Diese Dissertation befasst sich mit der numerischen Behandlung dissipativer quantenmechanischer Prozesse im Rahmen der reduzierten Dichtematrix-Theorie. Zunächst werden Elektronen-Transferprozesse mit Hilfe einer hierarchischen Methode zur Lösung der Bewegungsgleichung der System-Dichtematrix untersucht. Hier liegt der Fokus auf der Untersuchung des Konvergenzverhaltens der Hierarchie mit der Anzahl der berücksichtigten Ebenen bei unterschiedlichen Abbruchverfahren. Es wird gezeigt, dass die Konvergenz stark von der Abbruchmethode und der Observablen abhängt. Weiterhin wird das lineare Absorptionsspektrum des B850 Pigment-Rings von Rhodispirillum molischianum mit verschiedenen Methoden zur Berücksichtigung der Effekte eines angekoppelten Bades berechnet. Diese Methoden basieren auf störungstheoretischen Ansätzen in der System-Bad-Kopplung. Es gelang unter Verwendung der modifizierten Redfield Theorie (MRT) einen Ausdruck für das Absorptionsspektrum herzuleiten. Bei der MRT werden Teile der System-Bad-Wechselwirkung exakt behandelt. Diese Methode wird in zwei Varianten diskutiert und anderen Methoden gegenübergestellt. Modellrechnungen werden für verschiedene Spektraldichten angefertigt, darunter eine, die aus einer Molekulardynamik(MD)- Simulation stammt. Ebenso wird der Einfluss statischer Unordnung der Pigment-Energien auf die Form des Absorptionsspektrums diskutiert. Dazu werden Spektren sowohl einer einzelnen Realisierung als auch des Ensembles dargestellt. Im Falle der Spektraldichte aus der MD-Simulation werden die Ensemble-Spektren zusätzlich mit experimentellen Daten verglichen. Weiterhin wird eine Rechnung mit der Hierarchie zum Spektrum des B850 Rings und weitere zur Populationsdynamik eines kleineren Systems diskutiert und mit Ergebnissen aus der MRT verglichen. Außerdem wird eine Methode zur stochastischen Propagation von mehrdimensionalen Wellenfunktionen entwickelt. Mit Hilfe von Sprung- Prozessen gelingt es die Freiheitsgrade des Systems zu entkoppeln, sodass mehrere eindimensionale Wellenfunktionen stochastisch propagiert werden können. Die exakte Wellenfunktion kann so als Ensemblemittel von Produkten eindimensionaler stochastischer Wellenfunktionen beschrieben werden.

V práci je popsán princip synchronní detekce (SD), který byl uplatněn při měření s biosenzory. Metoda SD umožňuje dosažení výrazně lepšího poměru signálu k šumu, vyššího limitu detekce a celkové zlepšení robustnosti měření. Uplatnění SD při měření s biosenzory umožní zlepšit analýzu jeho odezvy a umožní odstranění nežádoucích interferencí nebo šumů, které mohou být způsobeny například mícháním roztoku, elektromagnetickými vlivy nebo parazitními proudy. SD také umožňuje rozložit získaný signál na odezvu stimulace a na dlouhodobý signál jiného procesu, a dále také identifikovat jevy druhého řádu. Pro identifikaci stimulačního signálu ve výstupním signálu měření byl na základě lineárního statistického modelu vyvinut specializovaný software. SD byla ověřena na modelovém případu výstupního signálu biosenzoru s aplikovaným komplexem fotosystému II (PSII) a jeho odezvě na stimulaci světlem. Odezva PSII se řídí kinetikou prvního řádu a může být také ovlivněna inhibitory. Kinetické konstanty vazby herbicidu na PSII závisí lineárně na koncentraci herbicidu. To umožňuje jejich měření také při nízkých koncentracích herbicidu.

