No presente trabalho foram investigadas as propriedades de centros de cor, de centros paramagnéticos e de centros de termoluminescência e, sua dependência com os defeitos intrínsecos e extrínsecos do berilo cor de rosa (chamada morganita) natural. O berilo, de fórmula química Be3 Al2 Si6 O18 é um cristal de silicato de alumínio e berílio que, por ser natural, encerra na sua rede cirstalina inúmeras impurezas. A análise por fluorescência de raios X revelou Na, Fe, Mn, Co, K, como átomos estranhos à rede cristalina com maior concentração e outros elementos em menor quantidade. O presente trabalho mostrou que somente Fe, Mn e Na (talvez K) como impureza atuantes, as outras não tendo influência sobre as propriedades em estudo. Uma amostra de morganita, tratada em 600º por uma hora, depois de irradiada com raios gama de uma fonte de 60Co, apresentou picos termoluminescentes (TL) em torno de 160ºC, 220ºC, 340ºC. A altura do pico de 160ºC cresce muito rapidamente com a dose da radiação tal que, os dois outros ficam escondidos. Estes podem ser destacados submetendo a amostra irradiada a um recozimento em 160ºC por 5 a 10 minutos. Quando uma amostra natural é tratada termicamente entre 500ºC e 900ºC por cerca de uma hora, a irradiação subsequente (no caso com 2 kGy de dose), produz pico TL em 160ºC crescente com a temperatura, enquanto que os dois outros picos decrescem. O tratamento térmico provoca um rearranjo na rede cristalina que favoreceu o crescimento do pico TL em 160ºC. A irradiação com luz UV de uma lâmpada de Hg (e também de Xe) induziu termoluminescência. Como a energia de um fóton de UV é meno do que a largura da banda proibida, esse resultado foi interpretado à luz da absorção de dois fótons, mecanismo, estudado por Maria Göppert-Mayer em 1931. O espectro de EPR da morganita natural apresenta sinais típicos de Mn2+, Fe3+ e do átomo H0. O centro H0 cresce com irradiação - gama, mas, é destruída por calor, a intensidade do sinal decaindo entre 160ºC e 300ºC. Na região do campo magnético entre 3100 e 3500 Gauss, são observados varias linhas EPR. A linha em g 2,0143 foi identificado como sendo devido ao radical CO3 proveniente de CO-2.3->CO-3 +e- ou HCO-3->H0+ CO-3, ambas as reações ocorrendo durante a irradiação. As linhas entre 3350 Gauss e 3360 Gauss podem ser uma superposição das linhas de Fe3+ e CH4. O espectro de absorção ótica de uma amostra natural entre 200 nm e 3200 nm apresentou bandas intensas de H2O, que são encontradas grande quantidade nos canais do berilo. O limiar de absorção UV situa-se em cerca de 350 nm. Bandas muito fracas são observadas na região visível, entre elas, as conhecidas bandas de Maxixe. Uma banda típica devido a Fe2+ é observada em 820 nm, que não se altera muito quando a morganita natural sobre um tratamento térmico em 700ºC por uma hora. As outras bandas, exceto de H2O, sofrem decréscimos consideráveis. Esse tratamento térmico revela bandas em 205 e 235 nm na região de UV. Uma irradiação muito intensa até 68 kGy aumenta a absorção UV, incluindo as bandas em 205 e 235 nm, atingindo a intensidade de absorção constante e patamar entre 200 e 250 nm. Essas irradiações intensas aumentam ligeiramente a banda de Fe2+, mas, tornam evidentes as bandas de Maxixe e bandas em 430 nm e 555 nm. / The relationship between point defects in a natural pink beryl (morganite) and its optical absorption, electron spin resonance and thermoluminescensce properties has been investigated. Beryl with chemical formula Be3 Al2 Si6 O18, is an aluminum silicate of beryllium, it is found in nature containing several impurities. A X-ray fluorescence analysis revealed Na, Fe, Mn, Co and F as impurities with large concentration and others in smaller concentration. The present work has shown that only Na, Fe an Mn have influence on properties of interest while others have none. A sample of morganite heat treated at 600°C for one hour, after being irradiated with 60Co gamma-rays exhibits thermoluminescesce (TL) peaks at 160, 220 and 340°C. the TL peak at 160°C grown very fast with radiation dose and the two others become hidden. It is necessary to annel at 160°C for few minutes in order to allow the TL peaks at 220 and 340°C to be isolated. The UV light from Hg lamp (and also Xe lamp) induces TL. Since an UV photon has not enough energy to promote one electron from the valence band to the conduction band, this result was considered as the effect of two photon absorption. Reported long time ago by M. Goppert-Mayer. The EPR spectrum of natural morganite shows the typical signals of Mn2+, Fe3+ and H°-center. H°-center grows with irradiation, but it decreases with heat, its intensity decreases between 160 and 300°C. In the interval of 3100 and 3500 Gauss of magnetic field, several EPR lines are observed, being the g 2,0143 signal identified as the CO-3 radical. This comes from the reaction CO2-3 -> CO-3 +e - or from HCO-3 -> H° + CO-3, both of them induced by irradiation. The lines seen between 3350 and 3360 Gauss can be attributed to the superposition of Fe3+ and CH4 lines. The optical absorption spectrum between 200 and 3200 nm has presented very strong bands due to H2O which are found in large amount in the beryl channels. The UV absorption edge occurs at around 350 nm. Few weak bands are present in visible region, three of them are known as Maxixe bands. A band at 820 nm is characterized of Fe2+, this band changes little on irradiation or heating. Except the H2O bands, there are bands decaying considerably under heat treatment: at 500 to 800°C, 700°C for one hour annealing shows clearly absorption bands at 205 and 235 nm, both in UV region. Irradiations up to about 70 kGy (very strong) increase highly the UV absorption including 205 and 325 nm. Such absorption is observed as a constant plateau located between 200 and 250 nm, for high dose. Strong irradiation changes slightly the Fe2+ band, and the other bands in the visible grow considerably.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

