Trichomonas vaginalis is flagelated microaerophilic protozoan parasite from Excavata group, which causes trichomoniasis, the most common nonviral sexually transmitted disease in the world. It causes vaginitis in women and uretritis in man and it can also cause problems for example during pregnancy. This thesis is aimed on the characterisation of hydrogenosomal iron-sulfur flavoproteins (ISF) from Trichomonas vaginalis, proteins, which were only recently discovered in the proteome of hydrogenosome of T. vaginalis. Specifically, we have focused on characterisation of ISF3 which is, according to our data, active homodimer and binds flavin mononucleotide (FMN) and iron-sulphur centre in its active site. The iron- sulphur centre is not characterised yet. ISF3 is able to reduce oxygen, hydrogen peroxide, sodium nitrate and metronidazole also in the enzymatic system with PFO and ferredoxin. Next, I tried to reduce ammonium sulphate with ISF3, but unsuccessfully. These results correspond with the activities obtained for ISF from Methanosarcina thermophila, where ISF reduces oxygen and hydrogen peroxide to water. In addition, ISF3 is able to reduce nitrogen compounds. It is important according to the fact, that metronidazole is a drug from the group of 5−nitroimidazoles. The other results show the decrease...

Flavinmononukleotid (FMN) und Flavin-Adenin-Dinukleotid (FAD) gehören zu den wichtigsten Redox-Coenzymen und sind an zentralen Stoffwechselprozessen aller Organismen beteiligt. Sie entstehen aus Riboflavin (Vitamin B2). Das bifunktionale RIBA Enzym, das Peptiddomänen für die GTP Cyclohydrolase II (GCHII) und 3,4-Dihydroxy-2-Butanon-4-Phosphat-Synthase (DHBPS) Aktivität umfasst, führt die beiden ersten Schritte der Riboflavin Biosynthese in Pflanzen durch. Die RIBA Proteine werden durch drei Gene in Arabidopsis thaliana und anderen Blütenpflanzen kodiert. Eines der Ziele war es, die physiologischen Rollen der drei RIBA Isoformen aufzuklären. Detaillierte enzymatische Untersuchungen wurden mit rekombinanten RIBA Proteinen IN VITRO durchgeführt. Es wurde gezeigt, dass RIBA2 und RIBA3 jeweils die GCHII oder die DHBPS Aktivität verloren haben. Des Weiteren konnte die Phosphorylierung von RIBA1, sowie die Hemmung von dessen GCHII Aktivität durch FMN nachgewiesen werden. Ein Knockout von RIBA1 führte zu Embryoletalität. Die schrittweise Reduzierung des RIBA1 Proteingehaltes in Arabidopsis, welches zu einem verringerten Flavingehalt führte, ergab einen schwerwiegenden Bleichungsphänotyp. Die konstitutive Antisense-Mutante, sowie das Verfahren des Virus-induzierten Gen-„Silencing“ wurden verwendet, um den durch RIBA1-Mangel hervorgerufenen Flavin-Defizienz-Effekt zu beschreiben. Eine Metabolomics Analyse ergab, dass die Abnahme des Flavingehaltes zu einer Deregulierung verschiedener Zitronensäurezyklus assoziierten Flavoenzyme und zu einer starken Reduktion der katabolischen Kapazität diverser Aminosäuren führt, während flavinabhängige Stickstoffassimilationsprozesse eher priorisiert wurden. Diese Arbeit leistet einen Beitrag zur Erweiterung des Kenntnisstands über den pflanzlichen Riboflavin Biosyntheseweg, sowie zum Verständnis über die Folgen von Flavinmangel in Pflanzen. Darüber hinaus wird hier der erste Versuch zu Erhöhung des Vitamin B2-Gehalt in Pflanzen beschrieben. / Flavin mononucleotide (FMN) and Flavin adenine dinucleotide (FAD) belong to the main redox coenzymes and are involved in central metabolic processes. They derive from riboflavin also referred to as vitamin B2. The bifunctional RIBA enzyme, which comprises peptide domains for GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) activity, performs the two initial steps of riboflavin biosynthesis in plants. Three genes encode RIBA proteins in Arabidopsis thaliana and many other flowering plants. One of the main aims of this study was to elucidate the physiological roles of the three RIBA isoforms. Detailed enzymatic studies were performed with recombinant RIBA proteins IN VITRO. It revealed for RIBA2 and RIBA3 the loss of either GCHII or DHBPS activity, respectively. The phosphorylation of RIBA1 as well as the inhibition of its GCHII activity by FMN could be demonstrated. A knockout of RIBA1, encoding the dominant RIBA isoform, led to embryo lethality. The gradual reduction of the RIBA1 protein content in Arabidopsis was associated with reduced flavin amounts and a severe bleaching phenotype that was caused by the gradual loss of pigments during leaf development. Flavin deficiency effects caused by RIBA1 depletion were characterised with a constitutive antisense mutant and the virus induced gene silencing method. A comprehensive metabolite profiling, revealed that the loss of almost one third of total flavin content led to a deregulation of several citric acid cycle-associated flavoenzymes. Moreover, a severe reduction of the catabolic capacity of numerous amino acids is seen, while seemingly flavin-dependent processes of the nitrogen assimilation are prioritised. In summary, this thesis contributes to the extended knowledge about the riboflavin biosynthesis pathway as well as to the understanding about consequences of flavin deficiency in plants. Moreover, the first attempt to increase the vitamin B2 content in plants is presented.