<p dir="ltr">Photosynthesis is the basis of primary productivity on Earth. Oxygenic photosynthesis utilizes the nearly inexhaustible energy of radiant solar light to fix atmospheric carbon dioxide into usable forms of chemical energy and produces dioxygen as a product. Central to this process are several large hetero-oligomeric protein complexes that comprise the photosynthetic electron transport chain. Photosystem II (PSII) initiates electron transport through the light-driven oxidation of water, in-turn relinquishing protons and oxygen. Through this reaction, electrons are used to form the reductant NADPH, while protons form a proton-motive gradient that is used to drive synthesis of ATP. As a result of this highly energetic reaction, PSII is often subject to oxidative photodamage due to the production of reactive oxygen species. Inevitably, accumulation of oxidative photodamage disrupts the catalytic activity of PSII, resulting in a loss of photosynthetic activity. To deal with the nearly constant incurred photodamage to PSII, oxygenic photoautotrophs undergo a disassembly and repair cycle that results in the complete turnover of the damaged D1 subunit of PSII. Due to its high tendency for damage, the D1 subunit has a half-life of under one hour in high light intensity. Despite our current understanding of photoinhibition and PSII repair, it is still unclear how D1 is replaced so rapidly in response to damaging conditions. Previous research has indicated a role for phosphorylation of PSII in D1 turnover, however the mechanism has not been totally resolved. In the first chapter of this thesis, our current understanding of PSII phosphorylation and oxidative damage is reviewed in the context of PSII repair. In the second chapter, the role of protein phosphorylation in the PSII repair cycle is investigated in the model organism <i>Arabidopsis</i>. Using several PSII phosphorylation mutants, we demonstrate that phosphorylation seems to mediate disassembly of large PSII supercomplexes and dimers into smaller subcomplexes. In the third chapter, the role of oxidative photodamage is investigated in mediating PSII disassembly. Here, we use several <i>in vitro</i> assays to demonstrate that photodamage is sufficient to induce the disassembly of smaller PSII subcomplexes. In the fourth chapter, a technique for determining the stoichiometry of photosynthetic complexes is examined, with implications for understanding PSII repair. Finally, in the fifth chapter, several conclusions and unanswered questions from this thesis are discussed.</p>

Os herbicidas aplicados em pré-plantio das mudas pré-brotadas (MPB) de cana-de-açúcar podem lixiviar e serem absorvidos pelas raízes da planta e causar fitotoxicidade, implicando em perdas de produtividade. Por isso, estudos da lixiviação do diuron e hexazinone amplamente utilizados na cana-de-açúcar associados ou não ao aminocyclopyrachlor fornecem informações sobre os possíveis impactos na seletividade à cultura. Assim, o objetivo foi avaliar a seletividade dos herbicidas diuron, hexazinone e aminocyclopyrachlor isolados e associados em MPB de cana-de-açúcar cultivar RB966928 em diferentes doses, a lixiviação em solo argiloso em campo e a lixiviação na presença de palha em condições de laboratório. Para o estudo da seletividade foram realizados dois experimentos, um em campo e outro em casa de vegetação. Os herbicidas diuron, hexazinone e aminocyclopyrachlor isolados e associados foram aplicados em quatro diferentes doses em pré-plantio das MPBs de cana-de-açúcar cultivar RB966928. As avaliações foram realizadas no tempo para as variáveis fitotoxicidade, índice SPAD, altura e número de perfilhos. Ao término do experimento em casa de vegetação foi avaliado a massa seca da parte aérea e raiz e no experimento em campo avaliado a produtividade. O experimento de lixiviação em campo foi realizado simultaneamente ao experimento de seletividade, na mesma área. Foram coletadas amostras de solo aos 30, 60 e 90 dias após a aplicação (DAA) dos herbicidas nas profundidades de 0-10, 10-20 e 20-30 cm