In the present thesis the density functional B3LYP has been used to study reactions of NO and O2 in redox active enzymes. Reduction of nitric oxide (NO) to nitrous oxide (N2O) is an important part in the bacterial energy conservation (denitrification). The reduction of NO in three different bimetallic active sites leads to the formation of hyponitrous acid anhydride (N2O22-). The stability of this intermediate is crucial for the reaction rate. In the two diiron systems, respiratory and scavenging types of NOR, it is possible to cleave the N-O bond, forming N2O, without any extra protons or electrons. In a heme-copper oxidase, on the other hand, both a proton and an electron are needed to form N2O. In addition to being an intermediate in the denitrification, NO is a toxic agent. Myoglobin in the oxy-form reacts with NO forming nitrate (NO3 -) at a high rate, which should make this enzyme an efficient NO scavenger. Peroxynitrite (ONOO-) is formed as a short-lived intermediate and isomerizes to nitrate through a radical reaction. In the mechanism for pumping protons in cytochrome oxidase, thermodynamics, rather than structural changes, might guide protons to the heme propionate for further translocation. The dioxygenation of arachidonic acid in prostaglandin endoperoxide H synthase forms the bicyclic prostaglandin G2, through a cascade of radical reactions. The mechanism proposed by Hamberg and Samuelsson is energetically feasible.

This thesis deals with the use of electric fields for evaluation and control of chemical systems. An electric field can result in the flow of charge across an interface between a metal and a solution, by means of chemical reactions. This interplay between electricity and chemistry, i.e. electrochemistry, is a field of crucial importance both within research and industry. Applications based on electrochemical principles encompass such diverse areas as batteries and fuel cells, pH electrodes, and the glucose monitor used by people suffering from diabetes.A major part of the present work concerns the use of static electric fields in solutions containing a non-contacted metal surface. In such a setup it is possible to control the extent of electrochemical reactions at different positions on the metal. This allows the formation and evaluation of various types of gradients on electrodes, via indirectly induced electrochemical reactions. This approach is a new and simple way of forming for instance molecular gradients on conducting surfaces. These are very advantageous in biomimetic research, because a gradient contains a huge amount of discrete combinations of for example two molecules. The basis for the technique is the use of bipolar electrochemistry. Briefly, a surface can become a bipolar electrode (an electrode that acts as both anode and cathode) when the electric field in the solution exceeds a certain threshold value, thereby inducing redox reactions at both ends. In our experiments, the driving force for these reactions will vary along the electrode surface. Since the result of an electrochemical reaction can be the deposition or removal of material from an electrode, bipolar electrochemistry can be used to create gradients of that material on a surface. In order to gain a deeper understanding of these processes, the potential and current density distributions at bipolar electrodes were investigated with different methods. Especially the use of imaging techniques was important for the visualization and analysis of the gradients. Using this knowledge, the formation of more complex gradients was facilitated, and the results were further compared to simulations based on simple conductivity models. These simulations also provided us with means to predict the behavior of new and interesting setup geometries for pattering applications.The other major part is more application driven and deals with the use of alternating electric fields for chemical analysis, a technique known as electrochemical impedance spectroscopy (EIS). In this work, EIS has been applied for the analysis of engine oils and industrial cutting fluids. Emphasis was placed on practical aspects of the measurement procedure, and on the evaluation of the results using statistical methods. It was for example shown that it was possible to simultaneously determine the amount of different contaminants in low conducting solutions. Generally, EIS is used to measure the impedance of a solution or a solid, often as a function of the frequency of the alternating electric field. The impedance of a system is closely correlated to its complex dielectric constant, and EIS can therefor be used to examine many chemical and physical processes. It is further well suited for characterizing low conducting media with little or no redox-active species. The evaluation of impedance data is often a quite complex task, which is why we have made use of statistical methods that drastically reduce the effort and quickly reveal significant intrinsic parameters.

Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

Photoinduced electron transfer is involved in a number of photochemical and photobiological processes. One example of this is photosynthesis, where the absorption of sunlight leads to the formation of charge-separated states by electron transfer. The redox equivalents built up by successive photoabsorption and electron transfer is further used for the oxidation of water and reduction of carbon dioxide to sugars. The work presented in this thesis is part of an interdisciplinary effort aiming at a functional mimic of photosynthesis. The goal of this project is to utilize sunlight to produce renewable fuels from sun and water. Specifically, this thesis concerns photoinduced electron transfer in donor(D)-photosensitizer(P)-acceptor(A) systems, in mimic of the primary events of photosynthesis. The absorption of a photon typically leads to transfer of a single electron, i.e., charge separation to produce a single electron-hole pair. This fundamental process was studied in several molecular systems. The purpose of these studies was optimization of single electron transfer as to obtain charge separation in high yields, with minimum losses to competing photoreactions such as energy transfer.Also, the lifetime of the charge separated state and the confinement of the electron and hole in three-dimensional space are important in practical applications. This led us to explore molecular motifs for linear arrays based on Ru(II)bis-tridentate and Ru(II)tris-bidentate complexes. The target multi-electron catalytic reactions of water-splitting and fuel production require a build-up of redox equivalents upon successive photoexcitation and electron transfer events. The possibilities and challenges associated with such processes in molecular systems were investigated. One of the studied systems was shown to accumulate two electrons and two holes upon two successive excitations, without sacrificial redox agents and with minimum yield losses. From these studies, we have gained better understanding of the obstacles associated with step-wise photoaccumulation of charge and how to overcome them.

<p>We have studied fundamental aspects of time-resolved two-photon excited fluorescence depolarisation. The thesis presents experimental as well as theoretical progress. We show that a multi-photon induced instrumental response function obtained from a suspension of gold nanoparticles is appropriate for the analysis of two-photon excited fluorescence decays obtained using time-correlated single photon counting detection. Theoretical expressions have been derived for the fluorescence anisotropy decay obtained upon two-photon excitation of various molecular systems in liquid solutions: a) an anisotropic rigid rotor that undergoes rotational diffusion in the presence of ultrafast unresolved restricted reorientations, e.g. librations. b) a molecular group covalently attached to a stationary macromolecule, and undergoing local reorientation in a uniaxial ordering potential. A new approach to the analysis of two-photon excited fluorescence depolarisation experiments was developed, which combines data obtained by using linearly and circularly polarised excitation light, in a global manner. In the analysis, knowledge about unresolved reorientations was obtained from one-photon excitation studies of the corresponding systems. By means of this procedure it has been possible to obtain quantitative information about the molecular two-photon absorption tensor for perylene and two of its derivatives. Thereby the symmetry of the final excited and intermediate vibronic states could be assigned. The analysis reveals that the two-photon transition studied with the 800 nm laser exhibits mixed character. An important finding from the experiments was that the two-photon absorption tensor appears to be solvent dependent. Furthermore, the thesis presents the first theoretical treatment of two-photon excited donor-donor energy migration in the presence of molecular reorientation and which applies the extended Förster theory. Explicit expressions for molecules that belong to the point groups D_2h, D₂ and C_2v are given. Preliminary experiments are finally also reported on a two-photon excited donor-donor energy migration system consisting of a bisanthryl-bisteroid. </p>

We have studied fundamental aspects of time-resolved two-photon excited fluorescence depolarisation. The thesis presents experimental as well as theoretical progress. We show that a multi-photon induced instrumental response function obtained from a suspension of gold nanoparticles is appropriate for the analysis of two-photon excited fluorescence decays obtained using time-correlated single photon counting detection. Theoretical expressions have been derived for the fluorescence anisotropy decay obtained upon two-photon excitation of various molecular systems in liquid solutions: a) an anisotropic rigid rotor that undergoes rotational diffusion in the presence of ultrafast unresolved restricted reorientations, e.g. librations. b) a molecular group covalently attached to a stationary macromolecule, and undergoing local reorientation in a uniaxial ordering potential. A new approach to the analysis of two-photon excited fluorescence depolarisation experiments was developed, which combines data obtained by using linearly and circularly polarised excitation light, in a global manner. In the analysis, knowledge about unresolved reorientations was obtained from one-photon excitation studies of the corresponding systems. By means of this procedure it has been possible to obtain quantitative information about the molecular two-photon absorption tensor for perylene and two of its derivatives. Thereby the symmetry of the final excited and intermediate vibronic states could be assigned. The analysis reveals that the two-photon transition studied with the 800 nm laser exhibits mixed character. An important finding from the experiments was that the two-photon absorption tensor appears to be solvent dependent. Furthermore, the thesis presents the first theoretical treatment of two-photon excited donor-donor energy migration in the presence of molecular reorientation and which applies the extended Förster theory. Explicit expressions for molecules that belong to the point groups D2h, D2 and C2v are given. Preliminary experiments are finally also reported on a two-photon excited donor-donor energy migration system consisting of a bisanthryl-bisteroid.