| 1. |
- de Lichtenberg, Casper, 1989-
(författare)
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Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II : water here, water there, water everywhere
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Nilsson, Håkan, 1970-
(författare)
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Substrate water binding to the oxygen-evolving complex in photosystem II
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxygenic photosynthesis in plants, algae and cyanobacteria converts sunlight into chemical energy. In this process electrons are transferred from water molecules to CO2 leading to the assembly of carbohydrates, the building blocks of life. A cluster of four manganese ions and one calcium ion, linked together by five oxygen bridges, constitutes the catalyst for water oxidation in photosystem II (Mn4CaO5 cluster). This cluster stores up to four oxidizing equivalents (S0,..,S4 states), which are then used in a concerted reaction to convert two substrate water molecules into molecular oxygen. The reaction mechanism of this four-electron four-proton reaction is not settled yet and several hypotheses have been put forward. The work presented in this thesis aims at clarifying several aspects of the water oxidation reaction by analyzing the mode of substrate water binding to the Mn4CaO5 cluster.Time-resolved membrane-inlet mass spectrometric detection of flash-induced O2 production after fast H218O labelling was employed to study the exchange rates between substrate waters bound to the Mn4CaO5 cluster and the surrounding bulk water. By employing this approach to dimeric photosystem II core complexes of the red alga Cyanidoschyzon merolae it was demonstrated that both substrate water molecules are already bound in the S2 state of the Mn4CaO5 cluster. This was confirmed with samples from the thermophilic cyanobacterium Thermosynechococcus elongatus. Addition of the water analogue ammonia, that is shown to bind to the Mn4CaO5 cluster by replacing the crystallographic water W1, did not significantly affect the exchange rates of the two substrate waters. Thus, these experiments exclude that W1 is a substrate water molecule.The mechanism of O-O bond formation was studied by characterizing the substrate exchange in the S3YZ● state. For this the half-life time of this transient state into S0 was extended from 1.1 ms to 45 ms by replacing the native cofactors Ca2+ and Cl- by Sr2+ and I-. The data show that both substrate waters exchange significantly slower in the S3YZ● state than in the S3 state. A detailed discussion of this finding lead to the conclusions that (i) the calcium ion in the Mn4CaO5 cluster is not a substrate binding site and (ii) O-O bond formation occurs via the direct coupling between two Mn-bound water-derived oxygens, which were assigned to be the terminal water/hydroxy ligand W2 and the central oxo-bridging O5.The driving force for the O2 producing S4→S0 transition was studied by comparing the effects of N2 and O2 pressures of about 20 bar on the flash-induced O2 production of photosystem II samples containing either the native cofactors Ca2+ and Cl- or the surrogates Sr2+ and Br-. While for the Ca/Cl-PSII samples no product inhibition was observed, a kinetic limitation of O2 production was found for the Sr/Br-PSII samples under O2 pressure. This was tentatively assigned to a significant slowdown of the O2 release in the Sr/Br-PSII samples. In addition, the equilibrium between the S0 state and the early intermediates of the S4 state family was studied under 18O2 atmosphere in photosystem II centers devoid of tyrosine YD. Water-exchange in the transiently formed early S4 states would have led to 16,18O2 release, but none was observed during a three day incubation time. Both experiments thus indicate that the S4→S0 transition has a large driving force. Thus, photosynthesis is not limited by the O2 partial pressure in the atmosphere.