e analisadas em laboratório por cromatografia líquida em detector de massas (LC/MS/MS) para determinação da concentração no tempo e em cada profundidade. Para a melhor compreensão da lixiviação do aminocyclopyrachlor, foi realizado um experimento em laboratório com aplicação de 14C-aminocyclopyrachlor sobre 0, 5, 10 e 20 t ha-1 de palha de cana-de-açúcar em colunas de solo e simulada precipitação. Foi determinada a lixiviação do 14C-aminocyclopyrachlor nas diferentes profundidades da coluna (0-30 cm) e no lixiviado. Como resultado dos experimentos, os herbicidas diuron, hexazinone e aminocyclopyrachlor associados foram seletivos às MPBs de cana-de-açúcar até a dose de 1299,50 + 345,00 + 80,50 g i.a. ha-1, com menor impacto na produtividade comparado aos herbicidas aplicados isolados. Em campo, o diuron associado ao hexazinone e ao aminocyclopyrachlor lixiviou menos e ficou mais retido de 0-10 cm em todas as avaliações. O hexazinone isolado ou associado lixiviou não havendo diferença entre os modos de aplicação. O aminocyclopyrachlor isolado ou associado lixiviou, com maior concentração na camada de 20-30 cm aos 90 DAA. O 14C-aminocyclopyrachlor foi detectado em todo o perfil do solo (0- 30 cm), independente da quantidade de palha de cana-de-açúcar. Contudo, 20 t ha-1 de palha aumentou a dificuldade do herbicida em ultrapassar a barreira formada pelo material. / The herbicides applied in pre-planting of the pre-sprouted seedlings (MPB) of sugarcane can be absorbed by the roots of the plant and cause phytotoxicity, implying yield loss. Therefore, studies of the leaching of diuron and hexazinone widely used in sugarcane associated or not to aminocyclopyrachlor provide information on the possible impacts to the environment as well as the effects on crop selectivity. Thus, the aim was to evaluate the selectivity of diuron, hexazinone and aminocyclopyrachlor herbicides isolated and associated in MPB of sugarcane cultivar RB966928 in different doses, the leaching in clay soil in the field and leaching in the presence of straw under laboratory conditions. For the study of the selectivity two experiments were carried out, one in the field and the other in a greenhouse, the herbicides diuron, hexazinone and aminocyclopyrachlor isolated and associated were applied in four different doses in pre-planting of the sugarcane MPBs RB966928. The evaluations were carried out in time for the variables SPAD index, height, number of tillers, phytotoxicity and at the end of the experiments dry mass of shoot and root and yield. The field leaching experiment was carried out simultaneously to the selectivity experiment, in the same area. Soil samples were collected at 30, 60 and 90 days after application (DAA) of the herbicides at depths of 0-10, 10-20 and 20-30 cm and analyzed in laboratory in LC/MS/MS for determination of concentration in time and in each depth. To better understand the leaching of aminocyclopyrachlor, a laboratory experiment was carried out with 14Caminocyclopyrachlor on 0, 5, 10 and 20 t ha-1 of sugarcane straw in soil columns and simulated precipitation. Was determined the leaching of 14C-aminocyclopyrachlor at different depths of the column (0-30 cm) and in the leachate. As result of the experiments, the herbicides diuron, hexazinone and aminocyclopyrachlor associated were selective to the sugarcane MPBs up to the dose of 1299.50 + 345.00 + 80.50 g a.i. ha-1, with less impact in the yield compared to the herbicides applied isolated. In the field, diuron associated to the hexazinone and aminocyclopyrachlor leached less and was more retained of 0-10 cm in all evaluations. Hexazinone isolated or associated leached without difference between the modes of application. Aminocyclopyrachlor isolated or associated leached, with a higher concentration in the 20-30 cm soil layer at 90 DAA. 14C-aminocyclopyrachlor was detected throughout the soil profile (0-30 cm), regardless of the amount of sugarcane straw. However, 20 t ha-1 of straw increased the difficulty of the herbicide in overcoming the barrier formed by the material.