Die Toluol-Dioxygenase von Pseudomonas putida F1 ist eine Rieske-Dioxygenase und besteht aus Reduktase-, Ferredoxin- und Oxygenase-Komponente. Sie katalysiert den ersten Schritt im aeroben Abbau von Toluol. Ein effizienter Elektronentransfer zur terminalen Oxygenase-Komponente - an der die Sauerstoffaktivierung und Umwandlung von Toluol zum cis-Toluol-Dihydrodiol stattfindet - setzt eine reibungslose Interaktion aller Komponenten voraus. Die Ergebnisse der Stopped-flow-Messungen in der reduktiven Halbreaktion zeigen, dass NADH die Reduktase mittels Hydridtransfer reduziert, wodurch ein stabiler Ladungstransfer-Komplex zwischen NAD+ und FADH- entsteht. In der oxidativen Halbreaktion wird dieser dann durch einen Elektronenakzeptor über das blaue Semichinon zum Chinon oxidiert. Dabei zeigt sich, dass der Ladungstransfer-Komplex die Reaktion der Reduktase mit Sauerstoff unterdrückt. Eine Erklärung hierfür liefert die Kristallstruktur des Ladungstransfer-Komplexes. Die Reaktion mit Sauerstoff wird dadurch unterdrückt, dass das NAD+ koplanar mit dem Isoalloxazinring ist und den reaktiven N5-C4a Teil des FADs schützt und zudem den Isoalloxazinring in eine planare, weniger sauerstoffempfindliche Konformation zwängt. Durch die Bildung des Reduktase-Ferredoxin-Komplexes wird ein effizienter Elektronentransfer folgendermaßen ermöglicht: a) das Ferredoxin bindet an die Reduktase aufgrund elektrostatischer Anziehung entgegengesetzter Oberflächenladungen beider Proteine, b) die hydrophobe Region, die die beiden Redoxzentren umgibt, fungiert als Ein- und Ausgang für Elektronen und c) die geringe Entfernung von 11.7 Å zwischen beiden Kofaktoren erlaubt einen schnellen Elektronentransfer. Die Ergebnisse dieser Arbeit zeigen, dass der Elektronentransfer zwischen Reduktase und Ferredoxin durch die Bildung eines stabilen Ladungstransfer- und Reduktase- Ferredoxin-Komplexes beeinflusst wird und dadurch das Problem einer ungewollten Reaktion mit Sauerstoff umgangen wird. / The toluene dioxygenase from Pseudomonas putida F1 is a three-component Rieske non-heme iron dioxygenase comprising of a reductase, ferredoxin and an oxygenase component. It catalyzes the initial step in the aerobic degradation of toluene to cis-toluene dihydrodiol. A smooth interaction between all three components needs to be ensured to efficiently transfer the electrons derived from NADH oxidation to the terminal oxygenase component where molecular oxygen is activated and used for the hydroxylation of toluene. The results of the kinetic studies of the reductive half reaction of reductase reveal that NADH reduces the reductase, resulting in the formation of a stable charge transfer complex between NAD+ and FADH-. Oxidation of the charge transfer complex by an electron acceptor proceeds via the neutral semiquinone to the quinone state of FAD. It is shown that the charge transfer complex suppresses the reaction of the reductase with dioxygen. An explanation for this change in reactivity can be deduced from the structure of the charge transfer complex. Its slower reaction with dioxygen results from NAD+ lying coplanar with the FAD shielding its reactive N5-C4a locus and the forced planarity of the isoalloxazine ring. The formation of the reductase-ferredoxin complex allows efficient electron transfer from reductase to ferredoxin because a) the oppositely charged interacting surfaces of both proteins facilitate the pre-orientation of the ferredoxin on the reductase, b) a hydrophobic region surrounding the two redox centers in the complex acts as an exit/entrance port for electrons and c) the short edge-to-edge distance between both cofactors of 11.7 Å guarantees a fast electron transfer. The results demonstrate that the electron transfer between reductase and ferredoxin is governed by the formation of a stable charge transfer and of a reductase-ferredoxin complex with which the problem of an unwanted side reaction with dioxygen is obviated.

