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No presente trabalho foram investigados vários sistemas moleculares contendo o dióxido de enxofre (SO2) como espécie elétron aceptora e diversas espécies elétron doadoras como aminas (alifáticas e aromáticas), complexos inorgânicos e líquidos iônicos. Estes compostos são chamados de complexos do tipo doador-aceptor, que no caso do SO2 são caracterizados por apresentarem baixas energias de ligação entre as espécies se comparada à maioria dos complexos desta categoria. A caracterização vibracional dos complexos e adutos de SO2, assim como do processo de transferência de carga (CT) intermolecular e da estabilidade destes sistemas consistem nos principais objetivos deste trabalho, tendo sido para tanto utilizadas as técnicas de espectroscopia eletrônica (UV-Vis), espectroscopia vibracional (Raman e infravermelho), cálculos de química quântica (DFT) e cálculos de dinâmica molecular clássica (MD). No caso de complexos entre aminas e SO2 foi observado que além da basicidade das aminas, o efeito estérico consiste em um fator crucial para a estabilidade. No caso de aminas aromáticas, complexos coloridos foram formados permitindo a obtenção de espectros Raman ressonante. No caso de um complexo inorgânico, com utilidade potencial como sensor de SO2foi observada a aplicação da espectroscopia Raman ressonante na caracterização do complexo de estequiometria 2:1. Já no caso da interação de SO2 e líquidos iônicos (LI) foi observada a capacidade de absorção de SO2 por LI e a conseqüente mudança das propriedades físico-químicas destes líquidos, o que abre a possibilidade, além de sua potencial utilização no contexto ambiental, de sintonizar propriedades de líquidos iônicos através da adição controlada de SO2. / In the present work molecular systems bearing the sulfur dioxide (SO2) as an electron acceptor species and several electron donor species as amines (aliphatic and aromatic), inorganic complexes and ionic liquids were investigated. Such complexes are commonly known as donor-acceptor complexes, and in particular, in the case of SO2 complexes are characterized by low binding energies when compared with the majority of charge transfer (CT) complexes. The vibrational characterization of the SO2 complexes, as well as of the intermolecular charge transfer (CT) process and of their stabilities are the main subjects of this work, and for such, electronic spectroscopy (UV-Vis), vibrational spectroscopy (Raman and infrared), quantum chemical (DFT) calculations and molecular dynamics (MD) simulations were used. In the case of complexes formed by amines and SO2 it was observed that besides the amine basicities, the steric effect plays a crucial role in their stabilities. In the case of aromatic amines, colored complexes were formed allowing the resonance Raman study. The use of Raman spectroscopy in the characterization of an inorganic complex (SO2 sensor) indicates the potential use of the resonance Raman effect for SO2 monitoring. In the case of the interaction between SO2 and ionic liquids it was observed the great capability of ionic liquids as SO2 absorbers, and the consequent change in the physical-chemical properties of these liquids, what opens the possibility, in addition to its potential use in the environmental context, for tuning the ionic liquids properties via the controlled addition of SO2.

Les études d'interaction protéine-ligand aident à mieux comprendre la structure et la fonction des protéines. Dans la première partie de la thèse, la cyt bd oxydase a été étudiée. La protéine d'E. coli a été immobilisée avec succès sur des électrodes modifiées par des nanoparticules d'or. Ainsi, un biocapteur électrochimique a été créé, permettant de tester certains inhibiteurs potentiels de cyt bd provenant d’E. coli. Le cyt bd issue de G. thermodenitrificans thermophile a également été étudié. En faisant appel aux spectroscopies IR et Raman ainsi que l’électrochimie, il a été démontré que la protéine est distincte du cyt bd d’E. coli. Une influence mutuelle du pH et de la température sur la catalyse a été aussi démontrée. La deuxième partie de la thèse portait sur la protéine mitochondriale mitoNEET. L'influence du pH et de divers ligands (pioglitazone, resvératrol, ions phosphates) a été examinée. / Protein-ligand interaction studies help to better understand the structure and function of proteins. In the first part of the thesis cyt bd oxidase was studied. The protein from E. coli was successfully immobilised at gold nanoparticles modified electrodes. Thus, an electrochemical biosensor was created allowing testing some potential inhibitors of cyt bd from E. coli. The cyt bd from thermophilic G. thermodenitrificans was also studied. By means of IR, Raman spectroscopy and electrochemistry the protein was shown to be distinct from cyt bd from E. coli. A mutual influence of pH and temperature was demonstrated on the electrochemical and catalytical properties. The second part of the thesis focused on mitochondrial mitoNEET protein. The influence of the pH and various ligands was studied.

