RESUMO
Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the principal axes of the g-tensor. Protons from His-E7 and Val-E11 residues are identified in the two symmetries, rhombic and axial, exhibited by MbNO. Our results indicate that both residues are present inside the heme pocket and help to stabilize one particular conformation.
Assuntos
Mioglobina/química , Animais , Fenômenos Biofísicos , Biofísica , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Heme/química , Histidina/química , Cavalos , Modelos Moleculares , Miocárdio/química , Conformação Proteica , Prótons , Valina/químicaRESUMO
Since the initial work of Ingram (8,10) Electron Paramagnetic Resonance contributed considerably to research in hemoglobins. Now, 40 years later we review some of the results of the application of EPR to nitrosylhemoglobin (HbNO), as an example of the diversity of information which this technique can provide.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hemoglobinas/química , Modelos Moleculares , Conformação ProteicaRESUMO
The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.
Assuntos
Hemoglobinas Glicadas/química , Hemeproteínas/química , Mioglobina/análogos & derivados , Óxido Nítrico/química , Fenômenos Biofísicos , Biofísica , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Mioglobina/química , Temperatura , TermodinâmicaRESUMO
The EPR spectra of nitrosyl hemoglobin and myoglobin in different conditions (native, denatured and lyophilized), as well as of hematin-NO were obtained in the temperature range of 80-280 K. There is a substantial and reversible decrease of the areas of the EPR spectra of all the hemoglobin samples above 150 K. The interpretation of the results implies the existence of two conformational states in thermal equilibrium, only one of which is EPR detectable. Thermodynamical parameters are determined for the hexa- and penta-coordinated cases.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Hemoglobinas/química , Conformação Proteica , Liofilização , Humanos , Desnaturação Proteica , Temperatura , TermodinâmicaAssuntos
Eritrócitos/metabolismo , Hemoglobinas/química , Animais , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Congelamento , Hemoglobinas/metabolismo , Humanos , Metemoglobina/análise , Metemoglobina/metabolismo , Óxido Nítrico , Oxiemoglobinas/química , Oxiemoglobinas/metabolismo , RatosRESUMO
The EPR spectrum of nitrosyl hemoglobin has been studied from 7.5 K to 104 K. It is composed of at least three components (A, B and C) which have a different dependence on temperature and power level. The A component decreases with increasing temperature. The B component disappears at around 30 K and is replaced by C. Relaxation of A follows the Orbach mechanism with an energy of 28 cm-1. This behavior can be attributed to phonon induced changes in the orientation of NO with respect to the heme plane.
Assuntos
Hemoglobinas , Fenômenos Biofísicos , Biofísica , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Micro-Ondas , TemperaturaRESUMO
Photolysis of HbNO has been studied from 6.2 K to 15.5 K by electron spin resonance during and after continuous illumination. Non-exponential kinetics of both dissociation and reassociation of NO was observed. The prolonged illumination separates the fast and slow ligands. This picture is consistent with NO tunnelling from two sites at different distances from the bound position. This result is obtained using a model of a sum of two exponentials or of conformational substates.
Assuntos
Hemoglobinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Cinética , Fotólise , Conformação Proteica , TemperaturaRESUMO
The mixture of low and high iron spin states is studied by electron spin resonance in methemoglobin and in metmyoglobin between 6K and 100K. The crystals contain iron (Fe3) exclusively in the high spin state, while powdered samples show a mixture of high and low spin iron. We detected, for the first time, the low spin state in metmyoglobin at low temperatures. The ratio of high to low spin concentrations (k-1) varies exponentially with inverse of temperature in both proteins, only the absolute value is greater in myoglobin. The slope of K-1 depends on the cooling rate and on the temperature range. The results are qualitatively explained assuming a temperature dependent distribution of crystical field around the cristal value, delta c.