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| 3. |
- Tomás Graça, André, 1994-
(författare)
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Light’EM up! : structural characterization of light-driven membrane protein complexes by cryogenic electron microscopy
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is probably the most important process for allowing life to develop into the diverse forms we see today. In this process, solar radiation is used to convert CO2 into biomass. From this process, we obtain oxygen to breathe, sources of food (plant biomass), and the potential for clean and sustainable energy. Photosystem II (PSII) – a key enzyme in photosynthesis –, is a protein complex located in the thylakoid membrane of photosynthetic organisms. PSII and its light-harvesting antennae capture light energy, driving a charge separation process, which leads to the extraction of electrons from water molecules, forming and releasing molecular oxygen. A PSII dimer is composed of more than 20 unique proteins and hundreds of cofactors which fine-tune the mechanisms of light-harvesting and water oxidation, and stabilize the whole complex. While the arrangement of most (but not all!) of these proteins and cofactors is known, their dynamics and individual contributions are not yet fully understood.In my thesis work, I took on the challenge of resolving the structure of large protein complexes, such as PSII complexes from various photosynthetic organisms, using a technique called cryogenic electron microscopy (cryo-EM). This PhD dissertation focuses on structurally describing these macromolecular assemblies and how their components (protein, cofactors, and substrate) interact with each other or with their immediate cellular environment.Among the several outcomes of my research on PSII, I would like to highlight the following findings: 1) the usage of digitonin as a detergent to solubilize PSII destroys the catalytic activity and changes LHCII pigment content, among other consequences; 2) PSII does not seem to incorporate chlorophyll (Chl) a molecules with a farnesyl tail, and the Chl tails’ flexibility justifies not resolving the full-length of some of these molecules in PSII structures. We concluded that flexibility may be an advantage to PSII function; 3) cryo-EM is a technique with the potential to reveal information about electron/proton transfer processes within PSII, and provided us with data, for instance, to suggest a pathway for the protonation of QB, the final electron acceptor in PSII.In another project, also using cryo-EM, I studied the structure of the S-layer Deinoxanthin Binding Complex (SDBC), a membrane protein complex from Deinococcus radiodurans. This complex is an essential part of the cell envelope, the outermost barrier of this bacterium, and it is known to bind a carotenoid called deinoxanthin, which has significant spectroscopic and antioxidant properties. Additionally, we studied the function of this complex and showed that the SDBC is a quencher of UVC-UVB radiation and reactive oxygen species, with superoxide dismutase activity. This complex has an α-β coiled-coil stalk long enough to reach the inner membrane of the cell envelope.In summary, visualizing the structural organization and chemistry within these complexes allowed us to gain a new understanding of their function and diversity. Furthermore, this work demonstrates the potential of cryo-EM as a method to render complementary information at resolution superior to state-of-the-art X-ray diffraction methods.
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| 4. |
- Kawde, Anurag Y., 1984-
(författare)
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Advanced silicon photoelectrodes for water splitting devices : design, preparation and functional characterization by photo-electrochemistry and high-energy X-ray spectroscopy
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- For the last century, mankind has been hugely dependent on fossil fuels to meet its energy needs. Harnessing energy from fossil fuels led to the emission of greenhouse gases. Greenhouse gases such as CO2 are a major contributor to global warming. Since the last decade, the global annual average temperature has increased by almost 1 oC, while the annual average temperature of Europe has increased by almost 1.7 oC. It is high time to find an alternative source of energy. Such an energy source must be renewable, sustainable, robust and free of greenhouse gases. Our earth has a non-stop supply of solar energy and water in oceans, harvesting energies from such resources will not only be clean but also inexpensive. Solar fuels such as H2 generated from sunlight and seawater using earth-abundant materials are expected to be a crucial component of a next generation renewable energy mix.My PhD research was thus focused on the use of solar energy to split water into molecular hydrogen and oxygen, a process that is referred to as ‘artificial photosynthesis’. This can be achieved with the help of semiconductor photocatalysts. As most of the earth crust has a high abundance of silicon (Si), I prepared my semiconductor photoelectrodes using Si. However, Si tends to degrade in an aqueous environment. Thus, my PhD research comprises the synthesis of microstructured Si photoelectrodes and their protection with a TiO2 inter layer followed by functionalization with various earth abundant co-catalysts. The study on the synthesis, morphology and elemental characterization of the photoelectrodes was carried out under the supervision of Prof. Dr. Johannes Messinger at the Chemistry Department of Umeå University. Deep insight on the electronic and atomic structure of the functionalized Si photoelectrodes was obtained by careful experiments at the European Synchrotron Radiation Facility (ESRF) under the supervision of Dr. Pieter Glatzel. I investigated the electronic and geometric structural properties of