Os herbicidas aplicados em pré-plantio das mudas pré-brotadas (MPB) de cana-de-açúcar podem lixiviar e serem absorvidos pelas raízes da planta e causar fitotoxicidade, implicando em perdas de produtividade. Por isso, estudos da lixiviação do diuron e hexazinone amplamente utilizados na cana-de-açúcar associados ou não ao aminocyclopyrachlor fornecem informações sobre os possíveis impactos na seletividade à cultura. Assim, o objetivo foi avaliar a seletividade dos herbicidas diuron, hexazinone e aminocyclopyrachlor isolados e associados em MPB de cana-de-açúcar cultivar RB966928 em diferentes doses, a lixiviação em solo argiloso em campo e a lixiviação na presença de palha em condições de laboratório. Para o estudo da seletividade foram realizados dois experimentos, um em campo e outro em casa de vegetação. Os herbicidas diuron, hexazinone e aminocyclopyrachlor isolados e associados foram aplicados em quatro diferentes doses em pré-plantio das MPBs de cana-de-açúcar cultivar RB966928. As avaliações foram realizadas no tempo para as variáveis fitotoxicidade, índice SPAD, altura e número de perfilhos. Ao término do experimento em casa de vegetação foi avaliado a massa seca da parte aérea e raiz e no experimento em campo avaliado a produtividade. O experimento de lixiviação em campo foi realizado simultaneamente ao experimento de seletividade, na mesma área. Foram coletadas amostras de solo aos 30, 60 e 90 dias após a aplicação (DAA) dos herbicidas nas profundidades de 0-10, 10-20 e 20-30 cm e analisadas em laboratório por cromatografia líquida em detector de massas (LC/MS/MS) para determinação da concentração no tempo e em cada profundidade. Para a melhor compreensão da lixiviação do aminocyclopyrachlor, foi realizado um experimento em laboratório com aplicação de 14C-aminocyclopyrachlor sobre 0, 5, 10 e 20 t ha-1 de palha de cana-de-açúcar em colunas de solo e simulada precipitação. Foi determinada a lixiviação do 14C-aminocyclopyrachlor nas diferentes profundidades da coluna (0-30 cm) e no lixiviado. Como resultado dos experimentos, os herbicidas diuron, hexazinone e aminocyclopyrachlor associados foram seletivos às MPBs de cana-de-açúcar até a dose de 1299,50 + 345,00 + 80,50 g i.a. ha-1, com menor impacto na produtividade comparado aos herbicidas aplicados isolados. Em campo, o diuron associado ao hexazinone e ao aminocyclopyrachlor lixiviou menos e ficou mais retido de 0-10 cm em todas as avaliações. O hexazinone isolado ou associado lixiviou não havendo diferença entre os modos de aplicação. O aminocyclopyrachlor isolado ou associado lixiviou, com maior concentração na camada de 20-30 cm aos 90 DAA. O 14C-aminocyclopyrachlor foi detectado em todo o perfil do solo (0- 30 cm), independente da quantidade de palha de cana-de-açúcar. Contudo, 20 t ha-1 de palha aumentou a dificuldade do herbicida em ultrapassar a barreira formada pelo material. / The herbicides applied in pre-planting of the pre-sprouted seedlings (MPB) of sugarcane can be absorbed by the roots of the plant and cause phytotoxicity, implying yield loss. Therefore, studies of the leaching of diuron and hexazinone widely used in sugarcane associated or not to aminocyclopyrachlor provide information on the possible impacts to the environment as well as the effects on crop selectivity. Thus, the aim was to evaluate the selectivity of diuron, hexazinone and aminocyclopyrachlor herbicides isolated and associated in MPB of sugarcane cultivar RB966928 in different doses, the leaching in clay soil in the field and leaching in the presence of straw under laboratory conditions. For the study of the selectivity two experiments were carried out, one in the field and the other in a greenhouse, the herbicides diuron, hexazinone and aminocyclopyrachlor isolated and associated were applied in four different doses in pre-planting of the sugarcane MPBs RB966928. The evaluations were carried out in time for the variables SPAD index, height, number of tillers, phytotoxicity and at the end of the experiments dry mass of shoot and root and yield. The field leaching experiment was carried out simultaneously to the selectivity experiment, in the same area. Soil samples were collected at 30, 60 and 90 days after application (DAA) of the herbicides at depths of 0-10, 10-20 and 20-30 cm and analyzed in laboratory in LC/MS/MS for determination of concentration in time and in each depth. To better understand the leaching of aminocyclopyrachlor, a laboratory experiment was carried out with 14Caminocyclopyrachlor on 0, 5, 10 and 20 t ha-1 of sugarcane straw in soil columns and simulated precipitation. Was determined the leaching of 14C-aminocyclopyrachlor at different depths of the column (0-30 cm) and in the leachate. As result of the experiments, the herbicides diuron, hexazinone and aminocyclopyrachlor associated were selective to the sugarcane MPBs up to the dose of 1299.50 + 345.00 + 80.50 g a.i. ha-1, with less impact in the yield compared to the herbicides applied isolated. In the field, diuron associated to the hexazinone and aminocyclopyrachlor leached less and was more retained of 0-10 cm in all evaluations. Hexazinone isolated or associated leached without difference between the modes of application. Aminocyclopyrachlor isolated or associated leached, with a higher concentration in the 20-30 cm soil layer at 90 DAA. 14C-aminocyclopyrachlor was detected throughout the soil profile (0-30 cm), regardless of the amount of sugarcane straw. However, 20 t ha-1 of straw increased the difficulty of the herbicide in overcoming the barrier formed by the material.

