Protein dynamics play a key role in enzyme-catalyzed reactions. Vibrational spectroscopy provides a method to follow these structural changes and thereby describe the reaction coordinate as a function of space and time. A vibrational spectroscopic technique, reaction-induced FTIR spectroscopy, has been applied to the study of the oxygen-evolving complex (OEC) of photosystem II (PSII). In plant photosynthesis, PSII evolves oxygen from the substrate, water, by the accumulation of photo-oxidizing equivalents at the OEC. Molecular oxygen and protons are the products of this reaction, which is responsible for the maintenance of an aerobic atmosphere on earth. The OEC is a Mn4CaO5 cluster with nearby bound chloride ions. Sequentially oxidized states of the OEC are termed the S states. The dark-stable state is S1, and oxygen is released on the transition from S3 to S0. Using short laser flashes, individual S states are generated, allowing vibrational spectroscopy to be used to study these different oxidation states of the OEC. In current X-ray crystal structures, hydrogen bonds to water molecules are predicted to form an extensive network around the Mn4CaO5 cluster. In the OEC, four peptide carbonyl groups are linked to the water network, which extends to two Mn-bound and two Ca-bound water molecules. This dissertation discusses a vibrational spectroscopic method that uses these peptide carbonyl frequencies as reporters of solvatochromic changes in the OEC. This technique provides a new, high-resolution method with which to study water and protein dynamics in PSII and other enzymes.

Biomimetic peptides are autonomously folding secondary structural units designed to serve as models for examining processes that occur in proteins. Although de novo biomimetic peptides are not simply abbreviated versions of proteins already found in nature, designing biomimetic peptides does require an understanding of how native proteins are formed and stabilized. The discovery of autonomously folding fragments of ribonuclease A and tendamistat pioneered the use of biomimetic peptides for determining how the polypeptide sequence stabilizes formation of alpha helices and beta hairpins in aqueous and organic solutions. A set of rules for constructing stable alpha helices have now been established. There is no exact set of rules for designing beta hairpins; however, some factors that must be considered are the identity of the residues in the turn and non-covalent interactions between amino acid side chains. For example, glycine, proline, aspargine, and aspartic acid are favored in turns. Non-covalent interactions that stabilize hairpin formation include salt bridges, pi-stacked aromatic interactions, cation-pi interactions, and hydrophobic interactions. The optimal strand length for beta hairpins depends on the numbers of stabilizing non-covalent interactions and high hairpin propensity amino acids in the specific peptide being designed. Until now, de novo hairpins have not previously been used to examine biological processes aside from protein folding. This thesis uses de novo designed biomimetic peptides as tractable models to examine how non-covalent interactions control the redox properties of tyrosine in enzymes. The data in this study demonstrate that proton transfer to histidine, a hydrogen bond to arginine, and a pi-cation interaction create a peptide environment that lowers the midpoint potential of tyrosine in beta hairpins. Moreover, these interactions contribute equally to control the midpoint potential. The data also show that hydrogen bonding is not the sole determinant of the midpoint potential of tyrosine. Finally, the data suggest that the Tyr 160D2-Arg 272CP47 pi-cation interaction contributes to the differences in redox properties between Tyr 160 and Tyr 161 of photosystem II.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

This thesis deals with the impact of hydrogen bonding on the properties of phenols. The possibility for tyrosine to form hydrogen bonds to other amino acids has been found to be important for its function as an electron transfer mediator in a number of important redox enzymes. This study has focused on modeling the function of tyrosine in Photosystem II, a crucial enzyme in the photosynthetic pathway of green plants. Hydrogen bonds between phenol and amines in both inter- and intramolecular systems have been studied with quantum chemical calculations and also in some solid-state structures involving phenol and imidazole. Different phenols linked to amines have been synthesized and their possibilities of forming intra- and intermolecular hydrogen bonds have been studied as well as the thermodynamics and kinetics of the generation of phenoxyl radicals via oxidation reactions. Since carboxylates may in principle act as hydrogen bond acceptors in a manner similar to imidazole, proton coupled electron transfer has also been studied for a few phenols intramolecularly hydrogen bonded to carboxylates with the aim to elucidate the mechanism for oxidation. Electron transfer in a new linked phenol—ruthenium(II)trisbipyridine complex was studied as well. The knowledge is important for the ultimate goal of the project, which is to transform solar energy into a fuel by an artificial mimic of the natural photosynthetic apparatus

