RESUMEN

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.

Asunto(s)

Biofisica/métodos , Superóxido Dismutasa/química , Aminoácidos/química , Dimerización , Sustancias Macromoleculares/química , Microscopía Fluorescente , Modelos Moleculares , Modelos Estadísticos , Mutación , Photobacterium/enzimología , Presión , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas/química , Proteínas Recombinantes/química , Programas Informáticos , Termodinámica , Triptófano/química

RESUMEN

Diffusion-controlled enzymes are characterized by second-order rate constants in the range 10(8)-10(10) M(-1)s(-1). These values are at the upper end of the observed rates of many enzyme-substrate reactions and have been predicted by theoretical studies on bimolecular reaction in solution. Such enzymes are considered to be perfect, since their rate-limiting step is not due to any chemical event but to the diffusional association rate between the enzyme and the substrate. Often the enzyme-substrate encounter is facilitated either through the presence of a strong attractive electric field, produced by charges on the enzyme surface, or through the reduction in the dimension of the search process. Here we provide a brief review of some of the enzymes characterized by a very fast second-order constant, focusing attention on triose phosphate isomerase and Cu,Zn superoxide dismutase taken as typical examples of such highly tuned enzymes.

Asunto(s)

Enzimas/química , Animales , Sitios de Unión , Catálisis , Difusión , Cinética , Modelos Químicos , Modelos Moleculares , Unión Proteica , Electricidad Estática , Especificidad por Sustrato , Superóxido Dismutasa/química , Factores de Tiempo , Triosa-Fosfato Isomerasa/química

RESUMEN

As part of the more general inquiry on the molecular basis of specific recognition between macromolecules, the subunit-subunit interface structure of dimeric superoxide dismutase from Photobacterium leiognathi has been probed selectively by the phosphorescence emission of Trp-73, located at the subunit contact region. Copper at the catalytic site was found to quench completely the delayed emission and therefore all studies were conducted with the copper-free or Cd(2+)-substituted protein. The spectrum at 140 K is diagnostic for an indole ring located in a hydrophobic environment whereas a degree of spectral broadening indicates that the local structure is not unique. Environmental heterogeneity is confirmed by the nonuniform phosphorescence decay in buffer, at 274 K, with lifetime components of 44 and 20 ms of practically equal amplitude. Information on the flexibility of the interface region was gathered from both the intrinsic lifetime and the accessibility of acrylamide to the site of the chromophore. The magnitude of the intrinsic lifetime, its temperature dependence, and the accessibility to solutes like acrylamide describe a tight dimeric structure in which hydrophobic interactions seem to play an important role. In particular the acrylamide bimolecular rate constant is 1.4 x 10(4) M(-1) s(-1) and indicates highly hindered diffusion of the solute through the interface region. Cd(2+) complexation to the apoprotein caused no detectable changes in protein conformation although the metal was able to influence the flexibility of the Trp-73 environment, indicating the occurrence of a long-range communication between the intersubunit surface and the active site, which is more than 16 A away.

Asunto(s)

Cobre/química , Photobacterium/enzimología , Superóxido Dismutasa/química , Triptófano/química , Cobre/deficiencia , Escherichia coli , Colorantes Fluorescentes/química , Mediciones Luminiscentes , Proteínas Recombinantes/química , Superóxido Dismutasa/genética , Temperatura

RESUMEN

The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

Asunto(s)

Cobre/metabolismo , Mutación/genética , Photobacterium/enzimología , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos/genética , Sitios de Unión , Catálisis/efectos de los fármacos , Quelantes/farmacología , Cristalografía por Rayos X , Dimerización , Ácido Edético/farmacología , Cinética , Modelos Moleculares , Photobacterium/genética , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Radiólisis de Impulso , Solventes , Análisis Espectral , Superóxido Dismutasa/genética , Temperatura , Factores de Tiempo

