Spectroscopic studies have been used to describe the mechanism of the decomposition of hydrogen peroxide by solutions of a dimeric Cu(II) complex of a dissymetric Schiff base, [CuSALAD]2.H2O, and imidazole or methyl substituted imidazoles, B, which form monomeric CuSALAD.B2 complexes, in aqueous ethanol solvent. Freezing point depression and vapor pressure lowering studies were carried out to confirm the dimeric nature of the [CuSALAD]2.H2O complex that had been previously reported. The stoichiometry of the [CuSALAD]2.H2O-imidazole equilibrium was extensively studied pointing to a 1:4 stoichiometry. The CuSALAD.B2 adducts exhibited certain catalytic properties that mimic those of catalase enzymes. The different imidazoles were buffered to acidic, neutral and basic pH media in order to investigate the pH effects of this reaction. Two charge transfer (CT) bands were observed near 420 and 450 nm upon addition of hydrogen peroxide to CuSALADB2 solutions, and were associated with two proposed intermediates (CuBOOH and CuBOOCu). A mechanism consistent with these results has been developed. First order dependence of the rate on CuSALAD.B2 was observed in the presence of excess CuSALAD.B2 over hydrogen peroxide, whereas second order dependence was observed with the latter in excess. The CuBOOCu intermediate was unstable in the presence of EDTA, and a first order dependence of rate of formation of intermediate on both CuSALAD.B2, and hydrogen peroxide was observed.

A scientific and cultural revolution occurred with the sequencing of the human genome. The information provided by this accomplishment has provided tools for researchers to test new ideas in silico and on the bench. In redox biology many of the genes, transcripts, proteins, and redox active species have been well characterized. However, the vast majority have not been quantitated in an absolute manner. This is a necessary step to provide the tools for mathematical modeling and systems biology approaches for predicting changes in the cellular redox environment and the biochemical and biological consequences. Here we demonstrate techniques for the absolute quantitation of human catalase, glutathione peroxidase, peroxiredoxin, thioredoxin, and superoxide dismutase within cells. These techniques can be parsed into two groups: detection of activity and detection of total amount of species. Methods for the absolute quantitation of active catalase, peroxiredoxins, and superoxide dismutase have been developed by utilizing specific characteristics of each enzyme. Catalase generates oxygen in the presence of hydrogen peroxide that can easily be detected with a Clark electrode (oxygen monitor); the data are fit to a single-exponential to determine the observed pseudo-first-order rate constant. From this the effective number of fully active catalase enzymes in the sample can be determined. Peroxiredoxin in the disulfide state can be reduced by thioredoxin; thioredoxin from E. coli loses fluorescence upon oxidation. The loss of fluorescence over time is mathematically fit to a single-exponential to determine the observed pseudo first-order rate constant from which the number of active enzymes can be determined. Using an inhibition assay to detect superoxide dismutase activity along with the rate constants at which superoxide reacts with the dismutase and the competing superoxide-reacting-indicator-molecule, the concentration of active superoxide dismutase can be determined. To detect the total amount of protein of an enzyme in a biological sample, an immunoassay was first implemented. This method utilized Bio-Plex® beads from Bio-Rad; however, it was problematic because the antibodies applied did not perform satisfactorily not allowing sufficient signal-to-noise to be deployed. Quantitative mass spectrometry was then implemented to detect total catalase, glutathione peroxidase 1, peroxiredoxin 2, and thioredoxin 1 in human red blood cells. With the absolute concentration of these enzymes and proteins along with data for oxygen consumption rates and peroxisomal hydrogen peroxide concentration for several cell lines, we hypothesize that a reasonable model of hydrogen peroxide and superoxide flux can be constructed. Quantitative data such as these provide the foundation for the new redox biology of the 21st century. Presented here is a roadmap for the obligatory first steps to dissect quantitatively the cellular and tissue metabolic pathways and redox networks that are the basis of all of biology.

Vitamin D is hydroxylated to form several active metabolites, of these, 1,25- dihydroxyvitamin D3 [1,25(OH)2D3] is the most studied stimulatory product. It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate homeostasis through its membrane receptor 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. Another metabolite, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] has been reported to be inhibitory with respect to calcium and phosphate absorption in intestine. Previous work in this laboratory has indicated that 24,25(OH)2D3 inhibits phosphate uptake in isolated intestinal cells and perfused duodenal loops and in vivo. This thesis further tested the hypothesis that the actions of 24,25(OH)2D3 on phosphate homeostasis are physiologically important. Catalase has been identified as a binding protein for 24,25(OH)2D3. We determined the localization of catalase in the presence and absence of steroid, monitored catalase mRNA levels related to gene 24,25(OH)2D3 gene transcription regulation. We studied the effects of the two isomers of 24,25(OH)2D3 on localization of catalase in chicken enterocytes over a time course of 15 sec to 60 min. It was demonstrated that 24R,25(OH)2D3 is the effective metabolite for catalase redistribution in vitro. We also studied the effects of vitamin D on catalase and phosphate uptake in chicken intestinal cells. It was once again demonstrated that 24R,25(OH)2D3 is the effective metabolite for decreasing phosphate uptake and catalase gene expression. These combined results lead us to conclude that 24,25(OH)2D3 is an important hormone in phosphate homeostasis in chick intestinal epithelial cells.

