RESUMEN
To establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PlSOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation. The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PlSOD is in reversible monomer-dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes.
Asunto(s)
Cobre/metabolismo , Photobacterium/enzimología , Salmonella typhimurium/enzimología , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión/genética , Cobre/deficiencia , Dimerización , Estabilidad de Enzimas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Photobacterium/genética , Salmonella typhimurium/genética , Superóxido Dismutasa/genética , Ultracentrifugación/métodos , Zinc/deficienciaRESUMEN
The infrared absorption spectra of ferric cyanide and ferrous carbonmonoxy Escherichia coli flavohemoglobin have been measured in order to probe the fine structural properties of the distal heme pocket, characterized by the presence of a tyrosine in position B10 and a glutamine in position E7. The stretching frequency of iron bound cyanide occurs at 2136 cm(-1), an unusually high value if compared to other heme proteins. The infrared spectrum of the CO bound derivative displays two peaks centered at 1960 cm(-1) and at 1909 cm(-1) respectively. H(2)O effects have been studied in both the ferric cyanide and ferrous CO derivatives in order to establish the presence of a distal hydrogen bonding to the iron bound ligand. The observed isotope shifts indicate that in the ferric cyanide derivative a hydrogen bond is donated from a residue in the distal pocket to the biatomic ligand whereas in the ferrous carbon monoxy derivative only the 1909 cm(-1) component is most likely hydrogen bonded to the phenolic group of TyrB10.
Asunto(s)
Dihidropteridina Reductasa , Proteínas de Escherichia coli/química , Hemo/química , Hemoproteínas/química , NADH NADPH Oxidorreductasas , Oxigenasas/química , Ferricianuros , Compuestos Ferrosos , Hierro/química , Oxidación-Reducción , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Sorcin is a 198 amino-acid Ca(2+)-binding protein that belongs to the penta-EF-hand family. Its Ca(2+)-binding domain (residues 33-198) has been crystallized in the absence of Ca(2+) in two different crystal forms. Two complete data sets have been collected on a synchrotron source under cryocooling conditions from crystals grown using ammonium sulfate as precipitant: monoclinic crystals in space group C2, with unit-cell parameters a = 130.93, b = 103.85, c = 78.55 A, beta = 118.0 degrees, diffracting to 2.1 A, and tetragonal crystals in space group P42(1)2, with unit-cell parameters a = b = 103.33, c = 79.15, diffracting to 2.7 A. Crystals were also grown using PEG 6000 as precipitating agent. They also belong to space group C2, diffract to 2.8 A and their unit-cell parameters are very similar to the first form. Structure determination by molecular replacement has been initiated. Structural information should be useful for elucidating the interaction of sorcin with membrane targets.
Asunto(s)
Proteínas de Unión al Calcio/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of beta chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.
Asunto(s)
Hemo/metabolismo , Hemoglobinas/metabolismo , Mutagénesis Sitio-Dirigida/genética , Mutación/genética , Albúminas/metabolismo , Sustitución de Aminoácidos/genética , Sitios de Unión , Cromatografía en Gel , Dimerización , Hemoglobinas/química , Hemoglobinas/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Oxígeno/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528).
Asunto(s)
Hemoglobinas/química , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Anguila Babosa , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.
Asunto(s)
Hemoglobinas/química , Perciformes/sangre , Animales , Carboxihemoglobina/química , Dimerización , Hemoglobina A/química , Hemoglobinas/aislamiento & purificación , Humanos , Enlace de Hidrógeno , Ligandos , Estructura Secundaria de Proteína , Solventes , Relación Estructura-Actividad , Propiedades de Superficie , UltracentrifugaciónRESUMEN
The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy-entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.
Asunto(s)
Hemoglobinas/química , Hemoglobinas/metabolismo , Moluscos/química , Regulación Alostérica , Animales , Dimerización , Hemo/química , Hemo/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Estructura Cuaternaria de Proteína , Termodinámica , Agua/metabolismoRESUMEN
The stability of the dodecameric Listeria innocua ferritin at low pH values has been investigated by spectroscopic methods and size-exclusion chromatography. The dodecamer is extremely stable in comparison to the classic ferritin tetracosamer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Below pH 2.0, dissociation into dimers occurs and is paralleled by the complete loss of tertiary structure and a significant decrease in secondary structure elements. Dissociation of dimers into monomers occurs only at pH 1.0. Addition of NaCl to the protein at pH 2.0 induces structural changes similar to those observed upon increasing the proton concentration, although dissociation proceeds only to the dimer stage. Addition of sulfate at pH values >/= 1.5 prevents the dissociation of the dodecamer. The role played by hydrophilic and hydrophobic interactions in determining the resistance to dissociation of L. innocua ferritin at low pH is discussed in the light of its three-dimensional structure.
