RESUMEN
We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k(cat), glucose S(0.5) and K(D) values and ATP K(M) while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with K(D)s of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.
Asunto(s)
Apoenzimas/química , Activadores de Enzimas/química , Glucoquinasa/química , Glucosa/química , Glicerol/química , Urea/química , Adenosina Trifosfato/química , Regulación Alostérica , Apoenzimas/genética , Estabilidad de Enzimas , Escherichia coli/genética , Glucoquinasa/genética , Humanos , Cinética , Modelos Moleculares , Mutación , Presión Osmótica , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Temperatura , Termodinámica , Agua/químicaRESUMEN
We report the use of infrared (IR) spectroscopy to detect ice crystals in biological solutions. The method is based on the temperature dependence of the OH bending and stretch bands of water. By using mixtures of D(2)O and H(2)O, water's absorption bands can be made to be on-scale in transmission mode. Water's stretch band moves to lower frequency and sharpens with freezing, and the bending band goes to higher frequency and becomes less sharp. The technique is demonstrated for the study of the hysteresis of freezing in the presence of glucosyl sugars, namely glucose, maltose, and trehalose.
Asunto(s)
Glucosa/química , Hielo , Maltosa/química , Espectrofotometría Infrarroja/métodos , Trehalosa/químicaRESUMEN
Water is required to hydrate molecules, but under cold conditions water freezes and under dehydrating conditions water evaporates--thus presenting a dilemma for organisms that live in extreme environments. Organisms have developed various strategies for protection against extreme temperatures and dehydration. In this review, we describe how the interaction of water and 2 natural cryoprotectants, namely glycerol and sugars, can be studied at the molecular level. Techniques using infrared spectroscopy and computation are described. In the case of glycerol, H-bonding of water to the OH groups of glycerol limits the amount of water available to form ice and prevents crystallization at low temperatures. For aldohexopyranose sugars, the different isomeric forms have different water H-bonding networks, which are consistent with isomeric-dependent activities. By studying the strategies used in nature, derivatives for use in food preservation can be more readily developed.
Asunto(s)
Carbohidratos/química , Crioprotectores/química , Glicerol/química , Enlace de Hidrógeno , Agua/química , Cristalización , Conservación de Alimentos/métodos , Espectrofotometría Infrarroja/métodosRESUMEN
The authors compared the spectral response of Zn-substituted horseradish peroxidase in a glycerol/water solvent to hydrostatic pressure at 2 K and ambient temperature. The low temperature experiments clearly demonstrate the presence of at least three different conformations with drastically different elastic properties. However, the main conformation, which determines the fluorescence spectrum at ambient temperature, did not show any significant difference between low and high temperature and pressure. The authors conclude that the local compressibility of the heme pocket of the protein depends only very weakly on temperature.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Temperatura , Zinc/química , Hemo/química , Presión Hidrostática , Estructura Terciaria de Proteína , Espectrometría de FluorescenciaRESUMEN
The iron of lactoperoxidase is predominantly high-spin at ambient temperature. Optical spectra of lactoperoxidase indicate that the iron changes from high-spin to low-spin in the temperature range from room temperature to 20 K. The transformation is independent of whether the enzyme is in glycerol/water or solid sugar glass. Addition of the inhibitor benzohydroxamic acid increases the amount of the low-spin form, and again the transformation is independent of whether the protein is in an aqueous solution or a nearly anhydrous sugar. In contrast to lactoperoxidase, horseradish peroxidase remains high-spin over the temperature excursion in both solvents and with addition of benzohydroxamic acid. We conclude that details of the heme pocket of lactoperoxidase allow ligation changes with temperature that are dependent upon the apoprotein but independent of solvent fluctuations. At low pH, lactoperoxidase shows a solvent-dependent transition; the high-spin form is predominant in anhydrous sugar glass, but in the presence of water, the low-spin form is also present in abundance. The active site of lactoperoxidase is not as tightly constrained at low pH as at neutrality, though the enzyme is active over a wide pH range.
Asunto(s)
Lactoperoxidasa/química , Sitios de Unión , Frío , Vidrio/química , Glicerol , Ácidos Hidroxámicos/química , Solventes , Espectrofotometría/métodos , Espectrofotometría Ultravioleta , Sacarosa/química , Trehalosa/químicaRESUMEN
We performed pressure-tuning hole-burning experiments on a modified cytochrome c protein in a glycerol/buffer glass. The shift and the broadening of the holes were investigated for various frequencies within the inhomogeneous band. On the basis of a simple model, we were able to estimate the interaction range between chromophore and protein. It is approximately 4.5 A. The parameters that enter the model are the compressibility, the static mean-square displacement, the inhomogeneous width, and the average spectral shift per pressure. From this result and from our experiments on pressure-induced denaturing, we conclude that water molecules have to be brought very close to the chromophore during the denaturation process.
Asunto(s)
Citocromos c/análisis , Citocromos c/química , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Sondas Moleculares/análisis , Sondas Moleculares/química , Espectrometría de Fluorescencia/métodos , Citocromos c/efectos de la radiación , Ambiente , Colorantes Fluorescentes/efectos de la radiación , Calor , Rayos Láser , Sondas Moleculares/efectos de la radiación , Presión , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Zinc/análisis , Zinc/química , Zinc/efectos de la radiaciónRESUMEN
The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.
Asunto(s)
Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Movimiento , Solventes/química , Arginina/química , Arginina/metabolismo , Sitios de Unión , Hemo/química , Hemo/metabolismo , Histidina/química , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Modelos Moleculares , Espectrofotometría Infrarroja , TemperaturaRESUMEN
Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Ácidos Hidroxámicos/química , Hemo/química , Conformación Proteica , Espectrofotometría , Espectrofotometría Infrarroja , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6.0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrational stretch frequency is at 1903 cm(-1). Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of approximately 320 cm(-1) in the Q(0,0) absorption band that is clearly evident at < 100 K and shifts the CO absorption to 1916 cm(-1). Increasing the pH to 9.3 also causes a split in the Q(0,0) optical band and elicits a shift in nu(CO) to a higher frequency (1936 cm(-1)). The splitting of the Q(0,0) band and the shifts in the IR spectra are both consistent with changes in the local electric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the distal histidine, although other effects are also considered. The larger effect on the Q(0,0) band with substrate at low pH and the shift of nu(CO) at high pH can be rationalized by the directionality of the field and the orientation dependence of dipolar interactions.
Asunto(s)
Monóxido de Carbono/química , Peroxidasa de Rábano Silvestre/química , Sitios de Unión , Hemoproteínas/química , Concentración de Iones de Hidrógeno , Ácidos Hidroxámicos/química , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad Estática , Especificidad por SustratoRESUMEN
We have recently demonstrated that metalloporphyrins are potent inhibitors of both human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2) reverse transcriptases (RTs) [Argyris, E.G., Vanderkooi, J.M., Venkateswaran, P.S., Kay, B.K., and Paterson, Y. (1999) J. Biol. Chem. 274, 1549-1556]. In addition, by screening a phage peptide library we discovered that a peptide with sequence similarity to residues 398-407 from the connection subdomain of HIV RTs binds heme. These findings suggested that this highly conserved region may be the binding site for metalloporphyrins and a novel site for inhibition of enzymatic activity. Our most recent data presented here confirm this suggestion. Screening of HIV-1 RT 398-407 peptide analogs by fluorescence assays demonstrates that Trp residues at positions 401 and 402 are important for heme binding. Furthermore, site-directed mutagenesis of these residues verified these findings and indicated that heme inhibits HIV-1 RT by binding on the connection subdomain of the p66 subunit of the enzyme but not on the p51 subunit. This was also confirmed by analyzing the binding affinities of heme for mutant HIV-1 RT heterodimers, using intrinsic fluorescence assays. The clear identification of the connection domain as a novel inhibition site is crucial in understanding the mechanism of heme binding and enzymatic inhibition and will facilitate the generation of novel porphyrin-based inhibitors of RT.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , Hemo/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Hemo/metabolismo , Metaloporfirinas/metabolismo , Metaloporfirinas/farmacología , Microscopía Fluorescente , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligopéptidos/metabolismo , Estructura Terciaria de Proteína , Inhibidores de la Transcriptasa Inversa/metabolismo , Triptófano/metabolismoRESUMEN
Cytoplasmic segments of the adult rat skeletal muscle sodium channel alpha-subunit (rSkM1) comprise a major portion (approximately 40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel's amino- and carboxy-termini and identify residues 1716-1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an alpha-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp1617.
Asunto(s)
Músculo Esquelético/química , Canales de Sodio/química , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dicroismo Circular , Cartilla de ADN/genética , Técnicas In Vitro , Proteínas de Unión a Maltosa , Oligopéptidos , Péptidos/química , Péptidos/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Eliminación de Secuencia , Canales de Sodio/genética , Espectrometría de FluorescenciaRESUMEN
We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes. In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed. The yeast species, on the other hand, displays a single electronic origin. Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space. The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes.
Asunto(s)
Grupo Citocromo c/química , Hemo/química , Animales , Fenómenos Biofísicos , Biofisica , Caballos , Técnicas In Vitro , Modelos Moleculares , Miocardio/química , Conformación Proteica , Saccharomyces cerevisiae/química , Análisis Espectral , Electricidad Estática , TermodinámicaRESUMEN
Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in the temperature range of 10-300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positions indicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increased hydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. The substrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-ray crystallography results. With removal of Ca, the protein maintained its overall helicity.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Calcio/química , Conformación Proteica , Solventes , Espectrofotometría Infrarroja , Especificidad por Sustrato , TemperaturaRESUMEN
Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately alpha-helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is approximately 3 kcal.
Asunto(s)
Grupo Citocromo c/química , Porfirinas/química , Secuencia de Aminoácidos , Animales , Grupo Citocromo c/metabolismo , Hemo/química , Concentración de Iones de Hidrógeno , Hierro/química , Hierro/metabolismo , Datos de Secuencia Molecular , Porfirinas/metabolismo , Pliegue de Proteína , Protones , Procesamiento de Señales Asistido por Computador , Espectrometría de Fluorescencia , Espectrofotometría Infrarroja , TemperaturaRESUMEN
Resonance Raman and UV-visible absorption spectra show that nickel(II) microperoxidase-11 (NiMP-11) is four-coordinate in aqueous solution in the pH range from 1.0 to 13.0. In aqueous solutions of NiMP-11 in the absence of cetyltrimethylammonium bromide (CTAB), NiMP-11 is aggregated. In CTAB micellar solutions, where aggregation of NiMP-11 does not occur, the Raman spectra of NiMP-11 are similar to that of nickel(II) cytochrome c (NiCyt-c). The presence of the peptide segment shifts the equilibrium heavily in favor of the nonplanar form, just as does the entire protein component in the case of NiCyt-c. This further elucidates the structural mechanism by which the protein segment ruffles the heme, most likely modulating the redox potential as indicated for the cytochromes c3 [Ma, J.-G., et al. (1998) Biochemistry 37, 12431-12442]. Furthermore, the hydrophobic environment that is provided by the CTAB micelle is found to be crucial to the native folding of the pentapeptide and formation of two hydrogen bonds in the peptide backbone. These two H-bonds act to contract the peptide segment exerting the force on the macrocycle that causes the ruffling and makes the redox potential more negative than if the heme were to remain planar. The structure of the heme and pentapeptide may also be associated with redox-linked triggering of the formation and release of cytochrome-protein complexes.
Asunto(s)
Grupo Citocromo c/química , Níquel/química , Peroxidasas/química , Hemo/química , Lípidos/química , Cómputos Matemáticos , Fragmentos de Péptidos/química , Porfirinas/química , Soluciones , Espectrofotometría Ultravioleta , Espectrometría Raman , AguaRESUMEN
We have shown that heme and zinc protoporphyrin inhibit both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs) and, in combination with other nucleoside and non-nucleoside inhibitors, exert an additive effect on HIV-1 RT inhibition. Screening of a phage peptide library against heme resulted in the isolation of a peptide with sequence similarity to sequence 398-407 from the connection subdomain of both HIV-1 and HIV-2 RTs, suggesting that this highly conserved region of HIV RTs corresponds to the binding site for metalloporphyrins and a new site for inhibition of enzyme activity. Inclusion of a synthetic peptide corresponding to the exact sequence 398-407 of HIV-1 RT in RT inhibition assays had a protective effect on metalloporphyrin inhibition, as it was able to reverse the inhibitory effect of both metalloporphyrins on HIV-1 RT activity. Furthermore, intrinsic fluorescence assays indicated that these metalloporphyrins bind to synthetic peptide 398-407 as well as to intact dimeric HIV-1 RT. The identification of this novel inhibition site will help to expand our understanding of the mode of action of metalloporphyrins in RT inhibition and will assist in the design and development of more potent metalloporphyrin RT inhibitors for the management of HIV infection.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Metaloporfirinas/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Aminoácidos , Bacteriófago M13 , Sitios de Unión , Dimerización , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Hemo/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conformación Proteica , Protoporfirinas/metabolismoRESUMEN
Three ways are generally used to visualize proteins: (1) a static model in which the atomic positions are defined, (2) a dynamic model taking into account fluctuations, and (3) a reactive model that reflects the internal and external electric fields of the molecule. The properties of chromophoric prosthetic groups can be probed by optical spectroscopy, and when high resolution techniques are used, the results reveal information about the local electric fields in proteins, as influenced and determined by atomic positions and dynamics.
Asunto(s)
Conformación Proteica , Análisis Espectral , Electricidad , Hemo/química , Hemoproteínas/química , Modelos Teóricos , Movimiento , PresiónRESUMEN
Fluorescence line narrowing is a high resolution spectroscopic technique that uses low temperature and laser excitation to optically select specific subpopulations from the inhomogeneously broadened absorption band of the sample. When applied to the study of fluorescent groups in proteins one can obtain vibronically resolved spectra, which can be analyzed to give information on spectral line shapes, vibrational energies of both the ground and excited state molecule, and the inhomogeneous distribution function of the electronic transitions. These parameters reveal information about the chromophoric prosthetic group and the protein matrix and are functions of geometric strains and local electric fields imposed by the protein. Examples of the use of fluorescence line narrowing are discussed in investigations of heme proteins, photosynthetic systems and tryptophan-containing proteins.
Asunto(s)
Proteínas/química , Espectrometría de Fluorescencia/métodos , Hemoproteínas/química , Rayos Láser , Modelos Químicos , Sondas Moleculares , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Triptófano/químicaRESUMEN
The photoexcited metastable triplet state of Mg(2+)-mesoporphyrin IX (MgMPIX) or Mg(2+)-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg2+ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8-110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MGPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is >> 150 cm-1, suggesting symmetry breaking from the anisotropy of intermal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527-16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q0.0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg2+ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.
Asunto(s)
Mioglobina/química , Mioglobina/efectos de la radiación , Animales , Fenómenos Biofísicos , Biofisica , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Hemo/química , Hemo/efectos de la radiación , Caballos , Técnicas In Vitro , Mesoporfirinas/química , Mesoporfirinas/efectos de la radiación , Fotoquímica , Protoporfirinas/química , Protoporfirinas/efectos de la radiación , Espectrofotometría , TermodinámicaRESUMEN
Resonance Raman spectra with Q-band excitation are reported for microperoxidase-11, the cytochrome c analog. Spectra were acquired in the mid-frequency range for the oxidized, and reduced forms of the undecapeptide, as well as for the imidazole and carbonyl complexes. Oxidation and spin state marker bands of the undecapeptides are consistent with a six-coordinate, low spin iron in both oxidation states. Porphyrin core size correlations yield a porphyrin-centre to pyrrole-nitrogen distance of 2.00 A for MP11, suggestive of a six-coordinate species in a distorted heme environment. Molecular dynamics results show that the non-planarity of the heme of the parent cytochrome is conserved in the microperoxidase and its carbonmonoxy analog.