Le monoxyde d'azote est exclusivement synthétisé chez les mammifères par une famille d’hémoprotéines, les NO-Synthases. Le cœur de l’activité des NO-Synthases est l’activation de l’oxygène c'est-à-dire l’activation de l’intermédiaire réactionnel FeIIO2. Cette étape est contrôlée par la réactivité intrinsèque du fer, par les transferts de proton et les transferts d’électron. Elle doit être parfaitement maîtrisée car elle contrôle le chemin catalytique emprunté et la nature du produit final. Comprendre l’étape d’activation de l’oxygène est essentiel à la compréhension du rôle biologique et/ou pathologique de la NO-Synthase de mammifère. Cette question s'étend aux NO-Synthases bactériennes pour lesquelles on ne connait ni le mécanisme moléculaire ni la fonction biologique. Ce manuscrit propose une analyse approfondie de l’étape d’activation de l’oxygène de la NO-Synthase. Dans un premier temps, nous avons étudié l’influence de l’environnement proximal sur la réactivité intrinsèque du fer et l’activation de l’oxygène. Nous avons généré des protéines mutées qui modifient les propriétés électroniques de la liaison proximale de l’hème. Ces protéines mutées ont été caractérisées par différentes spectroscopies (résonance paramagnétique électronique, Raman de résonance). Dans un second temps nous avons directement étudié le complexe FeIIO2, en présence d’analogues de substrat, grâce à des analyses de cinétique rapide en flux continu et en flux arrêté (stopped-flow). Dans un troisième temps, le rôle du cofacteur tetrahydrobioptérine dans le transfert de proton et d’électron a été étudié par une méthode de piégeage à des temps très courts : le freeze-quench. L'ensemble de nos résultats montrent que l’activation de l’oxygène est régulée par les propriétés électro-donneuses du ligand proximal et par le réseau de liaisons H distal. Nous mettons en évidence des différences dans le rôle redox du cofacteur tetrahydrobioptérine entre la NO-Synthase de mammifère et la NO-Synthase bactérienne. La difficulté majeure pour comprendre l’étape d’activation de l’oxygène de la NO-Synthase réside dans la complexité et la rapidité de la réaction catalytique. Dans ce contexte, nous avons cherché à adapter une méthodologie qui a prouvé son efficacité dans le cas des cytochromes P450 : la cryo-réduction couplée à des sauts en température. / Nitric oxide is exclusively synthesized by NO-Synthases in mammals. The heart of the NO-synthase activity is oxygen activation, which corresponds to the activation of the FeIIO2 intermediate. This step depends on the heme electronic properties and on the electron and proton transfers. Oxygen activation has to be well mastered to control exactly the nature of the end-product. Understanding the oxygen activation step is necessary to better understand the biological/pathological role of the mammalian NO-Synthases. Furthermore, bacterial NO-Synthases function and oxygen activation mechanism are unknown. This PhD work proposes a deep analysis of the oxygen activation step in NO-Synthases. First, proximal environment has been studied with mutated proteins. These mutations impact the electronic properties of the heme proximal bond. Spectroscopic analyses of these mutants have been done by electron paramagnetic resonance and resonance Raman. Then, we have studied the FeIIO2 intermediate with substrate analogs which has necessitated continuous flow and stopped-flow analyses. Finally, the role of the tetrahydrobiopterin cofactor in the electron and proton transfer has been studied and clarified thanks to a very fast trapping method : the freeze-quench. Our results show that the oxygen activation step is elaborately controlled by the proximal bond electron donation and the distal H bond network. At the same time we show some differences between mammalian and bacterial NO-Synthases concerning the redox role of the tetrahydrobiopterin cofactor. The major obstacle to understand the oxygen activation step resides in the complexity of the active site chemistry and the rate of catalytic reactions. For this reason, we propose to adapt an already successful protocol to trap some intermediates in the cytochromes P450 mechanism : cryo-reduction coupled with temperature jumps.

Sulfonated phthalocyanines were characterized using capillary electrophoresis and mass spectrometry. Derivatives investigated included the copper, cobalt, zinc and metal-free sulfonated phthalocyanines. The electropherograms of commercially available copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid and 4,4',4'',4'''-tetrasulfonic acid were very different, consistent with the latter compound having a structure that is not fully sulfonated. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were used to characterize the sulfonated phthalocyanines. Mass spectral evidence was obtained for a pentasulfonated species of both the metal-free phthalocyanine and zinc phthalocyanine when these species were made by sulfonation of the metal-free phthalocyanine (followed by zinc insertion in the latter case). Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The heme binding protein (HBP) of this transporter is SiaA (HtsA). Several biophysical techniques were used to determine the coordination state, and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggested that the heme is six-coordinate and low spin in both oxidation states of the protein, with methionine and histidine as axial ligands. The pKa of SiaA was determined, as were the reductive and oxidative midpoint potentials. Guanidinium titration studies of wild-type SiaA showed that the ferric state is less stable than the ferrous state. Free energy of unfolding values [ÄG(H2O)] for the oxidized and reduced proteins were 7.3 ± 0.8 and 16.0 ± 3.6 kcal mol−1, respectively. Denaturation of the histidine mutant H229A was not able to be followed via absorbance spectrometry, possibly due to the large amount of apoprotein present or to non-specific binding of the heme in the binding pocket. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

