Myoglobin (Mb) is a well characterized hemeprotein found in skeletal muscle. The dimethylester heme-substituted derivative of equine Mb (DME-Mb) was prepared to evaluate the involvement of the heme propionate groups in the electron transfer reactions of Mb. To achieve this goal, an efficient procedure to reconstitute and purify DME-Mb in high yield was developed. The near UV-visible absorption spectra of DME-Mb in various states of ligation and oxidation did not change significantly relative to those of native Mb. The ¹H NMR spectra obtained for native metMb (heme Fe(III) oxidation state) and metDME-Mb showed differences in the electromagnetic environment of their respective heme groups. The reactivity of DME-Mb was different from that of native Mb. For example the water ligand of metDME-Mb (Fe-H₂O) has a lower pKa than that of native metMb as determined by spectroscopic pH titrations. The autoxidation rate of oxyDME-Mb (Fe(II)-O₂) is faster than that of native oxyMb. MetDME-Mb apparently has a binding affinity for ferricyanide not evident in native metMb. Compared to native Mb, DME-Mb has decreased susceptibility to the oxidant hydrogen peroxide. The oxidation-reduction equilibrium of DME-Mb has been studied under a variety of solution conditions. At standard conditions (pH 7, I=0.1 M and 25°C) the midpoint reduction potential (Em) of DME-Mb is 100.0(2) mV vs. SHE, which is 39 mV higher than the Em of native Mb. Analysis of the pH dependence of Em showed differences in the identity or pKa between titratable groups found in native and DME-Mb. The ionic strength dependence of Em showed a higher net positive charge estimate for DME-Mb than native Mb consistent with the nature of the chemical modification involved. The temperature dependence of Em showed that DME-Mb has a greater difference in stability between oxidation states than native Mb. The kinetics of metDME-Mb reduction by Fe(EDTA)²⁻were also studied under a variety of conditions. At standard conditions, metDME-Mb reacted with the reductant Fe(EDTA)²⁻ at a second order rate constant (k₁₂) two orders of magnitude greater than that of native metMb. After correcting for the differences in reduction potential between reactants, metDME-Mb still reacted at a significantly faster rate than native metMb, indicating differences in their reaction mechanisms. The pH, temperature and ionic strength dependences of k₁₂ for DME-Mb and Fe(EDTA)²⁻ showed that DME-Mb had electrostatic and thermodynamic properties significantly different from that of native Mb. The functional differences between DME-Mb and native Mb can be attributed to the structural and electrostatics properties of the heme propionate groups. The interactions of these groups within the surrounding protein and the external environment are discussed with reference to the structure of Mb available from x-ray crystallographic studies. As a result, it is concluded that the heme propionate groups are involved in the structural stability, electron transfer specificity and reactivity of Mb. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Spectroscopic characterization of ferric soybean peroxidase with peroxides were studied to determine the ligand coordination and to characterize the structure of the heme active site and its intermediates (ferryl species). The lifetime, chemical reactivity and distinctive colors of the ferryl species (FeIV) formed during the oxidation of peroxidase (FeIII) by peroxides enabled structure, dynamics and reaction mechanisms to be studied. Resonance Raman spectroscopy was used as a means of characterizing the structure of the soybean peroxidase and its intermediates. Excitation in the Soret absorption band at 406.7nm with 2-5mW laser power was used for this study. Resonance Raman spectra in the 200 to 1700 cm-1 region were obtained for the soybean peroxidase. However, the focus of this study was on the vibrational region of the resonance Raman spectra from 900 to 500cm-1 where the FeIV=O stretching frequencies for heme compound II intermediates are expected. Several pH and pD (deuterium substitution) samples of the soybean peroxidase were analyzed using resonance Raman spectroscopy. The vibrational stretching frequencies of the ferryl peroxidases varied with varying pH/pD were observed within the 773–787cm-1 range. From the deuterium experiment, accompanied with changes in the vibrational frequencies of the iron-ligand, a 3cm-1 upshift and intense resonant enhancement of the peaks, we observed the ferryl nature of compound II intermediate for soybean peroxidase. Badger’s rule was used to estimate the bond distances that existed within Fe-O which offers additional insight into the structure of the ferryl species. The estimated bond distance for the soybean peroxidase was significantly less than Fe-O bond distances proposed by X-ray crystallographers for other peroxidases in the same family. Comparing the vibrational frequencies of the ferryl intermediates in soybean peroxidase to that in heme proteins portrayed the effect the protein environment has on the vibrational frequencies.

No description available.