<p>Amino acid radicals are key redox intermediates in several natural enzymes including Cytochrome c peroxidase, DNA photolyase, ribonucletide reductase, cytochrome c oxidase and photosystem II. Electron transfer from amino acids is often coupled to deprotonation and this thesis concerns the coupling of electron transfer from tyrosine and tryptophan to trisbipyridineruthenium(III) with deprotonation in model complexes. Specifically the mechanisms for these proton coupled electron transfer reactions have been studied and the controlling parameters have been identified, the possible mechanisms being stepwise electron transfer followed by deprotonation and deprotonation followed by electron transfer or concerted electron transfer/deprotonation.</p><p>Proton coupled electron transfer reactions have been studied using nano-second flash photolysis in water solution and the effect of pH, temperature, reaction driving force, deuteration and nature of the amino acid has been determined. I have shown that the rate constant for the concerted reaction depends intrinsically on the mixing entropy of the released proton and that the pH-dependence can be used as an experimental tool for mechanistic discrimination. Moreover I have shown that the concerted reaction inherently has a high reorganisation energy due to the coupling of the electron motion with deprotonation. Hydrogen bonding to the transferring proton however significantly reduces this reorganisation energy. The concerted reaction also has a relatively high driving force counteracting the high reorganisation energy in the competition between the concerted reaction and the stepwise electron transfer first reaction. The relative importance of the high reorganisation energy and the high driving force for the concerted reaction determines the mechanistic outcome of the reaction, the stepwise reaction being favoured by high over-all driving forces and the concerted reaction by high pH.</p><p>By comparing my results from model complexes with tyrosineZ oxidation in photosystem II, I give strong evidence for a concerted electron transfer/deprotonation mechanism.</p>

Amino acid radicals are key redox intermediates in several natural enzymes including Cytochrome c peroxidase, DNA photolyase, ribonucletide reductase, cytochrome c oxidase and photosystem II. Electron transfer from amino acids is often coupled to deprotonation and this thesis concerns the coupling of electron transfer from tyrosine and tryptophan to trisbipyridineruthenium(III) with deprotonation in model complexes. Specifically the mechanisms for these proton coupled electron transfer reactions have been studied and the controlling parameters have been identified, the possible mechanisms being stepwise electron transfer followed by deprotonation and deprotonation followed by electron transfer or concerted electron transfer/deprotonation. Proton coupled electron transfer reactions have been studied using nano-second flash photolysis in water solution and the effect of pH, temperature, reaction driving force, deuteration and nature of the amino acid has been determined. I have shown that the rate constant for the concerted reaction depends intrinsically on the mixing entropy of the released proton and that the pH-dependence can be used as an experimental tool for mechanistic discrimination. Moreover I have shown that the concerted reaction inherently has a high reorganisation energy due to the coupling of the electron motion with deprotonation. Hydrogen bonding to the transferring proton however significantly reduces this reorganisation energy. The concerted reaction also has a relatively high driving force counteracting the high reorganisation energy in the competition between the concerted reaction and the stepwise electron transfer first reaction. The relative importance of the high reorganisation energy and the high driving force for the concerted reaction determines the mechanistic outcome of the reaction, the stepwise reaction being favoured by high over-all driving forces and the concerted reaction by high pH. By comparing my results from model complexes with tyrosineZ oxidation in photosystem II, I give strong evidence for a concerted electron transfer/deprotonation mechanism.