No presente trabalho foram investigados vários sistemas moleculares contendo o dióxido de enxofre (SO2) como espécie elétron aceptora e diversas espécies elétron doadoras como aminas (alifáticas e aromáticas), complexos inorgânicos e líquidos iônicos. Estes compostos são chamados de complexos do tipo doador-aceptor, que no caso do SO2 são caracterizados por apresentarem baixas energias de ligação entre as espécies se comparada à maioria dos complexos desta categoria. A caracterização vibracional dos complexos e adutos de SO2, assim como do processo de transferência de carga (CT) intermolecular e da estabilidade destes sistemas consistem nos principais objetivos deste trabalho, tendo sido para tanto utilizadas as técnicas de espectroscopia eletrônica (UV-Vis), espectroscopia vibracional (Raman e infravermelho), cálculos de química quântica (DFT) e cálculos de dinâmica molecular clássica (MD). No caso de complexos entre aminas e SO2 foi observado que além da basicidade das aminas, o efeito estérico consiste em um fator crucial para a estabilidade. No caso de aminas aromáticas, complexos coloridos foram formados permitindo a obtenção de espectros Raman ressonante. No caso de um complexo inorgânico, com utilidade potencial como sensor de SO2foi observada a aplicação da espectroscopia Raman ressonante na caracterização do complexo de estequiometria 2:1. Já no caso da interação de SO2 e líquidos iônicos (LI) foi observada a capacidade de absorção de SO2 por LI e a conseqüente mudança das propriedades físico-químicas destes líquidos, o que abre a possibilidade, além de sua potencial utilização no contexto ambiental, de sintonizar propriedades de líquidos iônicos através da adição controlada de SO2. / In the present work molecular systems bearing the sulfur dioxide (SO2) as an electron acceptor species and several electron donor species as amines (aliphatic and aromatic), inorganic complexes and ionic liquids were investigated. Such complexes are commonly known as donor-acceptor complexes, and in particular, in the case of SO2 complexes are characterized by low binding energies when compared with the majority of charge transfer (CT) complexes. The vibrational characterization of the SO2 complexes, as well as of the intermolecular charge transfer (CT) process and of their stabilities are the main subjects of this work, and for such, electronic spectroscopy (UV-Vis), vibrational spectroscopy (Raman and infrared), quantum chemical (DFT) calculations and molecular dynamics (MD) simulations were used. In the case of complexes formed by amines and SO2 it was observed that besides the amine basicities, the steric effect plays a crucial role in their stabilities. In the case of aromatic amines, colored complexes were formed allowing the resonance Raman study. The use of Raman spectroscopy in the characterization of an inorganic complex (SO2 sensor) indicates the potential use of the resonance Raman effect for SO2 monitoring. In the case of the interaction between SO2 and ionic liquids it was observed the great capability of ionic liquids as SO2 absorbers, and the consequent change in the physical-chemical properties of these liquids, what opens the possibility, in addition to its potential use in the environmental context, for tuning the ionic liquids properties via the controlled addition of SO2.

Im ersten Teil der Arbeit werden Signalverstärkungsmechanismen für Raman-Spektroskopie erschlossen und evaluiert. Die als geeignet bewerteten Methoden finden im zweiten Teil ihre Anwendung zur Untersuchung der vibronischen Eigenschaften von dünnen Manganphthalocyaninschichten, die anschließend mit Kalium interkaliert werden. Hierbei sind verschiedene Phasen identifizierbar, die ein ganzzahliges Verhältnis von Kaliumatomen zu Manganphthalocyaninmolekülen besitzen. Im dritten Teil werden die elektrischen Eigenschaften durch die Verwendung dieses Materialsystems als aktives Medium eines Feldeffekttransistors untersucht.