Lors de la dégradation du peroxyde d'hydrogène en eau et dioxygène catalysée par les hémoenzymes à fer de type catalase et peroxydase, il se forme à l'échelle de la milliseconde un intermédiaire réactionnel radicalaire porté par la porphyrine. Dans le cas de l'enzyme modèle utilisée, la catalase de foie de bœuf, il a été montré par des études de spectroscopie RPE que ce radical est ensuite délocalisé sur un résidu tyrosyle de la chaîne polypeptidique. A ce jour, on ne connaît pas l'exacte localisation du résidu impliqué, donc le rôle de ce site d'oxydation alternatif. <br />Par ailleurs, il a été montré au laboratoire que l'identification et la quantification des radicaux formés sur les acides aminés d'une protéine par l'attaque de radicaux hydroxyle sont possibles. Cette méthode est basée sur le marquage au tritium des résidus acides aminés. Notre approche est basée sur la génération de radicaux hydroxyle par radiolyse de l'eau. Les radicaux hydroxyle formés arrachent un hydrogène sur la chaîne latérale des acides aminés et génèrent ainsi un radical carboné. Il est ensuite “réparé” in situ par un composé, le sel sodique de l'acide phénylphosphinique tritié, qui permet d'introduire un atome de tritium à la place de l'hydrogène précédemment arraché. Cet atome de tritium sert de marqueur pour détecter les sites de formation des radicaux. <br />Nous avons donc utilisé les propriétés de réparation du vecteur tritié pour identifier quelle est la tyrosine impliquée dans les transferts d'électrons de la BLC. Même s'il a été montré par RPE que la disparition du radical porté par la tyrosine est effective en présence de l'agent de réparation, les études de marquage n'ont pas abouti à déterminer l'exacte localisation du radical. Une des raisons invoquées est le manque d'efficacité de l'agent de réparation pour transférer son atome d'hydrogène. C'est pourquoi d'autres composés capables eux aussi de fournir un atome d'hydrogène par voie radicalaire ont été synthétisés puis testés sur ce système enzymatique par une étude de spectroscopie RPE.<br />En parallèle, nous avons voulu comprendre les mécanismes d'action des ces mêmes composés sur un système modèle en générant des radicaux sur la tyrosine en solution par radiolyse de l'eau. La méthode consiste à produire dans une solution aqueuse de tyrosine des radicaux hydroxyle, qui vont former les radicaux tyrosyle. Les radicaux ainsi générés peuvent être ensuite réparés par un atome de deutérium fourni par un donneur. L'incorporation en deutérium et la régiosélectivité de l'attaque sont ensuite analysées par spectrométrie de masse et RMN 2H. L'irradiation de solution de tyrosine en présence des différents composés choisis s'est révélée difficile à analyser, en raison notamment de la difficulté à déterminer la proportion de radicaux hydroxyle réagissant avec l'agent réparateur au lieu de la tyrosine, mais surtout en raison de l'incorporation inattendue de deutérium dans la tyrosine en l'absence de tout agent de transfert. Ce phénomène jusqu'alors inconnu a, dès lors, retenu toute notre attention. Nous avons alors focalisé nos travaux sur la compréhension des processus intervenant dans l'autoréparation de la tyrosine et ainsi proposé un mécanisme pour expliquer nos observations.