RESUMEN

A single mutation (Val29-->Gly) at the subunit interface of a Cu, Zn superoxide dismutase dimer leads to a twofold increase in the second order catalytic rate, when compared to the native enzyme, without causing any modification of the structure or the electric field distribution. To check the role of dynamic processes in this catalytic enhancement, the flexibility of the dimeric protein at the subunit interface region has been probed by the phosphorescence and fluorescence properties of the unique tryptophan residue. Multiple spectroscopic data indicate that Trp83 experiences a very similar, and relatively hydrophobic, environment in both wild-type and mutant protein, whereas its mobility is distinctly more restrained in the latter. Molecular dynamics simulation confirms this result, and provides, at the molecular level, details of the dynamic change felt by tryptophan. Moreover, the simulation shows that the loops surrounding the active site are more flexible in the mutant than in the native enzyme, making the copper more accessible to the incoming substrate, and being thus responsible for the catalytic rate enhancement. Evidence for increased, dynamic copper accessibility also comes from faster copper removal in the mutant by a metal chelator. These results indicate that differences in dynamic, rather than structural, features of the two enzymes are responsible for the observed functional change.

Asunto(s)

Photobacterium/enzimología , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Simulación por Computador , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida/genética , Photobacterium/genética , Conformación Proteica , Subunidades de Proteína , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Superóxido Dismutasa/genética , Termodinámica

RESUMEN

The Val28-->Gly single mutant at the subunit interface of Cu,Zn superoxide dismutase from Photobacterium leiognathi displays a k(cat)/K(M) value of 1.7x10(10) M(-1) s(-1), twice that of the native enzyme. Analysis of the three-dimensional structure indicates that the active site Cu,Zn center is not perturbed, slight structural deviations being only localized in proximity of the mutation site. The enzyme-substrate association rate, calculated by Brownian dynamics simulation, is identical for both enzymes, indicating that the higher catalytic efficiency of the Val28-->Gly mutant is not due to a more favorable electrostatic potential distribution. This result demonstrates the occurrence of an intramolecular communication between the mutation site and the catalytic center, about 18 A away and indicates a new strategy to encode extra efficiency within other members of this enzymatic family.

Asunto(s)

Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Ingeniería Genética , Concentración de Iones de Hidrógeno , Mutación , Photobacterium/enzimología , Estructura Terciaria de Proteína , Subunidades de Proteína , Especificidad por Sustrato , Superóxido Dismutasa/química , Superóxido Dismutasa/genética

RESUMEN

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.

Asunto(s)

Genes Bacterianos/genética , Salmonella typhimurium/enzimología , Salmonella typhimurium/patogenicidad , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cobre/metabolismo , Cristalización , Cristalografía por Rayos X , Dimerización , Campos Electromagnéticos , Espectroscopía de Resonancia por Spin del Electrón , Congelación , Concentración de Iones de Hidrógeno , Yoduros/metabolismo , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Protones , Salmonella typhimurium/genética , Alineación de Secuencia , Soluciones , Superóxido Dismutasa/genética , Temperatura , Virulencia/genética , Agua/metabolismo

RESUMEN

Tryptophan 83, a residue strongly involved in the intersubunit interaction of the Cu,Zn superoxide dismutases from Photobacterium leiognathi, has been selectively mutated to phenylalanine or tyrosine. The recombinant mutant enzymes expressed in Escherichia coli were purified in two well distinct and stable forms, one dimeric and fully active and the other monomeric and devoid of metals. In agreement, in vitro experiments indicate that the removal and addition of zinc in the mutant enzymes induces monomerization and dimerization, respectively, while does not perturb the dimeric association of the native protein. This is the first unambiguous experimental proof of a direct communication between the intersubunit interface and the metal active site.