abstract: This thesis focuses on serial crystallography studies with X-ray free electron lasers (XFEL) with a special emphasis on data analysis to investigate important processes in bioenergy conversion and medicinal applications. First, the work on photosynthesis focuses on time-resolved femtosecond crystallography studies of Photosystem II (PSII). The structural-dynamic studies of the water splitting reaction centering on PSII is a current hot topic of interest in the field, the goal of which is to capture snapshots of the structural changes during the Kok cycle. This thesis presents results from time-resolved serial femtosecond (fs) crystallography experiments (TR-SFX) where data sets are collected at room temperature from a stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an XFEL with the goal to obtain structural information from the transient state (S4) state of the cycle where the O=O bond is formed, and oxygen is released. The most current techniques available in SFX/TR-SFX to handle hundreds of millions of raw diffraction patterns are discussed, including selection of the best diffraction patterns, allowing for their indexing and further data processing. The results include two 4.0 Å resolution structures of the ground S1 state and triple excited S4 transient state. Second, this thesis reports on the first international XFEL user experiments in South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this new XFEL in a proof-of-principle experiment for the study of microcrystals of human taspase1 (an important cancer target) by SFX has been tested. The descriptions of experiments and discussions of specific data evaluation challenges of this project in light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å, are included in this report In summary, this thesis examines current techniques that are available in the SFX/TR-SFX domain to study crystal structures from microcrystals damage-free, with the future potential of making movies of biological processes. / Dissertation/Thesis / Masters Thesis Chemistry 2020

abstract: Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually. Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis. A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Role of PsbO isoforms in Arabidopsis thaliana Abstract Photosystem II (PSII) uses sunlight to catalyze water oxidation and reduce plastoquinone. Water oxidation takes place in oxygen evolving complex (OEC). OEC is stabilized by extrinsic subunits of PSII. The largest and most important of them is PsbO, manganese-stabilizing protein which can be found in all known oxygenic photosynthetic organisms. Model plant Arabidopsis thaliana expresses two isoforms of psbO gene, namely PsbO1and PsbO2.Mutants psbo1 and psbo2 lacking PsbO1 and PsbO2, respectively, recently brought new findings on the particular roles of isoforms in maintaining photosynthesis. PsbO1 is commonly considered as the main isoform facilitating water splitting, whereas PsbO2 is believed to be involved in PSII repair process (replacement of photodamaged D1 subunit). This work focuses on particular roles of Arabidopsis PsbO isoforms in maintaining photosynthesis with special focus on response to light stress. Mutants psbo1, psbo2 and wild type plants Col-0 were used for extensive biochemical investigation. Our aim was to find out what is the impact on overall thylakoid structure and composition in mutants. Furthermore, to investigate response to light stress in wild type regarding to yields of particular subcompartments, changes in photosystem II...

No description available.

Numerous pigment protein complexes exist in natural systems to harvest light energy such as photosystem II and Nanosalina xenorhodopsin. However, the mechanisms of these lipid embedded photo-active complexes have yet to be fully understood. Photosystem II is of interest due to being a compact complex which can perform the three initial key steps of photosynthesis: absorb light, transfer the excitation from the antennae to reaction center, and perform efficient charge separation. Despite considerable theoretical and experimental effort the exact mechanism of this process remains uncertain. Nanosalina xenorhodopsin is a more recently discovered inwards proton pump with minimal studies into the inwards proton pumping mechanism. Nanosalina xenorhodopsin is of interest as it contrasts with other known and well studied rhodopsins which serve as outwards proton pumps, moving H+ ions out of a cell. In this work, we use the Hamiltonian ensemble method to construct the first fully ab initio computational models of these systems which will be used to determine the mechanisms of these systems. To construct these models we first investigated the effect of the modeled surrounding membrane and simulated temperature. The effect of the extended modeled environment on calculated results is often overlooked but important for the construction of an accurate ab initio model. Our models showed that both membrane composition and temperature result in significant changes in the behavior of the extended membrane system, relative excitation energies of chromophores, and energy dynamics of a pigment protein complex. The absolute excitation energies of chromophores, absorption spectra, and linear dichroism spectra were comparatively insensitive to changes in the modeled environment. With the effect of the environment established, we present a preliminary method to extend our photosystem II model to include charge transfer states, and a preliminary model of Nanosalina xenorhodopsin which can determine the photocycle states through validation of calculated spectra against experimental results.

