Photoacclimation strategies in northeastern Atlantic seagrasses: Integrating responses across plant organizational levels

Seagrasses live in highly variable light environments and adjust to these variations by expressing acclimatory responses at different plant organizational levels (meadow, shoot, leaf and chloroplast level). Yet, comparative studies, to identify species' strategies, and integration of the relative importance of photoacclimatory adjustments at different levels are still missing. The variation in photoacclimatory responses at the chloroplast and leaf level were studied along individual leaves of Cymodocea nodosa, Zostera marina and Z. noltei, including measurements of variable chlorophyll fluorescence, photosynthesis, photoprotective capacities, non-photochemical quenching and D1-protein repair, and assessments of variation in leaf anatomy and chloroplast distribution. Our results show that the slower-growing C. nodosa expressed rather limited physiological and biochemical adjustments in response to light availability, while both species of faster-growing Zostera showed high variability along the leaves. In contrast, the inverse pattern was found for leaf anatomical adjustments in response to light availability, which were more pronounced in C. nodosa. This integrative plant organizational level approach shows that seagrasses differ in their photoacclimatory strategies and that these are linked to the species' life history strategies, information that will be critical for predicting the responses of seagrasses to disturbances and to accordingly develop adequate management strategies.Fundacao para a Ciencia e Tecnologia (FCT), Portugal [PTDC/MAR-EST/4257/2014

Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II : water here, water there, water everywhere

Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few. The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism. In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle. Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle. To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states. Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5  is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5. In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.ISBN för den tryckta versionen saknas i fulltext och spikblad: 978-91-7855-343-3.</p

Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II : water here, water there, water everywhere

Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few. The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism. In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle. Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle. To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states. Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5  is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5. In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.ISBN för den tryckta versionen saknas i fulltext och spikblad: 978-91-7855-343-3.</p

Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II : water here, water there, water everywhere

Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few. The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism. In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle. Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle. To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states. Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5  is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5. In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.ISBN för den tryckta versionen saknas i fulltext och spikblad: 978-91-7855-343-3.</p

Substrate water exchange in the S2 state of photosystem II is dependent on the conformation of the Mn4Ca cluster

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn4Ca cluster. During catalysis, the OEC cycles through five redox states, S0 to S4. In the S2 state, the Mn4Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H218O/H216O exchange between bulk water and the two substrate waters bound at the Mn4Ca cluster in the SLS2, SHS2, and the S3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10 times faster in the SHS2 than in the SLS2 state, and that the SLS2 → SHS2 conversion has at physiological temperature an activation barrier of 17 ± 1 kcal mol−1. Of the presently suggested SHS2 models, our findings are best in agreement with a water exchange pathway involving a SHS2state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S3 state is governed by a different equilibrium than in S2, and that the exchange of the fast substrate water in the S2 state is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining possibilities for O–O bond formation in PSII are discussed

The exchange of the fast substrate water in the S2 state of photosystem II is limited by diffusion of bulk water through channels - implications for the water oxidation mechanism.

The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the 'slow exchanging substrate water (Ws)', while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the 'fast exchanging substrate water (Wf)'. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H2 16O/H2 18O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution

The exchange of the fast substrate water in the S2 state of photosystem II is limited by diffusion of bulk water through channels - implications for the water oxidation mechanism

The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the 'slow exchanging substrate water (Ws)', while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the 'fast exchanging substrate water (Wf)'. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H2 16O/H2 18O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution

The D1-V185N mutation alters substrate water exchange by stabilizing alternative structures of the Mn4Ca-cluster in photosystem II

In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O2-release and strongly perturb the water network surrounding the Mn4Ca cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the H218O/H216O-exchange kinetics of the fast (Wf) and slow (Ws) exchanging substrate waters bound in the S1, S2 and S3 states to the Mn4Ca cluster of PSII core complexes isolated from wild type and D1-V185N strains of Synechocystis sp. PCC 6803. We found that the rate of exchange for Ws was increased in the S1 and S2 states, while both Wf and Ws exchange rates were decreased in the S3 state. Additionally, we used EPR spectroscopy to characterize the Mn4Ca cluster and its interaction with the redox active D1-Tyr161 (YZ). In the S2 state, we observed a greatly diminished multiline signal in the V185N-PSII that could be recovered by addition of ammonia. The split signal in the S1 state was not affected, while the split signal in the S3 state was absent in the D1-V185N mutant. These findings are rationalized by the proposal that the N185 residue stabilizes the binding of an additional water-derived ligand at the Mn1 site of the Mn4Ca cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.Originally included in thesis in manuscript form.</p

Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation