EPR spectroscopy and isotopic substitution were used to gain increased knowledge about the proton-coupled electron transfer (PCET) mechanism for the reduction of the tyrosine D radical (YD*) in photosystem II. pL dependence (where pL is either pH or pD) of both the rate constant and kinetic isotope effect (KIE) was examined for YD* reduction. Second, the manner in which protons are transferred during the rate-limiting step for YD* reduction at alkaline pL was determined. Finally, high field electron paramagnetic resonance (EPR) spectroscopy was used to study the effect of pH on the environment surrounding both the tyrosine D radical and the tyrosine Z radical (YZ*). At alkaline pL, it was determined that the proton and electron are both transferred in the rate-limiting step of YD* reduction. At acidic pL, the proton transfer occurs first followed by electron transfer. Proton inventory experiments indicate that there is more than one proton donation pathway available to YD* during PCET reduction at alkaline pL. Additionally, the proton inventory experiments indicate that at least one of those pathways is multiproton. High field EPR experiments indicate that both YD* and YZ* are hydrogen bonded to neutral species. The EPR gx component for YD* is invariant with respect to pH. Analysis of the EPR gx component for Yz* indicates that its environment becomes more electropositive as the pH is increased. This is most likely due to changes in the hydrogen bond strength

I have set for a challenging study that combined experimental and theoretical approaches in an attempt to resolve a role of small aminoacids in intermolecular interactions. First, I have proposed a hypothesis that described the interaction among individual aminoacids forming D helices of D1 and D2 proteins based on molecular dynamic simulations of a simplified model representing the reaction centre of photosystem II. Stability of the putative interhelical hydrogen bond network connecting D1 and D2 proteins was investigated experimentally with dynamic force spectroscopy using atomic force microscope. The results of both methods are in a full agreement with each other and reveal the key role of D1-Gly208 aminoacid in stability and functionality of photosystem II by providing milieu for weak interactions among three contact points at the cross of D helices: D1-Gly208 (O) and D2-Cys211 (O?), D1-Ser209 (O?) and D2-Ile204 (O), D1-Ser212 (O?) and D2-Gly207 (O). Mutation of the D1-Gly208 led to the increase in probability of the binding among the aforementioned aminoacids, undesirably strengthening the overall interactions among the proteins compromising photosynthetic capacity (D1-Ser208) or disabling of autotrophic growth (D1-Val208).

Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-10Bitstream added on 2014-06-13T20:47:02Z : No. of bitstreams: 1 graca_jp_me_jabo.pdf: 256015 bytes, checksum: 389bd2988baccfc63d2527a482a1e26f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A cana-de-açúcar (Saccharum spp.) é uma das principais culturas das regiões tropicais, cuja produtividade agrícola pode ser afetada pelo déficit hídrico. Para investigar o processo de tolerância e sensibilidade ao déficit hídrico, diferentes parâmetros fisiológicos foram avaliados em cultivares de cana-de-açúcar tolerantes (SP83-2847 e CTC15) e sensível (SP86-155) ao déficit hídrico. A deficiência hídrica afetou todo o aparato fotossintético das plantas de forma diferenciada dentro e entre as cultivares. A taxa fotossintética e condutância estomática diminuíram significativamente para todas as cultivares submetidas ao estresse. Nas plantas controle das cultivares (cv) tolerantes SP83-2847 e CTC15 observou-se que a taxa fotossintética apresentou valores mais altos em comparação a cultivar sensível SP86-155. Resultados do teor relativo de água mostraram que a cultivar CTC15 apresentou melhor condição hídrica durante o período de déficit hídrico. A eficiência quântica do fotossistema II da cultivar SP83-2847 mostrou maior estabilidade nos últimos dias do tratamento experimental, sugerindo que o decréscimo do teor relativo de água estimulou o ajuste da capacidade fotossintética frente às alterações da disponibilidade hídrica. De modo geral, as cultivares SP83-2847 e CTC15, consideradas tolerantes, sob déficit hídrico exibiram melhor desempenho em relação a cultivar sensível SP86-155. Os dados permitem sugerir que tais parâmetros fisiológicos podem ser empregados na avaliação e distinção de genótipos de cana-de-açúcar tolerantes e sensíveis ao déficit hídrico. / The sugarcane (Saccharum spp.) is one of the main crops cultivated in tropical areas, whose agricultural productivity can be affected by drought. To investigate the tolerance and sensitivity process to water deficit, various physiological parameters were evaluated in sugarcane cultivars considered tolerant (SP83-2847 and CTC15) and sensitive (SP86-155) to drought. The water deficit affected the entire photosynthetic apparatus of all plants in different manners, inside and among cultivars. The photosynthetic rate and stomatal conductance decreased significantly for all cultivars, submitted to water stress. In control plants of the tolerant cultivars SP83-2847 and CTC15, it was observed that the photosynthetic rate showed better values in comparison to sensitive cultivar SP86-155. According to relative water content results of the cultivar CTC15 showed better condition water performance during the drought. The quantum efficiency photosystem II of the cultivar SP83-2847 showed greater stability in recent days of the experimental treatment, suggesting that the decline in the relative water content stimulated the adjustment of photosynthetic capacity to face the changes in water availability. Thus, cultivars SP83-2847 and CTC15, considered tolerant under water deficit, showed better performance in comparison to sensitive cultivar SP86-155. The data suggest that these physiological parameters can be used in the evaluation and distinction of drought tolerant and sensitive sugarcane genotypes.

Resumo: O trabalho objetivou avaliar o efeito do suprimento de potássio no desempenho de clones de eucalipto submetidos a duas condições de disponibilidade hídrica em casa de vegetação utilizando características biométricas e fisiológicas. Cinco genótipos de eucalipto foram submetidos a dois níveis de adubação potássica (K0 - sem complementação potássica e K1 - adição de 166 mg.dm-3 de K) e a dois regimes de irrigação (RI1 - irrigação diária, até o solo atingir 60% dos poros preenchidos com água (PPA), ou seja, plantas sem restrição hídrica, e RI2 - plantas irrigadas até o solo atingir 60% PPA, com posterior suspensão da irrigação, até o aparecimento de sintomas iniciais de deficiência hídrica). Foram conduzidos sete ciclos de suspensão de irrigação, sendo a quantidade de água reposta em cada vaso determinada pelo método gravimétrico. No início e ao final do experimento, foram avaliados o diâmetro do coleto, a altura, o número de folhas, a área foliar e a massa de matéria seca de folhas, caule, raízes, da parte aérea e total. Nos momentos de máximo estresse hídrico, foram avaliadas as seguintes características fisiológicas: teor relativo de clorofila total (ICC), medida da eficiência quântica do fotossistema II (Fv/Fm), taxa de assimilação líquida (A), condutância estomática (gs), taxa de transpiração (E), eficiência intrínseca do uso da água (EUAintr=A/E), eficiência instantânea do uso da água (EUAinst=A/gs), conteúdo relativo de água (CRA) e, ao final do experimento foi determinado o potencial hídrico foliar ( f). O experimento foi estabelecido no delineamento de blocos ao acaso, em esquema fatorial 5 x 2 x 2 (5 genótipos, 2 regimes de irrigação e 2 níveis de adubação potássica), com cinco repetições por tratamento. As médias de tratamentos foram comparadas pelo teste de Tukey a 5% de probabilidade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study aimed to evaluate the supply potassium effect on the performance of the five eucalypt clones subjected to two water availability conditions in greenhouse from biometric and physiological traits. Five eucalypt genotypes were subjected to two levels of potassium supply (K0 - no potassium supplementation and K1 - addition of 166 mg dm-3 K) and two irrigation regimes (RI1- daily irrigation until the soil reaches 60% of pores filled with water (PFW), that is plants no water restriction, and RI2 - plants watered until the soil reaches 60% (PFW), with subsequent water suspension until the appearance of early water stress symptoms). Seven cycles of irrigation suspension were conducted, and the amount water replaced in each pot was determined by gravimetric method. At the beginning and the end of the experiment, were measured the collar diameter, height, leaf number, leaf area and dry weight mass of leaves, stems, roots, aerial parts and total. In the moment of maximum water stress were evaluated the following physiological traits: the chlorophyll content index (CCI), the quantum efficiency of photosystem II (Fv/Fm), net assimilation rate (A), stomatal conductance (gs), transpiration (E), intrinsic water use efficiency (WUEintr = A/E) and instantaneous water use efficiency (WUEinst = A/gs), relative water content (RWC) and at the end of the experiment was determined the leaf water potential ( f). The design experiment was established in the randomized blocks in factorial scheme 5 x 2 x 2 (5 genotypes, two irrigation regimes and two levels of potassium supplies), with five replicates. The treatment means were compared by Tukey test at 5% probability. Eucalypt clones present reduction in the biometric and physiological traits values under water limitations. For the physiological traits, the potassium supply caused effect reduction of the water stress. To the biometric... (Complete abstract click electronic access below) / Orientador: Rinaldo Cesar de Paula / Coorientadora: Nádia Figueiredo de Paula / Banca: Mara Cristina Pessôa da Cruz / Banca: Miguel Luiz Menezes Freitas / Mestre

The overall structure of photosynthetic pigment-protein complexes and thylakoid membranes of various photosynthetic organisms was studied using electron microscopy.

I have set for a challenging study that combined experimental and theoretical approaches in an attempt to resolve a role of small aminoacids in intermolecular interactions. First, I have proposed a hypothesis that described the interaction among individual aminoacids forming D helices of D1 and D2 proteins based on molecular dynamic simulations of a simplified model representing the reaction centre of photosystem II. Stability of the putative interhelical hydrogen bond network connecting D1 and D2 proteins was investigated experimentally with dynamic force spectroscopy using atomic force microscope. The results of both methods are in a full agreement with each other and reveal the key role of D1-Gly208 aminoacid in stability and functionality of photosystem II by providing milieu for weak interactions among three contact points at the cross of D helices: D1-Gly208 (O) and D2-Cys211 (O?), D1-Ser209 (O?) and D2-Ile204 (O), D1-Ser212 (O?) and D2-Gly207 (O). Mutation of the D1-Gly208 led to the increase in probability of the binding among the aforementioned aminoacids, undesirably strengthening the overall interactions among the proteins compromising photosynthetic capacity (D1-Ser208) or disabling of autotrophic growth (D1-Val208).

La photosynthèse permet de convertir l’énergie solaire en énergie chimique. Ce processus met en jeu un grand nombre de protéines et complexes protéiques. Le premier complexe de la chaîne photosynthétique est le photosystème II où a lieu l’oxydation de l’eau. Le PSII est composé des protéines D1 et D2. Chez la cyanobactérie thermophile Thermosynechococcus elongatus, il y a trois gènes qui codent trois protéines D1 différentes. La première partie de la thèse décrit le développement d’outils protéomiques basés sur les gels d’électrophorèse 2D pour étudier le protéome des trois différents variants, qui expriment chacun une seule protéine pour D1. Peu de différences ont été trouvées. Toutefois, un seul des variants exprime Tll0287. La deuxième partie de la thèse décrit la caractérisation de Tll0287 avec différents techniques : spectroscopies d’absorption UV-visible ou de résonance Raman et spectro-électrochimie. Tll0287 a été identifié comme un cytochrome de type c, mais il présente beaucoup de caractéristiques inattendues. Les spectres d’absorption UV-visible et de résonance Raman de Tll0287 réduit montrent une dépendance vis-à-vis du pH. Deux formes d’hèmes sont présents dans chacun des états oxydé et réduit. Un changement du ligand cystéine a été observé quand l’hème est réduit. Les titrages redox présentent de multiples potentiels à pH 10 et pH 5. Tll0287 peut fixer une molécule de CO à pH 7,6. Ces caractéristiques suggèrent que Tll0287 pourrait être une protéine senseur. De plus, la structure cristallographique montre que Tll0287 n’a pas un repliement classique d’un cytochrome de type c mais celui d’une protéine senseur. Les mutants de délétion du gène tll0287 ont été construits et aideront à comprendre la fonction de ce nouveau cytochrome. La troisième partie décrit l’étude de PsbV2 : un autre cytochrome de type c. Afin d’obtenir en quantité suffisante la protéine pour permettre sa caractérisation, elle a été surexprimée