<p>Cytochrome <i>cd</i>₁ nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the <i>d</i>₁ haem iron of the enzyme.</p><p>Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions.</p><p>Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction.</p><p>Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty.</p><p>Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.</p>

Cytochrome cd1 nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the d1 haem iron of the enzyme. Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions. Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction. Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty. Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.

Artificial systems involving water oxidation and solar cells are promising ways for the conversion of solar energy into fuels and electricity. These systems usually consist of a photosensitizer, an electron donor and / or an electron acceptor. This thesis deals with the synthesis and photoinduced electron transfer of several donor-sensitizer-acceptor supramolecular systems. The first part of this thesis describes the synthesis and properties of two novel dinuclear ruthenium complexes as electron donors to mimic the donor side reaction of Photosystem II. These two Ru2 complexes were then covalently linked to ruthenium trisbipyridine and the properties of the resulting trinuclear complexes were studied by cyclic voltammetry and transient absorption spectroscopy. The second part presents the synthesis and photoinduced electron transfer of covalently linked donor-sensitizer supramolecular systems in the presence of TiO2 as electron acceptors. Electron donors are tyrosine, phenol and their derivatives, and dinuclear ruthenium complexes. Intramolecular electron transfer from the donor to the oxidized sensitizer was observed by transient absorption spectroscopy after light excitation of the Ru(bpy)32+ moiety. The potential applications of Ru2-based electron donors in artificial systems for water oxidation and solar cells are discussed. In the final part, the photoinduced interfacial electron transfer in the systems based on carotenoids and TiO2 is studied. Carotenoids are shown to act as both sensitizers and electron donors, which could be used in artificial systems to mimic the electron transfer chain in natural photosynthesis.

This thesis presents briefly the application of quantum mechanics on systems ofchemical interest, i.e., the field of quantum chemistry and computational chemistry.The molecules of the ten-electron series, hydrogen fluoride, water, ammonia,methane and neon, are taken as computational examples. Some applications ofquantum chemistry are then shown on these systems, with emphasis on the natureof the molecular bonds. Conceptual methods of chemistry and theoreticalchemistry for these systems are shown to be valid with some restrictions, as theseinterpretations does not represent physically measurable entities.The orbitals and orbital energies of neon is studied, the binding van der Waalsinteractionresulting in a Ne2 molecule is studied with a theoretical bond lengthof 3.23 °A and dissociation energy of 81.75 μEh. The equilibrium geometries ofFH, H2O, NH3 and CH4 are studied and the strength and character of the bondsinvolved evaluated using bond order, dipole moment, Mulliken population analysisand L¨owdin population analysis. The concept of electronegativity is studied in thecontext of electron transfer. Lastly, the barrier of inversion for NH3 is studied, withan obtained barrier height of 8.46 mEh and relatively constant electron transfer.

One electron oxidation of DNA has been studied extensively over the years. When a charge is injected into a DNA duplex, it migrates through the DNA until it reaches a trap. Upon further reactions, damage occurs in this area and strand cleavage can occur. Many works have been performed to see what can affect this damage to DNA. Netropsin is a minor groove binder that can bind to tracts of four to five A:T base pairs. It has been used in the studies within to determine if it can protect DNA against oxidative damage, caused by one-electron oxidation, when it is bound within the minor groove of the DNA. By using a naphthacenedione derivative as a photosensitizer, several DNA duplexes containing netropsin binding sites as well as those without binding sites, were irradiated at 420 nm, analyzed, and visualized to determine its effect on oxidative damage. It has been determined netropsin creates a quenching sphere of an average of 5.8 * 108 Šwhether bound to the DNA or not. Herein we will show netropsin protects DNA against oxidative damage whether it is free in solutions or bound within the minor groove of a DNA duplex.

Cations are essential for ribonucleic acids (RNA), as they neutralize the negatively charged phosphate backbone. Divalent metals play important roles in the folding and function of RNA. The relationship between RNA and divalent cations magnesium (Mg(II)) and iron (Fe(II)) has been investigated. Mg(II) is involved in tertiary interactions of many large RNAs, and necessary for ribozyme activity. The influence of Mg(II) on RNA secondary and tertiary structure is investigated experimentally. Mg(II) binding to A-form RNA is accompanied by changes in CD spectra, indicating that Mg-RNA interactions influence the helical structure of RNA duplexes and helical regions of unfolded RNAs. Quantum mechanics calculations are used to probe the energetics of Mg(II)-chelation with phosphate oxygen atoms of nucleic acids. We identify the specific forces that contribute to stability of Mg(II)-chelation complexes in RNA. Fe(II) can serve as a substitute for Mg(II) in RNA folding and function. Fe(II) was abundant on early earth, it is plausible that RNA folding and function was mediated by Fe(II) instead of, or in combination with, Mg(II) in the anoxic environment of early earth. We have investigated oxidoreductase catalytic activity observed in RNA when in combination with Fe(II). This activity, only observed in the presence of Fe(II) and absence of Mg(II)appears to be a resurrection of ancient RNA capabilities that were extinguished upon the depletion of Fe(II) from the environment during the rise of oxygen after the great oxidation event. Finally, metal-ion based cleavage of RNA is used to identify the binding sites of Mg(II) and Fe(II). We observe that both metals cleave RNA in similar positions, providing further support for Fe(II) as a substitute for Mg(II) in RNA.

Heme-copper oxidases are transmembrane proteins that are found in aerobic and anaerobic respiratory chains. During aerobic respiration, these enzymes reduce dioxygen to water. The energy released in the reaction is used to transport protons across a biological membrane. Stored as proton electrochemical gradient, the energy can be used to regenerate ATP. It is known that aa3 oxidases, which are the most common oxidases, transport pumped protons and protons used for the catalytic reaction using two proton pathways. However, the molecular mechanism of pumping is still being debated. When oxygen is available in very small quantities, oxygen reductases with high affinity for oxygen are expressed by organisms like Thermus thermophilus. The proton pumping mechanism in the ba3 oxidase is slightly different from that of aa3 oxidases as this enzyme only uses a single proton uptake pathway. Here we analyzed the reaction mechanism of ba3 oxidase and found evidence that the first proton taken up by the four-electron reduced ba3 oxidase is transferred to a site distant from the catalytic site, the pump site, and that only every second proton taken up from solution is pumped. Data obtained from studies using site-directed mutagenesis and flow-flash spectroscopy suggest a probable location of the pump site. Under anaerobic conditions, some organisms are able to generate a proton- motive force using nitrate and nitrite as electron acceptors. In this process, the cytotoxic reaction intermediate nitric oxide is produced. Nitric oxide reductase (NOR), a deviant heme-copper oxidase that reduces NO to the rather harmless N2O, does not pump any protons. The catalytic mechanism of nitric oxide reduction by NOR is very poorly understood. Here we demonstrate that substrate inhibition, which occurs in NOR from Paracoccus denitrificans above 5 μM NO, can already be observed before the electrons from the low-spin hemes re-distribute to the active site. Furthermore, we found that a single specific proton pathway is used for proton-transfer leading from the periplasm to the active site.

