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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 - /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 /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.)

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.

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.

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 Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 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 (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-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 S1 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 Mn4CaO5 cluster in the S2 and S3 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.

Abstract: 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 NH2NH2 at 20 °C in the CO2/HCO3 −-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 NaHCO3, 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. [Copyright &y& Elsevier]