dans un système homologue en utilisant le promoteur de l’enzyme de la rubisco. Le potentiel redox de PsbV2 a été déterminé, comme étant très bas, inférieur à -460 mV (vs SHE, pH 5). Le spectre d’absorption UV-visible de la forme réduite a été caractérisé. La structure cristallographique de PsbV2 a été résolue et a révélé une cystéine comme ligand axial et un repliement proche de celui de cytochromes connus de T.elongatus. Bien que Tll0287 et PsbV2 présentent une cystéine comme ligand axial, leurs structures et leurs propriétés physico-chimiques suggèrent que leurs fonctions sont bien différentes. Une contribution majeure de cette thèse est la caractérisation d’un nouveau senseur à hème de type c chez les cyanobactéries et le développement d’outils nécessaires pour son étude. / Photosynthesis converts solar energy into chemical energy. This process involves a large number of proteins and protein complexes. The first protein complex in the photosynthetic chain is Photosystem II within the oxidation of water takes place. PSII is composed of the D1 and D2 proteins. In the thermophile cyanobacterium Thermosynechococcus elongatus, three genes encoded three different D1 proteins. The first part of this thesis describes the development of proteomics tools based on 2D gel-electrophoresis to study the proteome of three different variants, each expressing a single different D1 protein. Very few differences were found. However, only one expressed the protein Tll0287. The second part of the thesis describes the characterization of Tll0287. It was characterized using different techniques: electronic absorption and Raman resonance spectroscopies and spectro-electrochemistry. Tll0287 has been previously identified as a c-type cytochrome, but it presents some unexpected characteristics. The UV-visible absorption and Raman resonance spectra of reduced Tll0287 show a pH dependence. The reduced and oxidized states each had two different forms of the heme. A switch of ligands from a cysteine to histidine was observed in the reduced state. Redox titration showed multiple midpoints at pH 10 and 5. Tll0287 was shown to fix a CO molecule at pH 7.6. These physical properties suggested that Tll0287 could be a sensor. The crystallographic studies reveal that Tll0287 does not have a classical c-type cytochrome fold and is similar to other known sensor proteins, strengthening the hypothesis that it is a sensor. Deletion mutants were constructed that will help to better understand the function of this new cytochrome. The third part describes a study of the PsbV2, another c-type cytochrome. In order to obtain sufficient quantities to carry out characterization of this protein, it was overexpressed in a homologous system using the promotor of the rubisco enzyme. The redox midpoint potential of PsbV2 was found to be very low, below -460 mV (vs SHE, pH 5). The UV-visible absorption spectrum of the reduced form was determined. The crystallographic structure of PsbV2 was solved and reveals an axial cysteine ligand. Although both Tll0287 and PsbV2 share this feature, their different structures and physico-chemical properties suggest that their functions are unlikely to be similar. A major contribution of this thesis is the characterization of a new c-type cytochrome sensor in cyanobacteria and the development of proteomic tools required to study it.

<p>Metalloproteins, or proteins with a metal ion cofactor, are essential for biological function of both lower and higher level organisms. These proteins provide a multitude of functions from molecular transport, such as the hemoglobin transport of oxygen, to biologically important catalytic processes. As an example case, photosystem II (PSII) is studied as a representative metalloprotein. It was chosen based on the potential impact in the energy sector due to its ability to perform water oxidation using solar based energy. Understanding mechanisms by which the Mn₄Ca cluster inside PSII, also known as the oxygen evolving complex (OEC), can store energy as redox equivalents for splitting water will be essential for future development of analogous artificial systems. By using time resolved x-ray spectroscopy, the electron structure of the metal in the protein was probed through the catalytic cycle. While the applications mentioned herein are based on PSII from spinach, the developments in time-resolved x-ray spectroscopy techniques are also applicable to other metalloproteins.</p><p></p><p>By creating a new x-ray spectrometer we were able to capture the difference in x-ray emission spectra between two compounds differing in a single metal bound ligand, i.e. Mn^IV-OH and Mn^IV=O. This both establishes the functionality of the x-ray emission spectrometer and provides useful insight into the expected changes upon an oxygen double bond formation. This change in spectroscopic signal is discussed in context of the OEC which has been hypothesized to form a Mn^IV=O state.</p><p></p><p>A new sample delivery system and further developments to the x-ray spectrometer enabled both time-resolved x-ray absorption and time-resolved x-ray emission of PSII. These experiments show the potential of synchrotron sources for time-resolved x-ray spectroscopy. From our x-ray absorption measurements we were able to follow the electronic structure changes in time using a single incident photon energy. From the kinetic traces obtained, we show possible alternative interpretations of previous results showing a delay in reduction during the final step in water oxidation. From the x-ray emission spectroscopy (XES) measurements of PSII we were able to reproduce previous results within a limited collection time and give estimates for data size requirements for metalloproteins using this spectrometer. Between the results of both these measurements, we show the improved capability for time resolved measurements at synchrotrons.</p><p>The development of x-ray free electron lasers (XFELs) has also opened many opportunities for understanding faster electronic dynamics by providing femtosecond x-ray pulse durations with ~10¹² photons per pulse. While theoretical modeling of distortions to crystallographic data have been performed, little to no work has been done to understand under what conditions such an intense pulse will have on an impact on emission spectra. Here an atomistic model was developed, and data collected, to clarify the effects of sequential ionization, i.e. two single photons absorbed by the same atom at different times during a single pulse. Experimentally we found that XFELs easily achieve flux densities that invoke a different response than is classically observed for single photon absorption and emission for Mn^II which was used as a representative case for 3d transition metals in general. We also give parameters by which the onset of this damage can be predicted and an approximation to its effect on 3d transition metals. Additionally this work guides the work of future XFEL facilities as it shows that shorter pulses, currently believed to be able to escape x-ray induced distortions to crystallography data, is not a viable method for overcoming changes in x-ray emission spectra.</p><div><br></div>

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 4 uppsatser. Includes bibliographical references.

Adaptation to environmental changes is important for the survival of living organisms. Under extreme abiotic conditions, organic molecules (such as lipids, proteins and nucleic acids) are prone to damage. Under these conditions stress response mechanisms are activated, either to prevent the source of damage or to promote the rapid turnover of damaged molecules. Like all photoautotrophic organisms, cyanobacteria are sensitive to high light intensity and oxidative stress, which induces damage to the photosynthetic apparatus. My thesis is divided in two subjects related to particular stress responses in the cyanobacterium Synechocystis sp. PCC 6803: 1) the role of Deg/HtrA proteases and 2) investigations on the small CAB-like proteins. Deg/HtrA proteases are ATP-independent serine endopeptidases with a characteristic C-terminal PDZ domain. These proteases are largely dispersed among living organisms, with many different functions, mostly involved in protein quality control. The genome of Synechocystis sp. PCC 6803 contains three genes coding for Deg/HtrA proteases: HtrA, HhoA and HhoB. These proteases are essential for survival under high light and heat stress, and may overlap in their functions. During my Ph.D. studies I focused on the identification of the precise localization of the Deg/HtrA proteases in the cyanobacterial cell, analyzed the biochemical properties of recombinant Synechocystis Deg/HtrA proteases in vitro and adopted proteomic and metabolomic approaches to study the physiological importance of these proteases. My data show that Deg/HtrA proteases are not only important in stress response mechanisms under adverse conditions, but are also involved in the stabilization of important physiological processes, such as polysaccharides biosynthesis and peptidoglycan turnover. The small CAB-like proteins (SCPs) belong to the light harvesting-like family of stress induced proteins and are thought to be involved in the photoprotection of the photosynthetic apparatus. Five small CAB-like proteins where identified in Synechocystis sp. PCC 6803 (ScpA-E). In my studies I identified another relative to the SCPs, LilA, which I found to be co-transcribed with ScpD. I also focused on the subcellular localization and identification of potential interaction partners of the SCPs.