A detailed understanding of natural photosynthesis provides inspiration for the development of sustainable and renewable energy sources, i.e. a technology that is capable of converting solar energy directly into chemical fuels. This concept is called artificial photosynthesis. The work described in this thesis contains contributions to the development of artificial photosynthesis in two separate areas. The first one relates to light harvesting with a focus on the question of how electronic properties of photosensitizers can be tuned to allow for efficient photo-induced electron transfer processes. The study is based on a series of bis(tridentate)ruthenium(II) polypyridyl complexes, the geometric properties of which make them highly appealing for the construction of linear donor-photosensitizer-acceptor arrangements for efficient vectorial photo-induced electron transfer reactions. The chromophores possess remarkably long lived 3MLCT excited states and it is shown that their excited-state oxidation strength can be altered by variations of the ligand scaffold over a remarkably large range of 900 mV. The second area of relevance to natural and artificial photosynthesis that is discussed in this thesis relates to the coupled movement of protons and electrons. The delicate interplay between these two charged particles regulates thermodynamic and kinetic aspects in many key elementary steps of natural photosynthesis, and further studies are needed to fully understand this concept. The studies are based on redox active phenols with intramolecular hydrogen bonds to quinolines. The compounds thus bear a strong resemblance to the tyrosine/histidine couple in photosystem II, i.e. the water-plastoquinone oxidoreductase enzyme in photosynthesis. The design of the biomimetic models is such that the distance between the proton donor and acceptor is varied, enabling studies on the effect the proton transfer distance has on the rate of proton-coupled electron transfer reactions. The results of the studies have implications for the development of artificial photosynthesis, in particular in connection with redox leveling, charge accumulation, as well as electron and proton transfer. In addition to these two contributions, the excited-state dynamics of the intramolecular hydrogen-bonded phenols was investigated, thereby revealing design principles for technological applications based on excited-state intramolecular proton transfer and photoinduced tautomerization.

Daktaro disertacijoje apibendrintų mokslinių tyrimų tikslas - pritaikyti aukso nanodaleles, skirtingas gliukozės oksidazes bei elektrai laidų polimerą polipirolą elektrodo modifikavimui bei gliukozės amperometriniam nustatymui biologiniais jutikliais. Aukso nanodalelės (AuND) imobilizuotos grafito elektrodo paviršiuje kartu su tirpiu elektronų pernašos tarpininku užtikrina efektyvesnę elektronų pernašą nuo gliukozės oksidazės (GOx) aktyvaus centro elektrodui fermentinės gliukozės oksidacijos metu. Naudojant AuND modifikuotus elektrodus amperometriniais biologiniais jutikliais registruojami apie 2 kartus didesni maksimalūs analiziniai signalai lyginant su elektrodu be AuND. Biologinių jutiklių tiesinės priklausomybės nuo substrato koncentracijos intervalas yra iki 10 mmol/L gliukozės ir aptikimo riba 0,08 mmol/L analitės. Po 66 dienų biologiniu jutikliu naudojančiu elektrodą modifikuotą GOx užregistruotas analizinis signalas sudarė 43 % pradinės signalo reikšmės, tuo tarpu naudojant 13,0 nm AuND ir GOx jutiklio analizinis signalas sudarė tik 22 % pradinės reikšmės. AuND esančios tiriamajame tirpale užtikrina efektyvesnę elektronų pernašą nuo gliukozės oksidazės aktyviojo centro elektrodui, kai jų koncentracija yra nuo 0,01 iki 0,60 nmol/L ir tirpale yra N-metilfenazino metosulfato. Naudojant 13,0 nm skersmens AuND, biologinio jutiklio aptikimo riba yra 0,05 mmol/L gliukozės, o tiesiškumo intervalas nuo 0,1 iki 10 mmol/L substrato. Taip pat nustatyta, kad kuo didesnė AuND... [toliau žr. visą tekstą] / The amperometric and voltammetric biosensors, based on gold nanoparticles, were designed and applied for biochemical, clinical and environment applications. Electrochemical biosensors are very selective, sensitive, fast and reusable. Gold, silver, platinum and SiO2 particles in the range 1–100 nm often provide an ideal remedy for immobilized enzymes with minimum diffusion limitations, promotion of electrochemical reaction, maximum surface area per mass unit and high effective achievement of enzymes direct wiring to electrode surface. Also, nanoparticles increase electron transfer rate between enzyme and an electrode surface. The aim of the work was to to apply different size nanoparticles, different glucose oxidases and electroconductive polymer polypyrrole for graphite electrode modification and determine the analytical characteristics of enzymatic biosensors for determination of glucose. It is established, that gold nanoparticles immobilized on graphite electrode with electron transfer mediator provide more effective electron transfer from glucose oxidase to electrode. Also, gold nanoparticles present in solution provide more effective electron transfer from enzyme to electrode when concentration of gold nanoparticles is ˂ 0.06 nmol/L and concentration of electron transfer mediator PMS is 2 mmol/L. The higher concentrations of nanoparticles in the solution make the electron transfer in the same system less efficient if compared with lower concentrations of nanoparticles... [to full text]