Natural products are secondary metabolites produced by plants and microorganisms that often possess medicinal properties and are implicated in organismal defense. Drawbacks to utilizing natural products in the pharmaceutical industry are difficulties with isolation from biological sources and low yields that can lack stereospecificity from synthetic sources. It is paramount to solve these issues and to develop novel natural products to combat the growing antimicrobial resistance crisis, which was responsible for ~5 million deaths in 2019 alone. One approach is utilizing enzymes to synthesize existing natural products to improve the yields and stereospecificity issue. This dissertation is focused on the biochemical characterization of three enzymes-ZvFMO, OxaD, and CreE-that are implicated in the detoxification of natural products used for organismal defense or participate in the biosynthesis of novel natural products. Each of these enzymes belong to the flavin-dependent monooxygenase (FMO) family, which catalyze the oxygenation of a substrate, generating an oxidized product. ZvFMO, from the insect food crop pest, Zonocerus variegatus, was determined to catalyze a highly uncoupled oxygenation reaction of the nitrogen or sulfur atom of various substrates. OxaD, from Penicillium oxalicum F30, catalyzes novel sequential oxidation reactions of the indole nitrogen of roquefortine C. CreE, from Streptomyces cremeus, also catalyzes sequential nitrogen oxidation reactions to convert L-aspartate to nitrosuccinate en route to biosynthesis of cremeomycin. For each enzyme, the steady-state kinetics have been determined using an oxygen consumption assay and the rapid-reaction kinetics were measured using anaerobic time-resolved spectroscopy. All three enzymes feature a fast flavin reduction step and a slow flavin dehydration step. The oxygenation chemistry of each enzyme was found to proceed through a highly reactive oxygenating species, the C4a-hydroperoxyflavin. Site-directed mutagenesis efforts led to the identification of key active site residues involved in flavin motion and substrate binding, revealing important information about the active site architecture for enzyme engineering applications and drug discovery efforts. / Doctor of Philosophy / Natural products are compounds that are produced by many plants, fungi, and bacteria that have potent medicinal properties and can be used to defend the organism against pests. Unfortunately, using these compounds widely in the pharmaceutical industry is difficult because it is hard to isolate the compound of interest from the organism that produces it and attempts to produce it chemically can result in low yields. Additionally, the overuse of the current natural products, which are most of the antibiotics on the market today, has led to an extreme increase in the resistance of bacteria, fungi, and parasites to the natural product-based drug. Therefore, it is essential that a method is developed to produce novel natural products at high yields to combat the antimicrobial resistance crisis. One method is by using enzymes to generate the natural products of interest. Enzymes are biological catalysts that speed up reactions by ensuring that less energy is required to transition from a reactant to a product and are highly efficient. This dissertation focuses on the characterization of three enzymes that could aid in our understanding of natural product chemistry. All three enzymes insert an oxygen atom on a nitrogen of their respective reactant. The first enzyme ZvFMO, is from an insect and its reactivity causes the insect to become resistant to the natural product-based plant defense mechanism, demonstrating that ZvFMO is a great candidate for inhibitor design. OxaD is the second enzyme and is involved in producing natural products that have antimicrobial and anticancer properties. The last enzyme, CreE, is involved in generating the natural product, cremeomycin, which possesses potent antimicrobial and anticancer properties as well. The reactions of OxaD and CreE positions these enzymes as candidates to produce novel natural products and other efforts to expand their reactivity. The rates of each reaction step have been determined in this work. Key amino acids that contribute to the reaction chemistry and the uptake of the reactant have been identified, laying a solid foundation for drug discovery efforts.