Die pflanzliche Light-harvesting complex-Proteinfamilie besteht aus Proteinen mit vielfältigen Funktionen. Dabei ist die Funktion der Light-harvesting-like 3-Proteine (LIL3) sowie der One-helix-Proteine (OHPs) weitestgehend unbekannt. Im Rahmen dieser Arbeit wurde gezeigt, dass LIL3 nicht nur mit der Geranylgeranyl-Reduktase (CHLP), sondern auch mit der Protochlorophyllid-Oxidoreduktase (POR) interagiert. Sowohl CHLP als auch POR werden über die Interaktion zu LIL3 an die Thylakoidmembran gebunden und dadurch stabilisiert. Beide Enzyme liefern die direkten Vorstufen für den von der Chlorophyll-Synthase (CHLG) katalysierten finalen Chlorophyll-Syntheseschritt. Neben der Bestätigung der bereits früher gezeigten Chlorophyllbindung von LIL3 konnte eine Affinität zu den späten Intermediaten der Chlorophyllbiosynthese Proto IX, MgP, MgPMME und Pchlid nachgewiesen werden. Die größte Affinität bestand dabei gegenüber dem Substrat von POR, Pchlid. Basierend auf diesen Erkenntnissen wird LIL3 als Regulator der späten Chlorophyllbiosynthese-Schritte vorgeschlagen: LIL3 transportiert Substrate zwischen den Enzymen und ermöglicht durch die Bindung von CHLP und POR die Synthese der Chlorophyll-Edukte in räumlicher Nähe. Dadurch wird die Versorgung von CHLG mit dessen Edukten favorisiert. Beide OHP-Varianten (OHP1/2) bilden ausschließlich Heterodimere und binden Chlorophyll sowie Carotinoide im Verhältnis 3:1. Die Pigmentbindung basiert auf den konservierten Aminosäuren im Chlorophyllbindemotiv. An das OHP1-OHP2-Dimer bindet der PSII-Assemblierungsfaktor HCF244 und wird dadurch an der Membran verankert. HCF244 stabilisiert das OHP-Heterodimer und beide OHPs stabilisieren sich gegenseitig. Der heterotrimere OHP1-OHP2-HCF244-Komplex ist für die D1-Synthese wesentlich. Es wird vermutet, dass die OHPs an der co-translationalen Beladung von (p)D1 mit Pigmenten beteiligt sind sowie frühe Assemblierungsintermediate von PSII vor überschüssiger Anregungsenergie schützen. / The plant light-harvesting complex protein family comprises different members with a variety of functions. However, the function of the light-harvesting-like 3 proteins (LIL3) as well as the one-helix proteins (OHPs) is largely unknown. In this thesis, an interaction of LIL3 not only with geranylgeranyl-reductase (CHLP), but also with protochlorophyllide-oxidoreductase (POR) could be established. LIL3 tethers CHLP and POR to the thylakoid membrane, thereby conferring stability to both enzymes. Both CHLP and POR are synthesizing the direct chlorophyll precursors which are combined to chlorophyll by the subsequent chlorophyll synthase (CHLG). In addition to the chlorophyll binding ability of LIL3 reported earlier, an affinity of LIL3 towards the chlorophyll biosynthesis intermediates Proto IX, MgP, MgPMME, and Pchlide could be shown. Interestingly, the highest affinity of LIL3 was exerted towards Pchlide which is the substrate of POR. Therefore, LIL3 is postulated to shuffle the intermediates between enzymes and brings CHLP and POR in close proximity, which may help to supply CHLG with its substrates. Regarding the function of the OHPs an exclusive heterodimer formation of both the OHP1 and OHP2 variants could be shown. The OHP1-OHP2-heterodimer is able to bind chlorophyll and carotenoids in an approximate 3:1 ratio and pigment binding depends on dimer formation as well as the presence of the conserved amino acids in the chlorophyll binding motif. The PSII-assembly factor HCF244 is anchored to the thylakoid membrane by binding to both OHPs, thereby stabilizing the OHP-heterodimer. The heterotrimeric OHP1-OHP2-HCF244-complex is essential for D1 biosynthesis, although the exact molecular function of HCF244 is still unknown. It is suggested that the OHP-dimer is responsible for co-translational loading of (p)D1 with pigments as well as photoprotection of early PSII assembly intermediates.