Daktaro disertacijoje apibendrintų mokslinių tyrimų tikslas - pritaikyti aukso nanodaleles, skirtingas gliukozės oksidazes bei elektrai laidų polimerą polipirolą elektrodo modifikavimui bei gliukozės amperometriniam nustatymui biologiniais jutikliais. Aukso nanodalelės (AuND) imobilizuotos grafito elektrodo paviršiuje kartu su tirpiu elektronų pernašos tarpininku užtikrina efektyvesnę elektronų pernašą nuo gliukozės oksidazės (GOx) aktyvaus centro elektrodui fermentinės gliukozės oksidacijos metu. Naudojant AuND modifikuotus elektrodus amperometriniais biologiniais jutikliais registruojami apie 2 kartus didesni maksimalūs analiziniai signalai lyginant su elektrodu be AuND. Biologinių jutiklių tiesinės priklausomybės nuo substrato koncentracijos intervalas yra iki 10 mmol/L gliukozės ir aptikimo riba 0,08 mmol/L analitės. Po 66 dienų biologiniu jutikliu naudojančiu elektrodą modifikuotą GOx užregistruotas analizinis signalas sudarė 43 % pradinės signalo reikšmės, tuo tarpu naudojant 13,0 nm AuND ir GOx jutiklio analizinis signalas sudarė tik 22 % pradinės reikšmės. AuND esančios tiriamajame tirpale užtikrina efektyvesnę elektronų pernašą nuo gliukozės oksidazės aktyviojo centro elektrodui, kai jų koncentracija yra nuo 0,01 iki 0,60 nmol/L ir tirpale yra N-metilfenazino metosulfato. Naudojant 13,0 nm skersmens AuND, biologinio jutiklio aptikimo riba yra 0,05 mmol/L gliukozės, o tiesiškumo intervalas nuo 0,1 iki 10 mmol/L substrato. Taip pat nustatyta, kad kuo didesnė AuND... [toliau žr. visą tekstą] / The amperometric and voltammetric biosensors, based on gold nanoparticles, were designed and applied for biochemical, clinical and environment applications. Electrochemical biosensors are very selective, sensitive, fast and reusable. Gold, silver, platinum and SiO2 particles in the range 1–100 nm often provide an ideal remedy for immobilized enzymes with minimum diffusion limitations, promotion of electrochemical reaction, maximum surface area per mass unit and high effective achievement of enzymes direct wiring to electrode surface. Also, nanoparticles increase electron transfer rate between enzyme and an electrode surface. The aim of the work was to to apply different size nanoparticles, different glucose oxidases and electroconductive polymer polypyrrole for graphite electrode modification and determine the analytical characteristics of enzymatic biosensors for determination of glucose. It is established, that gold nanoparticles immobilized on graphite electrode with electron transfer mediator provide more effective electron transfer from glucose oxidase to electrode. Also, gold nanoparticles present in solution provide more effective electron transfer from enzyme to electrode when concentration of gold nanoparticles is ˂ 0.06 nmol/L and concentration of electron transfer mediator PMS is 2 mmol/L. The higher concentrations of nanoparticles in the solution make the electron transfer in the same system less efficient if compared with lower concentrations of nanoparticles... [to full text]