Asunto(s)

Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , Dimerización , Escherichia coli/genética , Metales/química , Mutagénesis Sitio-Dirigida , Photobacterium/enzimología , Photobacterium/genética , Mutación Puntual , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Superóxido Dismutasa/aislamiento & purificación

RESUMEN

The equilibrium unfolding process of human Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism and fluorescence spectroscopy as a function of increasing guanidinium hydrochloride concentration. The process occurs through the formation of a monomeric intermediate species following a three-state transition equilibrium. Comparison with the stability of the prokaryotic Cu,Zn SOD from P. leiognathi shows that the eukaryotic enzyme is more stable than the prokaryotic enzyme by approximately 3 kcal/mol. This difference is due to the monomer-to-unfolded equilibrium, while the dimer-to-monomer equilibrium is comparable for the two enzymes despite their different intersubunit interactions. These results are confirmed by the unfolding of the copper-depleted derivatives. The Cu,Zn superoxide dismutase represents a good example of how evolution has found two independent quaternary assemblies maintaining the same dimer stability.

Asunto(s)

Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Superóxido Dismutasa/química , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Photobacterium/enzimología , Pliegue de Proteína , Espectrometría de Fluorescencia , Termodinámica , Triptófano/química

RESUMEN

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.

Asunto(s)

Photobacterium/enzimología , Superóxido Dismutasa/química , Dicroismo Circular , Dimerización , Estabilidad de Enzimas , Escherichia coli/genética , Guanidina , Holoenzimas/química , Modelos Químicos , Photobacterium/genética , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Fluorescencia , Superóxido Dismutasa/genética , Termodinámica

RESUMEN

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the "boson peak" observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure.

Asunto(s)

Azurina/química , Cobre/química , Modelos Moleculares , Dispersión de Radiación , Proteínas Bacterianas/química , Simulación por Computador , Escherichia coli/química , Pseudomonas aeruginosa/química , Temperatura , Agua/química

RESUMEN

A Cu,Co derivative of the Cu,ZnSOD from Photobacterium leiognathi, in which cobalt has been selectively substituted for zinc, has been prepared and spectroscopically investigated. The derivative shows three bands in the visible region at 530, 566, and 600 nm when copper is in the oxidized state. Reduction or depletion of the copper ion produce a shift of the band absorbing at 600 to 590 nm because of the detachment from copper of the imidazolate bridging the two metals when copper is in the oxidized state. Numerous isotropically shifted 1H NMR lines are observed when copper is oxidized, confirming the presence of the imidazolate bridge between the two metals. Comparison of the optical and the NMR spectra with those observed for the eukaryotic enzyme reveals the occurrence of slight but unambiguous differences diagnostic of a different degree of distortion of the metal cluster between the prokaryotic and eukaryotic enzymes.

Asunto(s)

Cobalto , Cobre , Photobacterium/enzimología , Superóxido Dismutasa/química , Conformación Molecular , Células Procariotas/enzimología , Espectrofotometría

RESUMEN

Prokaryotic Cu,Zn superoxide dismutases are characterized by a distinct quaternary structure, as compared to that of the homologous eukaryotic enzymes. Here we report a newly determined crystal structure of the dimeric Cu,Zn superoxide dismutase from Photobacterium leiognathi (crystallized in space group R32, refined at 2.5 A resolution, R-factor 0.19) and analyse it in comparison with that of the monomeric enzyme from Escherichia coli. The dimeric assembly, observed also in a previously studied monoclinic crystal form of P. leiognathi Cu,Zn superoxide dismutase, is based on a ring-shaped subunit contact region, defining a solvated interface cavity. Three clusters of neighbouring residues play a direct role in the stabilization of the quaternary assembly. The present analysis, extended to the amino acid sequences of the other 11 known prokaryotic Cu,Zn superoxide dismutases, shows that at least in five other prokaryotic enzymes the interface residue clusters are under strong evolutionary constraint, suggesting the attainment of a quaternary structure coincident with that of P. leiognathi Cu,Zn superoxide dismutase. Calculation of electrostatic fields for both the enzymes from E. coli and P. leiognathi shows that the monomeric/dimeric association behaviour displayed by prokaryotic Cu, Zn superoxide dismutases is related to the distribution of surface charged residues. Moreover, Brownian dynamics simulations reproduce closely the observed enzyme:substrate association rates, highlighting the role of the active site neighbouring residues in determining the dismutase catalytic properties.

