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Oxygen evolution catalysed by calcium manganese and manganese-only oxides was studied in O-18-enriched water. Using membrane-inlet mass spectrometry, we monitored the formation of the different O-2 isotopologues O-16(2), (OO)-O-16-O-18 and O-18(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 (H2O2), hydrogen persulfate (HSO5-) 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 moxido manganese or mu-oxido calcium manganese sites. The studied processes show very similar O-18-labelling behaviour to the natural enzymes and are therefore interesting model systems for in vivo oxygen formation by manganese metalloenzymes such as PSII.

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 fo

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 Mn4OxCa 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 Si 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)OxCa 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.

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 Si state transition probabilities and/or the redox-potentials of the water oxidizing complex (WOC) in its different Si 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.