<p>Diiron carboxylate proteins constitute an important class of metall-containing enzymes. These proteins perform a multitude of reactions in biological systems that normally involve activation of molecular oxygen at the diiron site.</p><p>During activation and functioning of these proteins their diiron sites undergo redox changes in a rather wide range: from diferrous (FeII-FeII) to high potential intermediate Q(FeIV-FeIV). Two of these redox states are paramagnetic: (FeIV-FeIII), called high potential intermediate X, and (FeII-FeIII), called mixed-valent state of the diiron carboxylate proteins. In the present work it has been shown that these redox states are of functional relevance in two proteins with different functions.</p><p>Ribonucleotide reductase (RNR) from the human parasite<i> Chlamydia trachomatis</i> is a class I RNR. It is typical for class I RNR to initiate the enzymatic reaction on its large subunit, protein R1, by activation from a stable tyrosyl free radical in its small subunit, protein R2. This radical, in its turn, is formed through oxygen activation by the diiron center. In C. trachomatis the tyrosine residue is replaced by phenylalanine, which cannot form a radical. We have shown in the present work, that active <i>C. trachomatis</i> RNR uses the FeIII-FeIV state of the diiron carboxylate cluster in R2 instead of a tyrosyl radical to initiate the catalytic reaction.</p><p>The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants. The existence of the diiron carboxylate center in this protein was predicted on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. In experiments modeling the conditions of the enzyme catalytic cycle, i.e. reduction and reoxygenation of the overexpressed AOX in <i>Escherichia coli</i> membranes we were able to generate an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The alternative oxidase is the first membrane protein where the existence of the diiron carboxylate center has been shown experimentally.</p>

"MIT-3221-19." / Bibliography: p. 78-81. / Contract AT(30-1)-3221.

Based on a Ph.D. thesis in the Dept. of Electrical Engineering, 1963. / Bibliography: p. 81-83.

M.S. / Biochemistry / The EPR spectrum of the Tyr-Cys· free radical in oxidized apoGAOX has been investigated, using a combination of approaches. Power saturation analysis has been used to resolve two unique spectra through Evolving Factor Analysis (EFA) global fitting, indicating the presence of two distinct free radical species in the sample. The component that dominates at low microwave power arises from the Tyr-Cys· side chain, while the high power component has not yet been assigned. The experimental results show that the EPR spectrum collected at low power includes approximately 7% of the high power component. EPR spectra have been collected for ten different isotope derivatives of GAOX, including ²H-labeled, ¹³C-labeled, 17[superscript]O-labeled, and ³³S-labeled forms. XSophe simulation of the EPR spectra has been performed for the isotopically labeled samples in order to determine the spectroscopic parameters - g-values, hyperfine coupling constants, and linewidths. The g-values and the methylene proton hyperfine coupling constants obtained for the isotopically labeled samples are consistent with the literature values. The magnitude of the hyperfine coupling constants associated with each of the nuclei confirms that significant electron spin density is found on the methylene protons, the alternating carbon atoms within the aromatic π system and the 2p[subscript]z orbital of both sulfur and oxygen. Moreover, the rotation angle of the methylene protons to the phenoxyl ring around the C1-C7 bond has been evaluated based on the experimentally defined hyperfine coupling constants of the two methylene protons.