my photocatalysts using inner shell electron spectroscopy, which is also referred to as ‘X-ray spectroscopy’. Thus, my PhD thesis falls under the broad title of “Artificial Photosynthesis and X-Ray Spectroscopy”. With the motivation of developing a bias free photoelectrochemical device for overall water splitting, I first developed cost effective earth abundant photocathodes. The experimental data and detailed analysis of the photocathodes are presented in Paper I. The best photocathode obtained in Paper I (p-Si/TiO2/NiOx) was then coupled with a well-studied FTO/α-Fe2O3 photoanode in parallel-illumination mode. The two most significant information obtained in Paper II were: 1) p-Si/TiO2/NiOx outcompetes Pt as a counter electrode and 2) a space charge region in the pristine hematite can be enhanced using p-Si/TiO2/NiOx as photocathode without bias or using any dopant. The proof of concept device studied in Paper II was further optimized in Paper III by replacing the FTO substrate with the n-Si MW to a obtain n-Si MW/TiO2/α-Fe2O3 photoanode. A record high photocurrent density of 5.6 mA/cm2 was achieved for the undoped hematite photoanode. I also found out that the TiO2 inter layer plays a crucial role in enhancing the overall device performance. The role of TiO2 was thus further studied using valence to core X-ray emission spectroscopy, which opened a new avenue for identifying and investigating the prime components in such devices. Paper I to III discuss the role of TiO2 and of the co-catalysts towards solar water splitting and thus the only material left to study was the Si substrate. For paper IV, a detailed analysis on Si substrate was performed. The electronic structural changes on Si LII, III edge was studied using X-ray Raman spectroscopy. The X-ray spectroscopic studies presented in papers I to III were performed at the ID-26 beamline at ESRF, while the X-ray Raman Spectroscopy presented in Paper IV was performed at the ID-20 beamline at ESRF. The data presented in Paper IV is preliminary and needs to be processed and analyzed further.
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| 5. |
- Koroidov, Sergey, 1982-
(författare)
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Water splitting in natural and artificial photosynthetic systems
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is the unique biological process that converts carbon dioxide into organic compounds, for example sugars, using the energy of sunlight. Thereby solar energy is converted into chemical energy. Nearly all life depends on this reaction, either directly, or indirectly as the ultimate source of their food. Oxygenic photosynthesis occurs in plants, algae and cyanobacteria. This process created the present level of oxygen in the atmosphere, which allowed the formation of higher life, since respiration allows extracting up to 15-times more energy from organic matter than anaerobic fermentation. Oxygenic photosynthesis uses as substrate for the ubiquitous water. The light-induced oxidation of water to molecular oxygen (O2), catalyzed by the Mn4CaO5 cluster associated with the photosystem II (PS II) complex, is thus one of the most important and wide spread chemical processes occurring in the biosphere. Understanding the mechanism of water-oxidation by the Mn4CaO5 cluster is one of today’s great challenges in science. It is believed that one can extract basic principles of catalyst design from the natural system that than can be applied to artificial systems. Such systems can be used in the future for the generation of fuel from sunlight.In this thesis the light-induced production of molecular oxygen and carbon dioxide (CO2) by PSII was observed by membrane-inlet mass spectrometry. By analyzing this observation is shown that CO2 not only is the substrate in photosynthesis for the production of sugars, but that it also regulates the efficiency of the initial steps of the electron transport chain of oxygenic photosynthesis by acting, in form of HCO3-, as acceptor for protons produced during water-splitting. This finding concludes the 50-years old search for the function of CO2/HCO3− in photosynthetic water oxidation.For understanding the mechanism of water oxidation it is crucial to resolve the structures of all oxidation states, including transient once, of the Mn4CaO5 cluster. With this application in mind a new illumination setup was developed and characterized that allowed to bring the Mn4CaO5 cluster of PSII microcrystals into known oxidation states while they flow through a narrow capillary. The optimized illumination conditions were employed at the X-ray free electron laser at the Linac Coherent Light Source (LCLS) to obtain simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) at room temperature. This two methods probe the overall protein structure and the electronic structure of the Mn4CaO5 cluster, respectively. Data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. This approach opens new directions for studying structural changes during the catalytic cycle of the Mn4CaO5 cluster, and for resolving the mechanism of O-O bond formation.In two other projects the mechanism of molecular oxygen formation by artificial water oxidation catalysts containing inexpensive and abundant elements were studied. Oxygen evolution catalyzed by calcium manganese and manganese only oxides was studied in 18O-enriched water. It was concluded that molecular oxygen is formed by entirely different pathways depending on what chemical oxidant was used. Only strong non-oxygen donating oxidants were found to support ‘true’ water-oxidation. For cobalt oxides a study was designed to understand the mechanistic details of how the O-O bond forms. The data demonstrate that O-O bond formation occurs by direct coupling between two terminal water-derived ligands. Moreover, by detailed theoretical modelling of the data the number of cobalt atoms per catalytic site was derived.