Les catalases, les peroxydases et les catalase-peroxydases sont des hémoprotéines, dont l'activité enzymatique essentielle est la régulation de la concentration cellulaire du peroxyde d'hydrogène. Au cours de ce travail de thèse, nous avons mis en évidence et caractérisé, dans ces protéines, des intermédiaires réactionnels issus de l'oxydation de tyrosines et/ou de tryptophanes, alternatifs au composé I, [Fe(IV)=O Por•+]. La prise en compte de la formation de tels radicaux protéiques nous a amené à réexaminer le mécanisme des catalases et des peroxydases, jusque là centré sur l'oxydo-réduction de l'hème. Pour déterminer les structures électroniques et la réactivité des intermédiaires réactionnels, nous avons utilisé une approche multidisciplinaire combinant les spectroscopies de Résonance Paramagnétique Electronique (RPE) multifréquences (9-285 GHz), d'absorption électronique UV-visible par stopped-flow, ainsi que la mutagenèse dirigée et le marquage isotopique. L'étude comparative de huit catalases, provenant de souches bactériennes et de mammifères, nous a permis de démontrer que la température de réaction, le pH et l'excès d'oxydant favorisent la formation de l'intermédiaire [Fe(IV)=O Tyr•], résultant du transfert d'électron intramoléculaire entre la tyrosine et la porphyrine dans les catalases de foie de boeuf, d'érythrocytes humains, de B. abortus et de M. lysodeikticus. Nos études ont montré que l'intermédiaire [Fe(IV)=O Por•+] est prédominant à T ≥ 20°C. Ceci explique pourquoi les études cinétiques de la littérature, pour lesquelles seule l'absorption UV-visible à température ambiante a été utilisée, n'avaient pas permis de mettre en évidence la formation du radical tyrosyle. Nos résultats expliqueraient aussi les différences d'efficacité de dismutation du peroxyde d'hydrogène de ces catalases. En effet, le transfert spontané d'électron entre la tyrosine et la porphyrine, favorisé dans certaines catalases, serait en compétition avec la réaction entre l'intermédiaire [Fe(IV)=O Por•+] et le peroxyde d'hydrogène. Dans le cadre d'études spécifiques sur les catalase-peroxydases (KatGs) et en utilisant la catalase-peroxydase de B. pseudomallei comme cas d'étude, nous avons pu mettre en évidence la formation d'un radical tyrosyle et de deux intermédiaires tryptophanyles, dont l'un est formé sur le Trp330, équivalent au site radicalaire de la cytochrome c peroxydase. Nous avons démontré que l'intermédiaire [Fe(IV)=O Por•+] réagit avec l'ABTS et que le [Fe(IV)=O Trp330 •+] n'est pas l'intermédiaire réactif vis-à-vis de l'isoniazide (antibiotique utilisé dans le traitement de la tuberculose). Notre étude comparative de sept catalaseperoxydases a démontré que le site de formation du radical tryptophanyle est unique pour chaque enzyme, mais peut différer entre deux protéines, ce qui expliquerait en partie leurs différences de réactivité vis-à-vis de l'ABTS et de l'isoniazide. Nous avons montré que dans les catalase-peroxydases, les radicaux protéiques peuvent jouer le rôle de cofacteurs dans l'oxydation des substrats et que le mécanisme de la réaction de dismutation du peroxyde d'hydrogène diffère de celui des catalases monofonctionnelles. Afin de comprendre les propriétés physico-chimiques permettant et favorisant la formation des radicaux protéiques, en tant qu'intermédiaires alternatifs à l'intermédiaire [Fe(IV)=O Por•+], nous avons caractérisé une peroxydase de raifort reconstituée, dans laquelle l'hème à été remplacé par un hème lié par liaison covalente à une tyrosine ou à un tryptophane.

Scytalidium thermophilum is a termophilic fungus that effectively produces the extracellular enzyme catalase phenol oxidase (CATPO). The enzyme is distinct among catalases with its bifunctionality of oxidising phenolic compounds in the absence of H2O2. CATPO is capable of oxidizing catechol, chlorogenic acid, caffeic acid and catechin which are ortho &ndash / diphenolic compounds. Diphenolic compounds are known as strong antioxidants. Catalase is one of the important antioxidant enzymes. Therefore, in this thesis the effect of CATPO on the final antioxidant capacity of the oxidized products was analysed. Antioxidant capacity measurements of oxidized and unreacted phenolic compounds were done using the two widely used methods TEAC and FRAP. CATPO oxidized catechol showed 2.4 fold increase when compared to its nonoxidized form, which was highest among others. Catechol was followed by caffeic acid, chlorogenic acid, and catechin. This finding is new to the literature and may be of importance to the antioxidant mechanism of organisms. Results have also shown that the most well known phenol oxidases, laccase and tyrosinase, do not result in such high increases in antioxidant capacity upon oxidation of the substrates tested. Due to this finding, as a possible means of applying CATPO to increase the antioxidant capacity of products daily consumed, tea was selected. Tea is the second most consumed beverage after water and it is known to possess high amounts of flavanols. Green tea is rich in catechins whereas black tea is a rich source of theaflavins and thearubigins. Fermentation is a critical process for production of good quality tea and is the key step differing between green and black tea production. During this process phenol oxidases catalyze the oxidation of polyphenolic compounds present in tea leaves to their corresponding o-quinones. Utilization of CATPO in tea samples resulted in an increase in antioxidant capacity and its effect was enhanced by an increase in brewing time. Interestingly, the addition of sugar decreased antioxidant capacity. Laccase and tyrosinase were ineffective in increasing the antioxidant capacity of tea samples.

