficiency of photosystem II (PSII) membrane fragments isolated from spinach at flash frequencies between 1Hz and 50Hz in the presence of the commonly used exogenous electron acceptors potassium ferricyanide(III) (FeCy), 2,5-dichloro-p-benzoquinone (DCBQ), and 2-phenyl-p-benzoquinone (PPBQ). The data obtained clearly indicate that among the tested acceptors PPBQ is the best at high flash frequencies. If present at high enough concentration, the PSII turnover efficiency is unaffected by flash frequency of up to 30Hz, and at 40Hz and 50Hz only a slight decrease by about 5-7% is observed. In contrast, drastic reductions of the O(2) yields by about 40% and 65% were found at 50Hz for DCBQ and FeCy, respectively. Comparison with literature data reveals that PPBQ accepts electrons from Q(A)(-) in PSII membrane fragments with similar efficiency as plastoquinone in intact cells. Our data also confirm that at high flashing rates O(2) evolution is limited by the reactions on the electron-acceptor side of PSII. The relevance of these data to the evolutionary development of the water-splitting complex in PSII and with regard to the potential of artificial water-splitting catalysts is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cyanobacteria, or the blue-green algae as they used to be called until 1974, are the oldest oxygenic photosynthesizers. We summarize here adventures with them since the early 1960s. This includes studies on light absorption by cyanobacteria, excitation energy transfer at room temperature down to liquid helium temperature, fluorescence (kinetics as well as spectra) and its relationship to photosynthesis, and afterglow (or thermoluminescence) from them. Further, we summarize experiments on their two-light reaction - two-pigment system, as well as the unique role of bicarbonate (hydrogen carbonate) on the electron-acceptor side of their photosystem II, PSII. This review, in addition, includes a discussion on the regulation of changes in phycobilins (mostly in PSII) and chlorophyll a (Chl a; mostly in photosystem I, PSI) under oscillating light, on the relationship of the slow fluorescence increase (the so-called S to M rise, especially in the presence of diuron) in minute time scale with the so-called state-changes, and on the possibility of limited oxygen evolution in mixotrophic PSI (minus) mutants, up to 30 min, in the presence of glucose. We end this review with a brief discussion on the position of cyanobacteria in the evolution of photosynthetic systems.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oxygenic photosynthesis is the basis for aerobic life on earth. The catalytic Mn(4)O(x)CaY(Z) center of photosystem II (PSII), after fourfold oxidation, extracts four electrons from two water molecules to yield dioxygen. This reaction cascade has appeared as a single four-electron transfer that occurs in typically 1 ms. Inevitable redox intermediates have so far escaped detection, probably because of very short lifetime. Previous attempts to stabilize intermediates by high O(2)-back pressure have revealed controversial results. Here we monitored by membrane-inlet mass spectrometry (MIMS) the production of from (18)O-labeled water against a high background of in a suspension of PSII-core complexes. We found neither an inhibition nor an altered pattern of O(2) production by up to 50-fold increased concentration of dissolved O(2). Lack of inhibition is in line with results from previous X-ray absorption and visible-fluorescence experiments, but contradictory to the interpretation of previous UV-absorption data. Because we used essentially identical experimental conditions in MIMS as had been used in the UV work, the contradiction was serious, and we found it was not to be resolved by assuming a significant slowdown of the O(2) release kinetics or a subsequent slow conformational relaxation. This calls for reevaluation of the less direct UV experiments. The direct detection of O(2) release by MIMS shows unequivocally that O(2) release in PSII is highly exothermic. Under the likely assumption that one H(+) is released in the S(4) → S(0) transition, the driving force at pH 6.5 and atmospheric O(2) pressure is at least 220 meV, otherwise 160 meV.

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells., Methodology/principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited., Conclusions/significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.

From a chemical point of view methanol is one of the closest analogues of water. Consistent with this idea EPR spectroscopy studies have shown that methanol binds at-or at least very close to-the Mn(4)O(x)Ca cluster of photosystem II (PSII). In contrast, Clark-type oxygen rate measurements demonstrate that the O(2) evolving activity of PSII is surprisingly unaffected by methanol concentrations of up to 10%. Here we study for the first time in detail the effect of methanol on photosynthetic water-splitting by employing a Joliot-type bare platinum electrode. We demonstrate a linear dependence of the miss parameter for S( i ) state advancement on the methanol concentrations in the range of 0-10% (v/v). This finding is consistent with the idea that methanol binds in PSII with similar affinity as water to one or both substrate binding sites at the Mn(4)O(x)Ca cluster. The possibility is discussed that the two substrate water molecules bind at different stages of the cycle, one during the S(4) --> S(0) and the other during the S(2) --> S(3) transition.