Neste trabalho são apresentados estudos em três diferentes sistemas utilizando-se as técnicas de Ressonância Paramagnética Eletrônica (RPE) e absorção eletrônica. No primeiro estudou-se a formação dos possíveis complexos entre o aminoácido aliina, extraído do alho, e o íon cobre (II). Os dados de RPE e absorção eletronica no visível (400-1100 nm) foram utilizados para caracterizar os diferentes complexos obtidos. No segundo utilizou-se o monocristal do complexo dicloro bis (benzil difenil fosfinóxido) Cobre (II) e efetuaram-se medidas de RPE variando-se a direção de aplicação do campo magnético no monocristal. Os auto-valores encontrados para as componentes do tensor g&#175 foram: g1= 2,0891; g2= 2,4554 e g3= 2,0767. Com base nos dados de RPE e cristalográficos e usando a teoria de campo cristalino foi proposto dxy como estado fundamental para o íon cobre (II) neste complexo. Fez-se também um estudo preliminar observando-se as variações no centro ativo de hemoproteínas moduladas pelo grau de hidratação. Metahemoglobina bovina e metamioglobina eqüina liofilizadas foram usadas e o sinal de RPE do íon ferro (III) monitorado. Dos espectros de RPE das duas proteínas são observadas pelo menos duas simetrias para o íon ferro (III), uma axial caracterizada por um valor de g próximo de 6 e outra caracterizada por g= 4,3 correspondendo a simetria rômbica. A intensidade dos sinais mostrou-se muito sensíveis ao grau de hidratação. Metahemoglobina e metamioglobina apresentaram comportamentos distintos dos sinais de RPE e sugeriu-se serem eles devido as diferenças estruturais entre as duas proteínas / In this work studies in three different systems using Electron Paramagnetic Resonance (EPR) and eletronic absorption techniques are presented. The formation of possible complexes between the amino acid aliin, extracted of garlic, and the copper (II) íon was studied in the first work. EPR and visible eletronic absorption (400-1100 nm) data were utilized to characterize the different complexes obtained. In the second work a crystal of the complex dichloro bis (benzyl diphenyl phosphinoxide) copper (II) was used and the EPR measures were made for different directions of application of the magnetic field in the crystal. The eigenvalues obtained for the g&#175 tensor components were: g1= 2,0891; g2= 2,4554 and g3= 2,0767. Using EPR and crystallographic data and crystal field theory dxy was proposed as the ground state of the copper (II) íon in this complex. A preliminary study was also made observing changes at the active Center of the hemeproteins modulated by the hydration degree. Lyophilized bovine methemoglobin ande quine metmyoglobin were used and the EPR signal of the iron (III) íon monitored. Or both proteins the EPR spectra corresponding to at least two iron symmetries were observed, one axial characterized by a g value near 6.0 and another characterized by g= 4,3 corresponding to rhombic symmetry. The intensity of these signals were very sensitive to the hydration degree. Metmyoglobin and methemoglobin showed distint behaviors of the EPR signals and it was suggested that they were due to structural differences between the two proteins

Neste trabalho são apresentados estudos em três diferentes sistemas utilizando-se as técnicas de Ressonância Paramagnética Eletrônica (RPE) e absorção eletrônica. No primeiro estudou-se a formação dos possíveis complexos entre o aminoácido aliina, extraído do alho, e o íon cobre (II). Os dados de RPE e absorção eletronica no visível (400-1100 nm) foram utilizados para caracterizar os diferentes complexos obtidos. No segundo utilizou-se o monocristal do complexo dicloro bis (benzil difenil fosfinóxido) Cobre (II) e efetuaram-se medidas de RPE variando-se a direção de aplicação do campo magnético no monocristal. Os auto-valores encontrados para as componentes do tensor g&#175 foram: g1= 2,0891; g2= 2,4554 e g3= 2,0767. Com base nos dados de RPE e cristalográficos e usando a teoria de campo cristalino foi proposto dxy como estado fundamental para o íon cobre (II) neste complexo. Fez-se também um estudo preliminar observando-se as variações no centro ativo de hemoproteínas moduladas pelo grau de hidratação. Metahemoglobina bovina e metamioglobina eqüina liofilizadas foram usadas e o sinal de RPE do íon ferro (III) monitorado. Dos espectros de RPE das duas proteínas são observadas pelo menos duas simetrias para o íon ferro (III), uma axial caracterizada por um valor de g próximo de 6 e outra caracterizada por g= 4,3 correspondendo a simetria rômbica. A intensidade dos sinais mostrou-se muito sensíveis ao grau de hidratação. Metahemoglobina e metamioglobina apresentaram comportamentos distintos dos sinais de RPE e sugeriu-se serem eles devido as diferenças estruturais entre as duas proteínas / In this work studies in three different systems using Electron Paramagnetic Resonance (EPR) and eletronic absorption techniques are presented. The formation of possible complexes between the amino acid aliin, extracted of garlic, and the copper (II) íon was studied in the first work. EPR and visible eletronic absorption (400-1100 nm) data were utilized to characterize the different complexes obtained. In the second work a crystal of the complex dichloro bis (benzyl diphenyl phosphinoxide) copper (II) was used and the EPR measures were made for different directions of application of the magnetic field in the crystal. The eigenvalues obtained for the g&#175 tensor components were: g1= 2,0891; g2= 2,4554 and g3= 2,0767. Using EPR and crystallographic data and crystal field theory dxy was proposed as the ground state of the copper (II) íon in this complex. A preliminary study was also made observing changes at the active Center of the hemeproteins modulated by the hydration degree. Lyophilized bovine methemoglobin ande quine metmyoglobin were used and the EPR signal of the iron (III) íon monitored. Or both proteins the EPR spectra corresponding to at least two iron symmetries were observed, one axial characterized by a g value near 6.0 and another characterized by g= 4,3 corresponding to rhombic symmetry. The intensity of these signals were very sensitive to the hydration degree. Metmyoglobin and methemoglobin showed distint behaviors of the EPR signals and it was suggested that they were due to structural differences between the two proteins