A major sweet potato leaf-type catalase was detected and identified from fullyexpandedmature leaves using in-gel activity staining assay with native- andSDS-PAGEs. The putative catalase activity band was inhibited by a catalaseinhibitor 3-amino-1,2,4-triazole. The major leaf-type catalase activity wasoptimal over 8, and was significantly repressed by £]-mercaptoethanol. However,its activity was much less affected by temperature within the range of 5 to 450C.Temporal and spatial expression showed that it was specifically detected inleaves, but not in roots and stems. Its activity increased from the immature L2leaves, and reached the maximal at the fully-expanded mature L3 leaves, thenslightly decreased in partial yellowing senescent L4 leaves, and was almost notdetected in completely yellowing L5 leaves similar to folding unopenedimmature L1 leaves. The catalase level showed approximately inversecorrelation with the H2O2 amounts in leaves of different developmental stages.Dark and ethephon, an ethylene-releasing compound, also temporarily enhancedthe catalase activities from 6 h to 24 h, however, the enhanced activitydecreased from 24 h to 48 h in detached leaves after treatment. The catalaselevel also showed approximately negative correlation with the H2O2 amounts intreated leaves. The major leaf-type catalase activity was repressed by EGTA,and the repression can be reversed by exogenous CaCl2. The major leaf-typecatalase activity was also repressed by calmodulin inhibitor chlorpromazine,and the repression can be reversed by exogenous purified SPCAM calmodulinfusion protein. Chlorpromazine-treated leaves also elevated H2O2 amount.Based on these data we conclude that a major leaf-type catalase with maximalactivity in L3 leaf was identified in sweet potato. Its activity was temporarilyenhanced by dark and ethephon, and was modulated by external calcium ion(Ca2+) and calmodulin. A possible physiological role and function in associationwith cellular H2O2 homeostasis in cope with developmental and environmentalcues in sweet potato leaves is suggested.

In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the enzyme was shown to be a catalase, with an additional catechol oxidase activity. This novel bifunctional catalase-catechol oxidase (CCO) was purified 10 fold with 45 % yield by anion exchange and gel filtration chromatographies. CCO was determined as a tetrameric protein having total and subunit molecular weights of 320 and 80 kDa, respectively. Isoelectric point of CCO was verified as 5.0. CCO catalase and catechol oxidase activities were characterized in terms of their kinetic behavior at different pH and temperatures. Depending on the substrate specificity and inhibitor studies of CCO, the phenol oxidase activity was determined as catechol oxidase but not tyrosinase or laccase. The best crystallization condition for CCO was determined and X-ray diffraction data was collected at the Daresbury Synchrotron Radiation Source (United Kingdom) at 2.7 &Aring / resolution. The preliminary structure was solved by molecular replacement method using Penicilium vitale catalase structure. CCO was verified to have a tetrameric structure with two homodimers and a metal center in each polypeptide chain.

The aim of this study was to analyze the effect of hydrogen peroxide and selected phenolic compounds on growth and catalase production of Aspergillus fumigatus. As a result of growing A. fumigatus at different temperatures it was observed that, growth and catalase production of this species were highest at 37 &deg / C. Catalase production was highest in the presence of 1 mM H2O2, yielding a significant 3 fold increase with respect to the control. Biomass was also increased by 1,44 fold with respect to the control sample. H2O2 increased catalase production possibly by inducing oxidative stress as biomass production significantly increased after the depletion of H2O2. Both gallic acid and trans-resveratrol significantly enhanced biomass generation of A. fumigatus (1,17 fold increase at 10 mM gallic acid and 1,45 fold increase at 3 mM resveratrol with respect to controls) and decreased extracellular catalase production (4,33 fold at 25 mM gallic acid and 16,7 fold decrease at 3 mM resveratrol with respect to controls) especially in the first 5 or 6 days of the cultivation where the anti-oxidant activity of the compounds were possibly at their maximum. A sudden and significant rise was observed in extracellular catalase activity between 5th and 7th days of the cultivation in phenolic compound applied samples, possibly owing to the depletion of the antioxidant activity of gallic acid and resveratrol followed by fungal cells&rsquo / response to a sudden increase of oxidative stress by boosting catalase production.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.