Since the end of the 1950s hydrogencarbonate ('bicarbonate') is discussed as a possible cofactor of photosynthetic water-splitting, and in a recent X-ray crystallography model of photosystem II (PSII) it was displayed as a ligand of the Mn(4)O(x)Ca cluster. Employing membrane-inlet mass spectrometry (MIMS) and isotope labelling we confirm the release of less than one (~0.3) HCO(3)(-) per PSII upon addition of formate. The same amount of HCO(3)(-) release is observed upon formate addition to Mn-depleted PSII samples. This suggests that formate does not replace HCO(3)(-) from the donor side, but only from the non-heme iron at the acceptor side of PSII. The absence of a firmly bound HCO(3)(-) is corroborated by showing that a reductive destruction of the Mn(4)O(x)Ca cluster inside the MIMS cell by NH(2)OH addition does not lead to any CO(2)/HCO(3)(-) release. We note that even after an essentially complete HCO(3)(-)/CO(2) removal from the sample medium by extensive degassing in the MIMS cell the PSII samples retain > or =75% of their initial flash-induced O(2)-evolving capacity. We therefore conclude that HCO(3)(-) has only 'indirect' effects on water-splitting in PSII, possibly by being part of a proton relay network and/or by participating in assembly and stabilization of the water-oxidizing complex.

In this study, we probe the effects of bicarbonate (hydrogencarbonate), BC, removal from photosystem II in spinach thylakoids by measuring flash-induced oxygen evolution patterns (FIOPs) with a Joliot-type electrode. For this we compared three commonly employed methods: (1) washing in BC-free medium, (2) formate addition, and (3) acetate addition. Washing of the samples with buffers depleted of BC and CO2 by bubbling with argon (Method 1) under our conditions leads to an increase in the double hit parameter of the first flash (beta 1), while the miss parameter and the overall activity remain unchanged. In contrast, addition of 40-50 mM formate or acetate results in a significant increase in the miss parameter and to an approximately 50% (formate) and approximately 10% (acetate) inhibition of the overall oxygen evolution activity, but not to an increased beta 1 parameter. All described effects could be reversed by washing with formate/acetate free buffer and/or addition of 2-10 mM bicarbonate. The redox potential of the water-oxidizing complex (WOC) in samples treated by Method 1 is compared to samples containing 2 mM bicarbonate in two ways: (1) The lifetimes of the S0, S2, and S3 states were measured, and no differences were found between the two sample types. (2) The S1, S0, S(-1), and S(-2) states were probed by incubation with small concentrations of NH2OH. These experiments displayed a subtle, yet highly reproducible difference in the apparent Si/S(-i) state distribution which is shown to arise from the interaction of BC with PSII in the already reduced states of the WOC. These data are discussed in detail by also taking into account the CO2 concentrations present in the buffers after argon bubbling and during the measurements. These values were measured by membrane-inlet mass spectrometry (MIMS).

Acaroychloris (A.) marina is a unique oxygen evolving organism that contains a large amount of chlorophyll d (Chl d) and only very few Chl a molecules. This feature raises questions on the nature of the photoactive pigment, which supports light-induced oxidative water splitting in Photosystem II (PS II). In this study, flash-induced oxygen evolution patterns (FIOPs) were measured to address the question whether the S(i) state transition probabilities and/or the redox-potentials of the water oxidizing complex (WOC) in its different S(i) states are altered in A. marina cells compared to that of spinach thylakoids. The analysis of the obtained data within the framework of different versions of the Kok model reveals that in light activated A. marina cells the miss probability is similar compared to spinach thylakoids. This finding indicates that the redox-potentials and kinetics within the WOC, of the reaction center (P680) and of Y(Z) are virtually the same for both organisms. This conclusion is strongly supported by lifetime measurements of the S(2) and S(3) states. Virtually identical time constants were obtained for the slow phase of deactivation. Kinetic differences in the fast phase of S(2) and S(3) decay between A. marina cells and spinach thylakoids reflect a shift of the E(m) of Y(D)/Y(D)(ox) to lower values in the former compared to the latter organisms, as revealed by the observation of an opposite change in the kinetics of S(0) oxidation to S(1) by Y(D)(ox). A slightly increased double hit probability in A. marina cells is indicative of a faster Q(A)(-) to Q(B) electron transfer in these cells compared to spinach thylakoids.