Asunto(s)
Ferritinas/química , Listeria/química , Aniones , Cromatografía , Dicroismo Circular , Dimerización , Electroforesis en Gel de Poliacrilamida , Ferritinas/metabolismo , Concentración de Iones de Hidrógeno , Hierro/metabolismo , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Sulfatos/metabolismo , Sulfatos/farmacología , Rayos UltravioletaRESUMEN
Iron deposition in the unusual 12-subunit ferritin from thebacterium Listeria innocua proceeds in three phases: a rapidfirst phase in which Fe(2+) binds to the apoprotein, P(Z) of charge Z, according to the postulatedreaction 2Fe(2+)+P(Z)-->[Fe(2)-P](Z+2)+2H(+), where[Fe(2)-P](Z+2) represents adinuclear iron(II) complex formed at each of the 12 ferroxidase centresof the protein; a second phase corresponding to oxidation of thisputative complex, i.e. [Fe(2)-P](Z+2)+1/2 O(2)-->[Fe(2)O-P](Z)+2H(+);and a third phase of iron(II) oxidation/mineralization, i.e. 4Fe(2+)+O(2)+8H(2)O-->8FeOOH((s))+8H(+) [where FeOOH((s)) represents the hydrous ferric oxidemineral that precipitates from the solution], which occurs when iron isadded in excess of 24Fe(2+)/protein. In contrast with otherferritins, the ferroxidation reaction in L. innocua ferritinproceeds more slowly than the oxidation/mineralization reaction. Wateris the final product of dioxygen reduction in the 12-subunit L.innocua ferritin (the present work) and in the 24-subunit Escherichia coli bacterioferritin, whereas H(2)O(2) is produced in 24-subunit mammalian ferritins. Possible reasonsfor this difference are discussed.
Asunto(s)
Ferritinas/química , Hierro/química , Listeria/química , Hidrólisis , Oxidación-ReducciónRESUMEN
Surface plasmon resonance experiments show that at neutral pH the stability of the complex between sorcin and annexin VII (synexin) increases dramatically between 3 and 6 microM calcium; at the latter cation concentration the K(D) value is 0.63 microM. In turn, the lack of complex formation between the sorcin Ca(2+) binding domain (33-198) and synexin maps the annexin binding site to the N-terminal region of the sorcin polypeptide chain. Annexin VII likewise employs the N-terminal domain, more specifically the first 31 amino acids, to interact with sorcin [Brownawell, A.M. and Creutz, C.E. (1997) J. Biol. Chem. 272, 22182-22190]. The interaction may involve similar structural motifs in the two proteins, namely GGYY and GYGG in sorcin and GYPP in synexin.
Asunto(s)
Anexina A7/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calcio/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/inmunología , Ácido Egtácico/farmacología , Concentración de Iones de Hidrógeno , Sueros Inmunes/inmunología , Cinética , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.
Asunto(s)
Bivalvos/química , Compuestos Férricos/química , Hemo/genética , Hemoglobinas/química , Mutación Puntual , Sustitución de Aminoácidos/genética , Animales , Bivalvos/genética , Dimerización , Hemo/química , Hemoglobinas/genética , Histidina/genética , Concentración de Iones de Hidrógeno , Leucina/genética , Fenilalanina/genética , Espectrofotometría , Espectrometría Raman , Ultracentrifugación , Valina/genéticaRESUMEN
Ferritin is characterized by a highly conserved architecture that comprises 24 subunits assembled into a spherical cage with 432 symmetry. The only known exception is the dodecameric ferritin from Listeria innocua. The structure of Listeria ferritin has been determined to a resolution of 2.35 A by molecular replacement, using as a search model the structure of Dps from Escherichia coli. The Listeria 12-mer is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely the negatively charged channels along the three-fold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition. The electron density map shows 12 iron ions on the inner surface of the hollow core, at the interface between monomers related by two-fold axes. Analysis of the nature and stereochemistry of the iron-binding ligands reveals strong similarities with known ferroxidase sites. The L. innocua ferritin site, however, is the first described so far that has ligands belonging to two different subunits and is not contained within a four-helix bundle.