Asunto(s)

Evolución Molecular , Photobacterium/enzimología , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Animales , Cobre , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Células Procariotas , Conformación Proteica , Electricidad Estática , Superóxido Dismutasa/clasificación , Xenopus laevis , Zinc

RESUMEN

The active-site copper ion of the prokaryotic Cu,Zn superoxide dismutase from P. leiognathi is found to undergo reversible reduction upon irradiation of the protein solution with a high-intensity X-ray beam from a third-generation synchrotron source. The same phenomenon is observed for the enzyme crystals, whose diffraction pattern has been obtained from synchrotron sources. In this case the active-site copper-ligand coordination bond lengths and in particular the Cu-NE2(His61) distance are consistent with a copper ion in the reduced state. These results are in line with previous studies on the eukaryotic Cu,Zn superoxide dismutases and suggest the conservation of an identical catalytic mechanism in both the prokaryotic and eukaryotic enzymes.

Asunto(s)

Photobacterium/enzimología , Superóxido Dismutasa/química , Animales , Sitios de Unión , Bovinos , Cobre/química , Cristalografía por Rayos X , Escherichia coli/enzimología , Células Eucariotas , Ligandos , Modelos Moleculares , Oxidación-Reducción , Fotoquímica , Células Procariotas , Conformación Proteica , Análisis Espectral , Superóxido Dismutasa/efectos de la radiación , Rayos X

RESUMEN

The catalytic rate constant of recombinant Photobacterium leiognathi Cu,Zn superoxide dismutase has been determined as a function of pH by pulse radiolysis. At pH 7 and low ionic strength (I = 0.02 M) the catalytic rate constant is 8.5 x 10(9) M-1 s-1, more than two times the value found for all the native eukaryotic Cu,Zn superoxide dismutases investigated to date. Similarly, Brownian dynamics simulations indicate an enzyme-substrate association rate more than two times higher than that found for bovine Cu,Zn superoxide dismutase. Titration of the paramagnetic contribution to the water proton relaxation rate of the P. leiognathi with increasing concentration of halide ions with different radii indicates that the proteic channel delimiting the active site is wider than 4.4 A. This is at variance with that found on the eukariotic enzymes, and provides a rationale for the high catalytic rate of the bacterial enzyme. Evidence for solvent exposure of the active site different from that observed in the eukaryotic enzyme is suggested from the pH dependence of the water proton relaxation rate and of the EPR spectrum line shape, which indicate the occurrence of a prototropic equilibrium at pH 9.1 and 9.0, respectively. The pH dependence of the P. leiognathi catalytic rate has a trend different from that observed in the bovine enzyme, indicating that groups differently exposed to the solvent are involved in the modulation of the enzyme-substrate encounter.

Asunto(s)

Proteínas Bacterianas/química , Photobacterium/enzimología , Superóxido Dismutasa/química , Animales , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Bovinos , Cobre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Protones , Relación Estructura-Actividad , Especificidad por Sustrato , Superóxido Dismutasa/metabolismo , Termodinámica , Zinc

RESUMEN

Binding of azide to the native and arginine-modified bovine Cu,Zn superoxide dismutase in the oxidized and reduced form and to the copper-free derivative has been investigated by Fourier transform infrared spectroscopy. The antisymmetric stretching band of the azide is shifted to higher energy upon coordination to the copper atom of the oxidized form of the native enzyme. Similar spectral changes occur upon interaction of the anion with the Cu-diethylenetriamine model compound. On the other hand, interaction of azide with the native reduced form of the enzyme results in a band shift toward lower energy with respect to the free anion band. The same shift is observed after reaction of the azide with free lysine or arginine but not when it is reacted with other amino acid residues. The antisymmetric band of the azide is not perturbed by addition of the reduced arginine-modified enzyme; it is likely shifted toward higher energy upon addition of oxidized arginine-modified enzyme while it is again shifted toward lower energy in the presence of the copper-free derivative of the unmodified enzyme. It is concluded that azide does not directly coordinate to the copper in the reduced form of Cu,Zn superoxide dismutase but it remains in the active-site pocket in electrostatic interaction with the guanidinium group of Arg141, which is an invariant residue in this class of enzymes.