Histidinkinase-Rhodopsine (HKRs) können als besondere Gruppe der Hybrid-Histidinkinasen beschrieben werden, deren N-terminale sensorische Domäne ein mikrobielles Rhodopsin ist. HKR-codierende Sequenzen konnten in den Genomen verschiedener Algen, Pilze und Amoeben gefunden werden doch ihre Aufgaben und Wirkungsweisen sind bisher ungeklärt. Im Rahmen dieser Arbeit wurden die rekombinanten Rhodopsin-Domänen von zwei HKRs mit verschiedenen spektroskopischen Techniken charakterisiert. Sie zeigten mehrere Besonderheiten. Das Rhodopsin-Fragment von Cr-HKR1 aus Chlamydomonas reinhardtii kann durch alternierende kurzwellige und langwellige Belichtung zwischen zwei stabilen Absorptionsformen konvertiert werden: einer Blaulicht-absorbierenden (Rh-Bl) und einer UVA-Licht-absorbierenden Form (Rh-UV). Dies resultiert aus der ungewöhnlichen thermischen Stabilität des Zustandes mit deprotonierter Schiff’scher Base. Das zweite charakterisierte HKR, die Os-HKR-Rhodopsin-Domäne aus der marinen Picoalge Ostreococcus tauri, zeigt eine Dunkelabsorption von 505 nm. Auch Os-HKR ist photochrom und die deprotonierte Spezies kann effizient akkumuliert werden. Diese P400-Absorptionsform ist jedoch nicht völlig stabil sondern es kommt nach Belichtungsende zur langsamen Dunkelzustands-Regeneration. Überraschenderweise konnte die Bindung sowie die transiente Abgabe eines Anions während des Os-HKR-Photozyklus festgestellt werden. Somit beeinflusst nicht nur das Licht, sondern auch das Salz in der Umgebung die Os-HKR-Reaktionen. Aufgrund ihrer photochromen Eigenschaften werden die HKRs als wirksame lichtinduzierte Schalter für die C-terminalen Signaltransduktionsdomänen postuliert. Schwingungsspektroskopische Analysen deckten eine Heterogenität hinsichtlich der im Protein gebundenen Retinal‐Konfiguration sowie die Existenz von zwei parallelen Photozyklen auf. Jeder dieser Photozyklen geht aus einer der beiden Retinal-Isomere hervor. / Histidine kinase rhodopsins (HKRs) can be described as hybrid histidine kinases with a microbial rhodopsin as N-terminal sensory domain. HKR-encoding sequences were found in the genomes of various unicellular organisms such as algae, fungi and amoeba but their mechanistic and physiologic function is unknown. During this work the absorptive properties of the recombinant rhodopsin domains of two HKRs were studied by the usage of different spectroscopic techniques. Both HKRs showed unusual characteristics. The rhodopsin fragment of Cr‐HKR1 from Chlamydomonas reinhardtii can be interconverted between two stable absorbance forms by the alternate application of short‐ and long‐wavelength light: a blue light-absorbing dark form (Rh-Bl) and a UVA light-absorbing form (Rh-UV). This unusual photocycle results from the uncommon thermal stability of the absorbance state with a deprotonated retinal Schiff base. The second studied HKR, the Os‐HKR rhodopsin domain from the marine picoalga Ostreococcus tauri, shows an absorbance maximum at 505 nm in darkness. Likewise Cr‐HKR1 the Os‐HKR is photochromic and the deprotonated form P400 can be efficiently accumulated. But the Os-HKR P400-form is not completely stable. A slow dark state recovery occurs. Surprisingly the dark state absorbance of Os‐HKR was found to be dependent on anion binding in the protein. Furthermore during the photocycle the transient anion release occurs and therefore not only light but also salt impacts the Os-HKR-reactions. Due to their pronounced photochromic properties, the HKRs are postulated to act as effective molecular switches for the C-terminal signal transduction domains in response to the light conditions. Vibrational spectroscopy revealed the heterogeneity with regard to the retinal configuration bound in the HKRs suggesting the existence of two parallel photocycles. Either of these photocycles originates from one of the two retinal isoforms.

All life on earth depends mainly on the presence of oxygen. Largest producers of oxygen are green plants, cyanobacteria and algae. Oxygen is released from the oxygenevolving complex of photosystem II during photosynthesis and it is used in cellular respiration of all life complexes. The oxygen-evolving complex of photosystem II has the same function in each photosynthetic organism, but it has a different composition and organization of extrinsic proteins; only PsbO protein is ubiquitous in all known oxyphototrophs. Until now only low resolution electron microscopy structural models of plant PSII and crystal structures of cyanobacterial PSII are available. Higher plant extrinsic proteins (PsbP, PsbQ and PsbR) are structurally unrelated, non-homologues to the cyanobacterial extrinsic proteins (PsbO, PsbU and PsbV) and this is the reason why it is not possible to predict arrangement of these proteins on the lumenal site of higher plant PSII. Recently, models differ mainly in the structure of the oxygen-evolving complex, which could be resolved by determination of the exact binding sites for extrinsic proteins. An other question evolves: if the difference in the oxygen-evolving complex composition is the result of evolution or adaptation of photosynthetic organisms to their environment. Structural knowledge of extrinsic proteins that could help to resolve the location and subsequently the function of extrinsic proteins is still incomplete. From this case,structural analysis, interactions and probably arrangement of proteins PsbP and PsbQ was studied and is described in detail in this thesis.