Complex I (NADH:ubiquinone oxidoreductase) couples electron transfer to proton translocation across the inner mitochondrial membrane, to drive the synthesis of ATP. Its distinctive L-shaped structure comprises 45 subunits, encoded by both the mitochondrial and nuclear genomes, which are assembled by a complicated modular pathway. Complex I genetic defects are the most common cause of mitochondrial disorders and often present in early childhood, with high mortality rates. Recent high-resolution electron cryo-microscopy structures of mammalian complex I provide a foundation for both interpreting biochemical and biomedical data and understanding the catalytic mechanism. First, this thesis explores how the flavin cofactor is inserted into the NADH-binding (N-) domain of complex I. Genetic manipulation of cultured human cells, to starve them of flavin, revealed a hierarchal impact on the mitochondrial flavoproteome. High riboflavin content in the growth media ameliorated observed phenotypes, requiring cell conditioning in low riboflavin conditions. CRISPR knockout of the putative mitochondrial flavin transporter SLC25A32 demonstrated the severe impact of decreased flavin on complexes I and II, and mass spectrometry 'complexome' analyses suggest that the N-domain is still assembled onto complex I in the absence of the flavin. Second, the model organism Yarrowia lipolytica was used to assess the importance of residues in the quinone-binding site of complex I. Three residues with proposed roles in binding the quinone head-group were targeted. One variant was catalytically inactive, while two retained some activity. They showed decreased ability to reduce physiologically-relevant, long chain quinones, but their ability to reduce short-chain analogues was affected less severely. The results suggest a complicated picture in which interactions between the protein and both the hydrophilic quinone head-group and hydrophobic isoprenoid chain contribute to quinone-binding affinity and catalysis. Finally, a model for human complex I, generated from a recent high-resolution structure of mouse complex I, was used to investigate whether the pathogenicity of human variants could be predicted. Structural information on variant residues, including their secondary structure, proximity to key features and surface exposure, was collated and the power of each property to predict pathogenicity investigated. The analysis was then extended to the whole structure, to identify potential pathogenic hotspots in the enzyme, inform future studies of functionally important regions in complex I, and aid the diagnosis of clinically relevant pathogenic variants.

A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power than that of the reactants with less reducing power. Highly reducing electrons at low reduction midpoint potential are required for life processes of both aerobic and anaerobic metabolic processes. For electron bifurcation to function, the semiquinone (SQ) redox intermediate needs to be destabilized in the protein to suppress its ability to trap electrons. This dissertation examines SQ suppression across a number of flavin systems for the purpose of better understanding the nature of SQ suppression within FBEB and elucidates potential mechanistic roles of SQ. The major achievement of this work is advancing the understanding of SQ suppression and its utility in flavoproteins with the capacity to bifurcate pairs of electrons. Much of these achievements are highlighted in Chapter 6. To contextualize these mechanistic studies, we examined the kinetic and thermodynamic properties of non-bifurcating flavoproteins (Chapters 2 and 3) as well as bifurcating flavoproteins (Chapters 4 and 5). Proteins were selected as models for SQ suppression with the aim of elucidating the role of an intermediate SQ in bifurcation. The chemical reactions of flavins and those mediated by flavoproteins play critical roles in the bioenergetics of all lifeforms, both aerobic and anaerobic. We highlight our findings in the context of electron bifurcation, the recently discovered third form of biological energy conservation. Bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I (Nfn) and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin were studied by transient absorption spectroscopy to compare electron transfer rates and mechanisms in the picosecond range. Different mechanisms were found to dominate SQ decay in the different proteins, producing lifetimes ranging over 3 orders of magnitude. The presence of a short-lived SQ alone was found to be insufficient to infer bifurcating activity. We established a model wherein the short SQ lifetime in Nfn results from efficient electron propagation. Such mechanisms of SQ decay may be a general feature of redox active site ensembles able to carry out bifurcation. We also investigated the proposed bifurcating electron transfer flavoprotein (Etf) from Pyrobaculum aerophilum (Pae), a hyperthermophilic archaeon. Unlike other Etfs, we observed a stable and strong charge transfer band (λmax= 724 nm) for Pae’s Etf upon reduction by NADH. Using a series of reductive titrations to probe bounds for the reduction midpoint potential of the two flavins, we argue that the heterodimer alone could participate in a bifurcation mechanism.