Diiron carboxylate proteins constitute an important class of metall-containing enzymes. These proteins perform a multitude of reactions in biological systems that normally involve activation of molecular oxygen at the diiron site. During activation and functioning of these proteins their diiron sites undergo redox changes in a rather wide range: from diferrous (FeII-FeII) to high potential intermediate Q(FeIV-FeIV). Two of these redox states are paramagnetic: (FeIV-FeIII), called high potential intermediate X, and (FeII-FeIII), called mixed-valent state of the diiron carboxylate proteins. In the present work it has been shown that these redox states are of functional relevance in two proteins with different functions. Ribonucleotide reductase (RNR) from the human parasite Chlamydia trachomatis is a class I RNR. It is typical for class I RNR to initiate the enzymatic reaction on its large subunit, protein R1, by activation from a stable tyrosyl free radical in its small subunit, protein R2. This radical, in its turn, is formed through oxygen activation by the diiron center. In C. trachomatis the tyrosine residue is replaced by phenylalanine, which cannot form a radical. We have shown in the present work, that active C. trachomatis RNR uses the FeIII-FeIV state of the diiron carboxylate cluster in R2 instead of a tyrosyl radical to initiate the catalytic reaction. The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants. The existence of the diiron carboxylate center in this protein was predicted on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. In experiments modeling the conditions of the enzyme catalytic cycle, i.e. reduction and reoxygenation of the overexpressed AOX in Escherichia coli membranes we were able to generate an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The alternative oxidase is the first membrane protein where the existence of the diiron carboxylate center has been shown experimentally.

Electron paramagnetic resonance (EPR) dosimetry using the well established dosimeter material alanine is a generally accepted dosimetric method for measurements of high absorbed doses. Alanine EPR dosimetry is however not sensitive enough for high precision measurements of low (&lt; 5 Gy) absorbed doses using reasonably measurement times and small dosimeters. It has therefore not been possible to fully exploit the benefits of EPR dosimetry for applications in radiation therapy. The aim of this thesis was to show that sensitive EPR dosimetry is a competitive method for applications in radiation therapy fulfilling the requirements of measurement precision. Our strategy for reaching this goal was to search for new, more sensitive, EPR dosimeter materials fulfilling the criteria of being tissue equivalent, having a high radical yield and having a narrow EPR spectrum suitable for dosimetry. The best materials were found among formates and dithionates. Doping with small amounts of metal ions and recrystallisation in D2O were tested to further increase the sensitivity. Four promising candidate materials were tested regarding radical stability and dose response and among them lithium formate was chosen for dosimetry in radiation therapy applications. A high precision EPR dosimetry method was developed using lithium formate. The method included the development of a production method for EPR dosimeters with very homogenous shape, mass and composition. A read-out process was developed with maximal measurement precision for reasonably short measurement times. The method also included a dosimeter quality control before actual dose measurements. Measurement accuracy was controlled for every new dosimeter batch. This high precision lithium formate EPR dosimetry method was evaluated for pretreatment verifications of intensity modulated radiation therapy (IMRT) treatment plans. The precision and accuracy was shown to be sufficient (&lt; 5 %) for measurements of doses above 1.5 Gy using one single dosimeter and a measurement time of 15 minutes. The described evaluation is therefore a demonstration of the improved precision at low dose determinations that is available with our sensitive EPR dosimeter materials. While the EPR signal intensity is proportional to absorbed dose, the signal shape is in some cases dependent on the radiation quality. A new method is presented for simultaneous measurements of beam LET (linear energy transfer) and absorbed dose in heavy charged particle beams using potassium dithionate EPR dosimetry. The study shows that when irradiating a dosimeter with 35 MeV carbon ions, the ratio of the signal amplitudes from two radicals in potassium dithionate vary along the track indicating a dependence on linear energy transfer, LET. Potassium dithionate may therefore be a promising EPR dosimeter material for simultaneous measurements of absorbed dose and LET in heavy charged particle radiation fields.

Mixed-valence compounds are of interest as model systems for the study of electron transfer reactions. The intramolecular electron transfer processes and patterns of charge delocalization in such compounds depend on the interplay between the electronic (V) and the vibronic (L) coupling. One can obtain both parameters from a Hush analysis of the intervalence band that arises upon optical intramolecular electron transfer if the band is intense and well-separated from other bands. This is quite often the case for mixed-valence triarylamines. As such, both Hush analysis and simulation of the intervalence band are widely used to classify these compounds as charge localized (class-II) or delocalized (class-III). Yet one must estimate the diabatic electron transfer distance (R) to calculate V in the Hush formalism. For mixed-valence triarylamines, R is commonly taken as the N-N distance; we show this to be a poor approximation in many cases. The activation barrier to thermal intramolecular electron transfer in a class-II mixed-valence compound is also related to the parameters V and L. Thus, if one can capture the rate of thermal electron transfer at multiple temperatures, then two experimental methods exist by which to extract the microscopic parameters. One technique that is widely used for organic mixed-valence compounds is variable-temperature electron spin resonance (ESR) spectroscopy. But this method is only rarely used to determine thermal electron transfer rates in mixed-valence triarylamines, as the electron transfer in most of the class-II compounds with distinct intervalence bands is too fast to observe on the ESR timescale. We show, for the first time, that one can use ESR spectroscopy to measure thermal electron transfer rates in such compounds. Simulation of ESR spectra based on density functional theory calculation and comparison with optical data also uncover the nature (i.e., adiabatic or nonadiabatic) of the electron transfer process.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xi, 148 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.