This thesis describes an investigation of the molecular mechanism underlying solar conversion processes that occur in Type I photosynthetic reaction centers, in which P700 plays a central role. Static Fourier transform infrared (FTIR) difference spectroscopy (DS) was used to probe the electronic and structural organization of P700 and P700+. In combination with isotope labeling and site directed mutagenesis we have investigated how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. Comparison of (P700+-P700) FTIR difference spectra (DS) obtained using wild type and mutant PS I led us to suggest that the 131 keto carbonyl group of PA is essentially free from hydrogen bonding in the ground state. Upon cation formation, this hydrogen bonding becomes stronger, probably because of a cation induced reorientation of the hydroxyl group of a nearby threonine residue. We also tentatively suggested that a difference band at 1639(-)/1660(+) cm-1 in (P700+-P700) FTIR DS might be due to a C=C mode of the imidazole side chain of the ligating histidine residues. Most of this thesis is geared towards investigating the validity of this interpretation. (P700+-P700) FTIR DS obtained using mutant PS I particles in which hydrogen bonding to P700 is altered can be reconciled within the context of our new interpretation. (P700+-P700) FTIR DS obtained using uniformly 2H, 15N, and 13C labeled PS I particles also support our new interpretation, and indicate that the difference band at 1639(-)/ 1660(+) cm-1 cannot be associated with a strongly hydrogen bonded keto carbonyl group of PA. To investigate if the imidazole side-chain of ligating histidine residues could contribute to bands in (P700+-P700) FTIR DS vibrational mode frequencies and intensities for several protonation forms of 4-methylimidazole were calculated. The calculations suggest that the 1639(-)/1660(+) cm-1 band in (P700+-P700) FTIR DS may not be due to a C=C mode of the imidazole side chain of the ligating histidine residues. Thus we have produced data that suggests neither of the proposed interpretations alone can adequately explain the origin of the 1639(-)/1660(+) cm-1 difference band in (P700+-P700) FTIR DS. The origin of the 1639(-)/1660(+) cm-1 difference band in (P700+-P700) FTIR DS is therefore still an open question.

Proton coupled electron transfer reactions often involve tyrosine residues, because when oxidized, the phenolic side chain deprotonates. Tyrosine Z (YZ) is responsible for extracting electrons in a stepwise fashion from the oxygen evolving-complex in order to build enough potential to oxidize water. This process requires that each step YZ must deprotonate and reprotonate in order to maintain the high midpoint potential that is necessary to oxidize the oxygen-evolving complex, which makes YZ highly involved in proton coupled electron transfer reactions. In this thesis YZ has been studied within oxygen-evolving photosystem II utilizing electron paramagnetic resonance spectroscopy to monitor the tyrosyl radical that is formed upon light excitation. Kinetic analysis of YZ has shed light on the factors that are important for PSII to carry out water oxidation at the oxygen-evolving complex. Most notably the strong hydrogen-bonding network and the midpoint potential of YZ have been shown to be integral aspects of the water splitting reactions of PSII. By studying YZ within oxygen-evolving PSII, conclusions are readily applied to the native system.

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