2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.

Choline oxidase (E.C. 1.1.3.17) from Arthrobacter globiformis catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an enzyme-bound intermediate. In each oxidative half-reaction, molecular oxygen acts as electron acceptor and is converted into hydrogen peroxide. Biochemical, structural, and mechanistic studies on the wild-type and a number of mutant variants of choline oxidase have recently been carried out, allowing for the depiction of the mechanism of alcohol oxidation catalyzed by the enzyme. Catalysis by choline oxidase is initiated by the removal of the hydroxyl proton of alcohol substrate by a catalytic base in the enzyme-substrate complex, yielding the formation of the alkoxide species. In this dissertation, the roles of His351 and conserved His466 were investigated. The results presented demonstrate that His351 is involved in the stabilization of the transition state for the hydride transfer reaction and contributes to substrate binding. His466 is likely to be a catalytic base in choline oxidase due to its dramatic effect on enzymatic activity. Comparison of choline oxidase and other enzymes within its superfamily reveals the presence of a conserved His-Asn pair within the active site of enzymes. Therefore, the role of the conserved Asn510 in choline oxidase was examined in this study. The results presented here establish the importance of Asn510 in both the reductive and oxidative half-reactions. The lost of ability to form a hydrogen bond interaction between the side chain at position 510 with neighboring residues such as His466 resulted in a change from stepwise to concerted mechanism for the cleavages of OH and CH bonds of choline, as seen in the Asn510Ala mutant. Finally, the steady-state kinetic mechanism of pyranose 2-oxidase in the pH range from 5.5 to 8.5 was investigated. It was found that pH exerts significant effects on enzyme mechanism. This study has established the involvement of the residues in the initiation of enzyme catalysis and the stabilization of the alkoxide intermediate in choline oxidase. In addition, this work demonstrates the first instance in which the kinetic mechanism of a flavin-dependent oxidase is governed by pH.

Choline oxidase catalyzes the oxidation of choline to glycine betaine. This reaction is of considerable medical and biotechnological applications, because the accumulation of glycine betaine in the cytoplasm of many plants and human pathogens enables them to counteract hyperosmotic environments. In this respect, the study of choline oxidase has potential for the development of a therapeutic agent that can specifically inhibit the formation of glycine betaine, and therefore render pathogens more susceptible to conventional treatment. The study of choline oxidase has also potential for the improvement of the stress resistance of plant by introducing an efficient biosynthetic pathway for glycine betaine in genetically engineered economically relevant crop plant. In this study, codA gene encoding for choline oxidase was cloned. The cloned gene was then used to express and purify the wild-type enzyme as well as to prepare selected mutant forms of choline oxidase. In all cases, the resulting enzymes were purified to high levels, allowing for detailed characterizations. The biophysical and biochemical analyses of choline oxidase variants in which the positively charged residue close to the flavin N(1) locus (His466) was removed (H466A) or reversed (H466D) suggest that in choline oxidase, His466 modulates the electrophilicity of the bound flavin and the polarity of the active site, and contributes to the flavinylation process of the covalently bound FAD as well as to the stabilization of the negative charges in the active site. Biochemical, structural, and mechanistic relevant properties of selected flavoproteins with special attention to flavoprotein oxidases, as well as the biotechnological and medical relevance of choline oxidase, are presented in Chapter I. Chapter II summarizes all the experimental techniques used in this study. Chapter III-VII illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter III), spectroscopic and steady state kinetics (Chapter IV), the catalytic roles of His466 and the effects of reversing the protein positive charge close to the flavin N(1) locus (Chapter V and VI), and the roles of His310 with a special attention to its involvement in a proton-transfer network (Chapter VII). Chapter VIII presents a general discussion of the data presented.

The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions.