RESUMEN
A spectroscopic study of soybean peroxidase (SBP) has been carried out using electronic absorption, resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopy in order to determine the effects of temperature on the heme spin state. Upon lowering the temperature a transition from high spin to low spin is induced in SBP resulting from conformational changes in the heme cavity, including a contraction of the heme core, the reorientation of the vinyl group in position 2 of the porphyrin macrocycle, and the binding of the distal His to the Fe atom. Moreover, the combined analysis of the data derived from the different techniques at both room and low temperatures demonstrates that at low temperature the quantum-mechanically admixed spin state (QS) of SBP has RR frequencies different from those observed for the QS species at room temperature.
Asunto(s)
Glycine max/enzimología , Peroxidasas/química , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón/métodos , Congelación , Hemo/química , Imidazoles/química , Porfirinas/química , Conformación Proteica , Teoría Cuántica , Proteínas Recombinantes/química , Espectrofotometría/métodos , Espectrometría Raman/métodos , TermodinámicaRESUMEN
Electronic absorption, resonance Raman and EPR spectra are reported for ferric horseradish peroxidase isoenzyme A2 at neutral and alkaline pH together with its imidazole complex at 12 K. The data are compared with those obtained at room temperature. At neutral pH, lowering the temperature induces conformational changes with the formation of two types of low-spin hemes, a bis-histidyl type and a hydroxo type. The transition induced by lowering the temperature is accompanied by a change in the orientation of a vinyl substituent which appears less conjugated to the porphyrin macrocycle than at room temperature. At low temperature the low-spin hemes coexist with a quantum admixed spin species. All the forms are characterized by extremely high resonance Raman frequencies, indicating a contraction of the core size from that of the room temperature species. At alkaline pH, only one low-spin species is observed at both room and low temperatures, with a hydroxo ligand bound to the heme iron. The v(Fe-OH) stretching mode has been assigned at 512 cm(-1), on the basis of the isotopic shift observed in D2O and H2(18)O. This relatively low frequency, together with the anomalous shift observed in deuterium, indicates that the hydrogen bonds between the oxygen atom and the distal residues are stronger than in metmyoglobin, but weaker than those of horseradish peroxidase isoenzyme C. This is in agreement with the lower tetragonality, determined from the EPR g values, of alkaline horseradish peroxidase isoenzyme A2 than of metmyoglobin.
Asunto(s)
Hemo/química , Peroxidasa de Rábano Silvestre/química , Arginina/química , Frío , Espectroscopía de Resonancia por Spin del Electrón , Histidina/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Isoenzimas/química , Conformación Proteica , Espectrofotometría InfrarrojaRESUMEN
Resonance Raman (RR) spectra have been obtained for single-crystal horseradish peroxidase isozyme C complexed with benzhydroxamic acid (BHA). The data are compared with those obtained in solution by both RR and electronic absorption spectroscopies at room and low (12-80 K) temperatures. Moreover, the analysis has been extended to Coprinus cinereus peroxidase complexed with BHA. The results obtained for the two complexes are very similar and are consistent with the presence of an aqua six-coordinate high-spin heme. Therefore it can be concluded that despite the rather long Fe-H2O distance of 2.6-2.7 A found by X-ray crystallography in both complexes, the distal water molecule can still coordinate to the heme iron.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/metabolismo , Coprinus/enzimología , Cristalización , Hemo , Hierro/química , Hierro/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Metamioglobina/química , Metamioglobina/metabolismo , Peroxidasa/química , Peroxidasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluciones , Espectrometría Raman , Temperatura , AguaRESUMEN
Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.
Asunto(s)
Hemo/química , Peroxidasas/química , Cristalografía por Rayos X , Bases de Datos Factuales , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Modelos Moleculares , Proteínas de Plantas/química , Espectrofotometría , Espectrometría Raman , TemperaturaRESUMEN
The pH dependence of the electronic absorption and resonance Raman (RR) spectra of FeIII and FeII forms of Coprinus cinereus peroxidase (CIP) and its Asp245-->Asn (D245N) mutant has been examined in detail. The spectral data were obtained in the pH range 3.8-12.0. These spectra were used to assess the spin and ligation states of the heme via the porphyrin marker band frequencies and the wavelengths of the absorption maxima, especially that of the band (CT1) due to the charge transfer from the porphyrin to the heme iron via the a' 2u(pi)-->eg (d pi) electronic transition. The RR spectra were obtained by using different excitation wavelengths and polarized light. The data obtained for ferric CIP show that two pH-induced structural transitions exist. At acid pH the Soret and the CT1 absorption maxima occur at 394 and 652 nm, respectively, compared with the values of 403 and 649 nm observed at neutral pH. The electronic data indicate that at acid pH the proximal Fe-Im bond might be weakened or ruptured, and the RR spectra show a new species (5-c HS) different from the normal neutral 5-coordinate high-spin (5-c HS) heme. At pH 12.0, the protein converts to a 6-coordinate low-spin (6-c LS) heme with a hydroxyl ligand coordinated in the sixth position of the heme iron and strongly hydrogen-bonded with the positively charged guanidinium group of the distal Arg51 residue. Replacement of the aspartate carboxylate group of Asp245, which acts as hydrogen-bond acceptor to the proximal His183 ligand of the heme Fe, with a carboxamide group of an asparagine residue has a profound influence on the heme coordination. The RR spectra of the Fe(II) form of this mutant at both neutral and alkaline pH values show a band at 204 cm-1 assigned to the Fe-His stretch associated with a fairly weak or non-hydrogen-bonded imidazole. The ferric form of the mutant shows a great variability in coordination and spin states upon pH titration. Between pH 8.8 and 3.8 the spectra are mainly characteristic of a 6-coordinate high-spin heme, presumably with a water molecule bound on the distal side of the Fe atom. The pKa of the alkaline transition of the mutant is much lower than that of the wild-type protein. At pH 10.0 the D245N mutant is in its final alkaline form, which markedly differs from that of the parent enzyme. The spectral data indicate that the majority of the protein has 5-coordinate high-spin heme (5-c HS), with the Fe-His 183 bond broken and the distal axial coordination site of the heme iron occupied by a hydroxyl group, which is strongly hydrogen-bonded with distal Arg51. Therefore, the Asp245-->Asn mutation on the proximal side results in the breakage of the Fe-His bond at alkaline pH.
Asunto(s)
Asparagina/genética , Ácido Aspártico/genética , Coprinus/enzimología , Hemo/química , Peroxidasas/química , Catálisis , Compuestos Ferrosos/química , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Peroxidasas/genética , Peroxidasas/metabolismo , Espectrometría RamanRESUMEN
Resonance Raman (RR) and electronic absorption spectra of the ferric and ferrous forms of the His175Glu mutant of cytochrome c peroxidase are reported. At 296 K, the FeIII form is five-coordinate high spin and the resonance Reman spectra are very similar to those obtained for the wild type enzyme, even though in the mutant the Fe atom is bound to an oxygen atom of the Glu residue. The only difference is that the bands due to the out-of-plane modes are very weak, indicating a less distorted heme plane compared to CCP. The absorption spectrum is similar to that of CCP, as far as the Soret and alpha, beta bands are concerned, but the charge-transfer band due to the a2u(pi)-->eg(d pi) transition is 8 nm blue-shifted relative to that of the wild type enzyme, indicating that a more negative ligand is bound to the heme iron. As the temperature is lowered, the five-coordinate heme converts to a six-coordinate high-spin form. The conversion is readily reversible. A temperature effect on the protein structure is proposed that permits the Fe atom to approach the heme plane and to bind the distal water molecule. The results are discussed in terms of the X-ray structure, which shows a different disposition of the distal water molecules in the Glu175 mutant. The RR spectra also show that the heme is more contracted and distorted at 19 K than at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Citocromo-c Peroxidasa/química , Citocromo-c Peroxidasa/metabolismo , Hemo/metabolismo , Histidina , Mutación Puntual , Secuencia de Aminoácidos , Sitios de Unión , Ácido Glutámico , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometría , Espectrometría Raman , Difracción de Rayos XRESUMEN
Absorption and resonance Raman spectra using Soret excitation of alkaline metmyoglobin (metMb), methemoglobin (metHb), and horseradish peroxidase (HRP) were obtained at room and low temperature. At 298 K both metMb and metHb exhibit two isotope-sensitive bands assigned to high- and low-spin nu(Fe-OH) stretching modes, respectively, which are correlated with the spin-state population. The low-spin stretch occurs 60 cm-1 to higher energy than the corresponding high-spin vibration. When the temperature is lowered, only the low-spin species is observed. HRP exhibits at both 298 and 20 K only the low-spin nu(Fe-OH) stretching mode, which occurs 50 cm-1 to lower energy than the corresponding modes observed in the globins. This is explained in the context of a strong hydrogen bond between the hydroxyl ligand and the distal His42 and/or Arg38. Lowering temperature causes in all of the examined proteins a strengthening of the Fe-OH bond and a contraction of the core of about 0.01 A, as determined by the upshifting of the low-spin nu(Fe-OH) stretching mode and the core size marker bands. Both effects are ascribed to an increase of the packing forces.
Asunto(s)
Peroxidasa de Rábano Silvestre/metabolismo , Hidróxidos/metabolismo , Metahemoglobina/metabolismo , Metamioglobina/metabolismo , Animales , Fenómenos Químicos , Química Física , Deuterio , Peroxidasa de Rábano Silvestre/química , Caballos , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Metahemoglobina/química , Metamioglobina/química , Espectrofotometría , Espectrometría Raman , TemperaturaRESUMEN
Resonance Raman (RR) spectra of the acidic form of FeIII horseradish peroxidase (HRP) were obtained at room and low temperatures using B- and Q-band excitation. At 296 K, HRP exhibits two sets of porphyrin skeletal stretching frequencies which are attributed to a thermal mixture of 5- and 6-coordinate high-spin FeIII states. When the temperature is lowered, the observed bands shift to higher frequencies, and these are assigned to intermediate- and low-spin states. Addition of 40% glycerol has no effect on the spectra at 296 K, but at 20 K, all four frequency sets are observed corresponding to the two forms observed at room and low temperature in the absence of glycerol. The 296 K RR spectrum of the HRP-hydroquinone complex is similar to that of free HRP, but conversion to the intermediate- and low-spin states is complete at a higher temperature than in the free enzyme. Addition of benzohydroxamic acid (BHA) to HRP shifts the RR frequencies to those corresponding to a 6-coordinate high-spin species at both room and low temperature. Two upsilon (C = C) stretching modes are observed for HRP and its donor complexes, indicating that the vinyl groups are inequivalent. On BHA binding, one of the vinyl modes and upsilon 37 (Eu) are enhanced, suggesting symmetry lowering of the heme site.
Asunto(s)
Compuestos Férricos/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Animales , Compuestos Férricos/química , Glicerol , Peroxidasa de Rábano Silvestre/química , Concentración de Iones de Hidrógeno , Hidroquinonas/metabolismo , Ácidos Hidroxámicos/metabolismo , Espectrometría Raman , Temperatura , Compuestos de Vinilo/metabolismoRESUMEN
Pre-resonance Raman spectra of actinomycin D have been measured using the exciting lines of an Ar+ laser. The analysis of the excitation profiles provided information on the origin of the electronic states; in particular, the absorption feature between 400 and 500 nm was interpreted as due to a vibrational structure of a single electronic state which is located at 450 nm. In addition, on the basis of the excitation profiles, the number of observed Raman bands, and their frequencies, it has been possible to propose a vibrational assignment of the chromophoric framework of the drug.