Liquid-Phase Measurements of Photosynthetic Oxygen Evolution.

This chapter compares two different techniques for monitoring photosynthetic O production: the widespread Clark-type O 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 evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Electrocatalytic Water Oxidation by MnO /C: In Situ Catalyst Formation, Carbon Substrate Variations, and Direct O /CO Monitoring by Membrane-Inlet Mass Spectrometry.

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 ) followed by sintering at 100-400 °C. During electrochemical measurements in neutral aqueous buffer, nearly all of the MnO /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 was deposited on graphitic carbon materials (faradaic O yields >90 %).

Evolution of the Z-scheme of photosynthesis: a perspective.

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.

Structure of photosystem II and substrate binding at room temperature.

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 MnCaO cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S to S), in which S is the dark-stable state and S is the last semi-stable state before O-O bond formation and O evolution.

Biogenesis of water splitting by photosystem II during de-etiolation of barley (Hordeum vulgare L.).

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.

Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

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.

Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii.

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.

Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation.

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.

Studying the oxidation of water to molecular oxygen in photosynthetic and artificial systems by time-resolved membrane-inlet mass spectrometry.

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.

Efficiency of photosynthetic water oxidation at ambient and depleted levels of inorganic carbon.

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.

Photosystem II and the unique role of bicarbonate: a historical perspective.

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.

Probing the turnover efficiency of photosystem II membrane fragments with different electron acceptors.

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.

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana.

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.

Calcium manganese oxides as oxygen evolution catalysts: O2 formation pathways indicated by 18O-labelling studies.

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+) .

Membrane-inlet mass spectrometry reveals a high driving force for oxygen production by photosystem II.

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.

Adventures with cyanobacteria: a personal perspective.

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.

Effects of methanol on the Si-state transitions in photosynthetic water-splitting.

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%.

Hydrogencarbonate is not a tightly bound constituent of the water-oxidizing complex in photosystem II.

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.

Interactions of photosystem II with bicarbonate, formate and acetate.

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.

Characterization of the water oxidizing complex of photosystem II of the Chl d-containing cyanobacterium Acaryochloris marina via its reactivity towards endogenous electron donors and acceptors.

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).

Effect of bicarbonate on the water-oxidizing complex of photosystem II in the super-reduced S-states.

It is shown that the hydrazine-induced transition of the water-oxidizing complex (WOC) to super-reduced S-states depends on the presence of bicarbonate in the medium so that after a 20 min treatment of isolated spinach thylakoids with 3 mM NH(2)NH(2) at 20 degrees C in the CO(2)/HCO(3)(-)-depleted buffer the S-state populations are: 42% of S(-3), 42% of S(-2), 16% of S(-1) and even formal S(-4) state is reached, while in the presence of 2 mM NaHCO(3), the same treatment produces 30% of S(-3), 38% of S(-2), and 32% of S(-1) and there is no indication of the S(-4) state. Bicarbonate requirement for the oxygen-evolving activity, very low in untreated thylakoids, considerably increases upon the transition of the WOC to the super-reduced S-states, and the requirement becomes low again when the WOC returns back to the normal S-states using pre-illumination. The results are discussed as a possible indication of ligation of bicarbonate to manganese ions within the WOC.