Asunto(s)

Inhibidores Enzimáticos/metabolismo , Azida Sódica/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Arginina/química , Sitios de Unión , Bovinos , Cobre/química , Cobre/metabolismo , Inhibidores Enzimáticos/química , Lisina/química , Oxidación-Reducción , Poliaminas/química , Azida Sódica/química , Espectroscopía Infrarroja por Transformada de Fourier , Superóxido Dismutasa/química

RESUMEN

Cu,Zn SOD from the bathophile teleost Lampanyctus crocodilus (LSOD) shows a high degree of homology with the sequence of the enzymes from other teleostean fish species. The catalytic properties of LSOD are very similar to those of the bovine enzyme, albeit with higher sensitivity to thermal denaturation. The apparent molecular mass of LSOD (37.6 KDa) is higher than the other Cu,Zn SOD variants studied. The aminoacid sequence of LSOD reveals interesting substitutions compared to the bovine enzyme. These are discussed in view of the particular environmental conditions to which L. crocodilus is adapted.

Asunto(s)

Peces , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Animales , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Datos de Secuencia Molecular , Mapeo Peptídico , Tiburones , Superóxido Dismutasa/metabolismo

RESUMEN

Cu,Zn superoxide dismutase from Photobacterium leiognathi has been cloned and expressed in Escherichia coli. The circular dichroism spectrum in the UV region of the recombinant protein indicates an higher content of random coil structure with respect to the eukaryotic enzymes. Investigation of the active site by optical, CD, and EPR spectroscopy indicates a different coordination geometry around the catalytic copper site with respect to the eukaryotic enzymes. In particular a different orientation of the metal bridging histidine is suggested. The pH dependence of the copper EPR spectrum shows the presence of a single equilibrium which is at least one unit lower than the pK value observed for the bovine enzyme. Despite such structural differences the catalytic rate of this enzyme is identical to that observed for the eukaryotic Cu,Zn superoxide dismutase, suggesting that the overall electric field distribution is similar to that observed in the eukaryotic enzymes.

Asunto(s)

Cobre/metabolismo , Proteínas Recombinantes/química , Superóxido Dismutasa/química , Animales , Sitios de Unión , Catálisis , Bovinos , Dicroismo Circular , Clonación Molecular , ADN Recombinante/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Photobacterium , Conformación Proteica , Superóxido Dismutasa/genética

RESUMEN

X-ray absorption spectroscopy has been carried out on the copper K edge in oxidized and reduced bovine Cu,Zn SOD in solution and in crystalline state. The results indicate that the copper coordination geometry is unaffected by the solution or by the crystalline state of the protein, in both oxidation states. Moreover the two oxidation states of the active copper ion are reflected under, all the experimental conditions, by distinct coordination spheres around the catalytic metal, which is four-coordinated and three-coordinated in the Cu(II) and in the Cu(I) enzyme, respectively.

Asunto(s)

Histidina , Superóxido Dismutasa/química , Absorciometría de Fotón , Animales , Bovinos , Cobre , Cristalización , Imidazoles , Oxidación-Reducción , Soluciones , Superóxido Dismutasa/metabolismo , Zinc

RESUMEN

The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectivity replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N3(-)-Cu(II), Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(II) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm-1; the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Addition of N3- to the Cu(II),Co(II) SOD introduces slight changes in all the Co(II) visible bands, indicating the occurrence of minor perturbations of the structural cobalt site upon anion binding to the catalytic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(II) enzymes indicates that the His61 imidazolate bridge is released from the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the derivatives. The cobalt site is characterized by a harmonic dynamics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Polticelli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degree of local microheterogeneity at the cobalt site is smaller than that observed for the copper site and increases in the order N3(-)-Cu(II),Co(II) approximately Cu(II),Co(II) < Cu(I),Co(II) < E,Co(II) indicating a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic behavior with respect to the catalytic, solvent-accessible, copper site is discussed.

Asunto(s)

Cobalto/química , Superóxido Dismutasa/química , Animales , Bovinos , Frío , Espectrofotometría