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| 6. |
- Pham, Long Vo, 1988-
(författare)
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Oxidation and reduction reactions of the water-oxidizing complex in photosystem II
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The oxygen that we breathe and food that we eat are products of the natural photosynthesis. Molecular oxygen is crucial for life on Earth owing to its role in the glycolysis and citric acid pathways that yield in aerobic organisms the energy-rich ATP molecules. Photosynthetic water oxidation, which produces molecular oxygen from water and sunlight, is performed by higher plants, algae and cyanobacteria. Within the molecular structure of a plant cell, photosynthesis is performed by a specific intracellular organelle – the chloroplast. Chloroplasts contain a membrane system, the thylakoid membrane, which comprises lipids, quinones and a very high content of protein complexes. The unique photosynthetic oxidation of water into molecular oxygen, protons and electrons is performed by the Mn4CaO5 cluster in photosystem II (PSII) complex. Understanding the mechanism of water oxidation by Mn4CaO5 cluster is one of the great challenges in science nowadays. When the mechanism of this process is fully understood, artificial photosynthetic systems can be designed that have high efficiencies of solar energy conversion by imitating the fundamental principle of natural system. These systems can be used in future for generation of fuels from sunlight. In this thesis, the efficiency of water-splitting process in natural photosynthetic preparations was studied by measuring the flash-induced oxygen evolution pattern (FIOP). The overall aim is to achieve a deeper understanding of oxygen evolving mechanism of the Mn4O5Ca cluster via developing a complete kinetic and energetic model of the light-induced redox reactions within PSII complex. On the way to reach this goal, the hydrogen peroxide that is electrochemically generated on surface of Pt-cathode was discovered. The chemical effect of electrochemically produced H2O2 that can interfere in the oxygen evolution pathway or change the observed FIOP data was demonstrated. Therefore, in order to record the clean FIOP data that are further characterized by global fitting program (GFP), H2O2 has to be abolished by catalase addition and by purging the flow buffer of the Joliot-type electrode with nitrogen gas. After FIOPs free of H2O2-induced effects were achieved, these clean data were then applied to a global fitting approach (GFP) in order to (i) result a comprehensive figure of all S-state decays whose kinetic rates were simultaneously analyzed in a high reliability and consistency, (ii) the dependence of miss parameter on S-state transitions and the oxidation state of tyrosine D (YD) can be tested, (iii) how dependent of all S-state re-combinations (to S1 state) on the various pH/pD values can be also determined in case of using Cyanidioschyzon merolae (C. merolae) thylakoids. Our data support previous suggestions that the S0 → S1 and S1 → S2 transitions involve low or no misses, while high misses occur in the S2 → S3 transition or the S3 → S0 transition. Moreover, the appearance of very slow S2 decay was clearly observed by using the GFP analysis, while there are no evidences of very slow S3 decay were recorded under all circumstances. The unknown electron donor for the very slow S2 decay which can be one of the substances of PSII-protective branch (i.e. cytochrome b559, carotenoid or ChlZ) will be determined in further researches.
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| 7. |
- Yeşilbaş, Merve, 1987-
(författare)
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Thin water and ice films on minerals : a molecular level study
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Minerals in Earth’s crust and suspended in the atmosphere form water or ice films as thin as a few nanometers to as thick as a few micrometers, and beyond. Mineral-bound water and ice films in terrestrial systems (e.g. vadose zones, permafrosts) can impact the bio(geo)chemistry of nutrients and contaminants, water cycling, as well as possible land-air exchanges in terrestrial environments. In the atmosphere, films are tied to clouds and rain formation, and can influence the absorption and scattering of solar radiation of dust mineral aerosols. Water films are, at the same time, of interest to technology. They are even of interest in the study of asteroids, comets, and planet Mars. Still, their formation on the various types of minerals common to the environment is misunderstood.The aim of this thesis is to gain fundamental insight on the roles that minerals play on forming and stabilising thin water and ice films. This work is separated in two parts, with Part A associated with Papers I-II, and Part B with Papers III-V of the appendix of this thesis.In Part A of this work (Papers I-II), water loadings and vibrational signatures of thin water films were collected on 21 different minerals (metal oxides, silicates, carbonates) relevant to terrestrial environments, atmosphere and perhaps outer-space. Measurements were made on minerals of varied (i) composition, (ii) structure, (iii) morphology, (iv) particle size and (v) surface roughness. Loadings, measured by a microgravimetric Dynamic Vapour Sorption technique, were of a few monolayers in sub-micrometer-sized particles but of several hundreds to thousands of water layers in micrometer-sized particles (Paper I). This was seen in the Fourier Transform Infrared (FTIR) spectra of sub-micrometer-sized particles with different hydrogen bonding environments than liquid water. Micrometer-sized particles formed liquid-like films regardless of the mineral. Similar observations were made in the spectra of the thinnest water films remaining on these minerals after long periods of sublimation of ice overcoatings at sub-freezing temperatures (Paper II).In Part B of this work (Papers III-V), focus on the expandable clay mineral montmorillonite was made to study (i) intercalated water, (ii) ice and cryosalt formation inside microporous gels, and (iii) its interactions with intercalated CO2. FTIR extracted spectral components reflecting interlayer hydration states of ~0W, 1W and 2W monolayers of water (Paper III). Thermal dehydration/dehydroxylation experiments showed that the driest forms of montmorillonite strongly retained low levels of crystalline water in its structure. FTIR also showed that frozen wet gels of montmorillonite form ice and the cryosalt mineral hydrohalite. Ice was seen in rigid gels and aggregated compact particles, as well as low particle density with low salt content. In contrast, concentrated (>> 10 g/L) saline gels host hydrohalite, probably between and/or near aggregated clay particle walls. Field-Emission Cryogenic Scanning Electron Microscopy showed that ice microcrystals form in micropores of the gels (Paper IV). Finally, release rates of CO2 trapped in interlayers of montmorillonite, monitored by FTIR spectroscopy, were larger in the presence of 1-2W. The activation energy of CO2 release from~0W montmorillonite (34 kJ/mol) is comparable to other mineral surfaces. This study highlights that the most stabilised CO2 occur in of dry and cold conditions.This thesis will hopefully serve as a springboard for further work exploring the chemistry and physics of water and ice films at minerals surfaces. It should contribute to improve our understanding of the geochemistry of Earth’s soils, processes in the atmosphere, and even of space chemistry.
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| 8. |
- D'Amario, Luca, et al.
(författare)
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Towards time resolved characterization of electrochemical reactions : electrochemically-induced Raman spectroscopy
- 2022
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Ingår i: Chemical Science. - : RSC Publishing. - 2041-6520 .- 2041-6539. ; 13:36, s. 10734-10742
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Tidskriftsartikel (refereegranskat)abstract
- Structural characterization of transient electrochemical species in the sub-millisecond time scale is the all-time wish of any electrochemist. Presently, common time resolution of structural spectro-electrochemical methods is about 0.1 seconds. Herein, a transient spectro-electrochemical Raman setup of easy implementation is described which allows sub-ms time resolution. The technique studies electrochemical processes by initiating the reaction with an electric potential (or current) pulse and analyses the product with a synchronized laser pulse of the modified Raman spectrometer. The approach was validated by studying a known redox driven isomerization of a Ru-based molecular switch grafted, as monolayer, on a SERS active Au microelectrode. Density-functional-theory calculations confirmed the spectral assignments to sub-ms transient species. This study paves the way to a new generation of time-resolved spectro-electrochemical techniques which will be of fundamental help in the development of next generation electrolizers, fuel cells and batteries.
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