Asunto(s)
Ferritinas/química , Ferritinas/metabolismo , Hierro/metabolismo , Listeria/química , Estructura Cuaternaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Escherichia coli/química , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Sorcin, a 21.6 kDa cytosolic EF-hand protein which undergoes a Ca(2+)-induced translocation from cytoplasm to membranes, has been assigned to the newly defined penta EF-hand family. A molecular model of the C-terminal Ca(2+)-binding domain has been generated using as a template the X-ray coordinates of the corresponding domain in the calpain light subunit, the family prototype [Lin, G., et al. (1997) Nat. Struct. Biol. 4, 539-546]. The model indicates that in sorcin the three-dimensional structure is conserved and in particular that of EF1, the novel EF-hand motif characteristic of the family. On this basis, two stable fragments have been obtained and characterized. Just like the native protein, the sorcin Ca(2+)-binding domain (residues 33-198) is largely dimeric, interacts with the ryanodine receptor at physiological calcium concentrations, and undergoes a reversible, Ca(2+)-dependent translocation from cytosol to target proteins on Escherichia coli membranes. In contrast, the 90-198 fragment (residues 90-198), which lacks EF1 and EF2, does not bind Ca(2+) with high affinity and is unable to translocate. Binding of calcium to the EF1-EF2 pair is therefore required for the activation of sorcin which uses the C-terminal calcium-binding domain for interaction with the ryanodine receptor, a physiological target in muscle cells.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/fisiología , Motivos EF Hand , Escherichia coli/química , Modelos Moleculares , Fragmentos de Péptidos/química , Canal Liberador de Calcio Receptor de Rianodina/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/química , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
FTIR spectra of native Scapharca homodimeric hemoglobin (HbI) and of the Phe97-->Ile mutant have been measured in the region 2400-2700 cm(-1) where the absorption of the sulfhydryl groups can be observed. In native HbI, the two Cys92 residues give rise to a relatively intense band centered at 2559 cm(-1) that is shifted to 2568 cm(-1) and strongly quenched upon ligand binding. In the Phe97-->Leu mutant, such ligand-linked changes are not observed and the strong peak at around 2560 cm(-1) persists in the liganded derivatives. In native HbI, the observed changes have been attributed to the decrease in polarity of the interface due to the ligand-induced extrusion of the Phe97 phenyl ring from the heme pocket to the interface and the subsequent release of several water molecules that are clustered in the vicinity of Cys92. In contrast, in the Phe97-->Leu mutant, the Leu residue does not leave the heme pocket upon ligand binding and the interface is unaltered. The Cys92/S-H infrared band, therefore, represents a sensitive probe of the structural rearrangements that take place in the intersubunit interface upon ligand binding to HbI. The heterotetrameric Scapharca hemoglobin HbII contains, in addition to the Cys92 residues in the interfaces, two extra sulfhydryl groups per tetramer (Cys9 in the B chain) that are exposed to solvent in the A helix. The frequency of the Cys9/S-H stretching vibration occurs at 2582 cm(-1) in the unliganded and at 2586 cm(-1) in the liganded derivative, pointing to the involvement of the A helix in the ligand-linked polymerization characteristic of HbII.
Asunto(s)
Bivalvos/química , Hemoglobinas/química , Compuestos de Sulfhidrilo/química , Animales , Cisteína/química , Dimerización , Hemoglobinas/metabolismo , Ligandos , Modelos Químicos , Unión Proteica , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad Estática , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Single crystals of ferritin extracted from Listeria innocua have been obtained by the vapour-diffusion method using PEG 1000 as precipitant. The crystals are orthorhombic, space group P212121, with unit-cell dimensions a = 87.7, b = 137.5, c = 173.1 A. The crystals diffract to 2.9 A resolution on a rotating-anode X-ray source and to 2.35 A resolution on a synchrotron X-ray source. The asymmetric unit contains one molecule formed by 12 subunits, corresponding to a packing density of 2.41 A3 Da-1
Asunto(s)
Ferritinas/química , Listeria/química , Cristalización , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
The polypeptide chain that assembles into the unusual dodecameric shell of Listeria innocua apoferritin lacks the ferroxidase centre characteristic of H-type mammalian chains, but is able to catalyse both Fe(II) oxidation and nucleation of the iron core. A cluster of five carboxylate residues, which correspond in part to the site of iron core nucleation typical of L-type mammalian ferritins, has been proposed to be involved in both functions. The features of the iron uptake kinetics and of Fe(II) autoxidation in the presence of citrate followed spectrophotometrically confirm this assignment. In Listeria the kinetics of iron uptake is hyperbolic at low Fe(II)-to-dodecamer ratios and becomes sigmoidal when iron exceeds 150 Fe(II) atoms per dodecamer, namely when a fast crystal growth phase follows a slow initial nucleation step. Iron autoxidation in the presence of citrate displays a similar behaviour. Thus the time course is sigmoidal at low citrate-to-Fe ratios at which Fe(III) polymerization is predominant, but is hyperbolic at ligand concentrations high enough to prevent polymerization. The marked inhibitory effect of Tb(III) on the kinetics of iron incorporation confirms that carboxylates provide the iron ligands in L. innocua apoferritin. Iron uptake followed in steady-state fluorescence experiments allows one to distinguish Fe(II) binding and oxidation from the subsequent movement of Fe(III) into the apoferritin cavity as in mammalian ferritins despite the different localization of the tryptophan residues.
Asunto(s)
Ferritinas/metabolismo , Hierro/metabolismo , Listeria/metabolismo , Apoferritinas/química , Catálisis , Citratos/química , Ferritinas/química , Concentración de Iones de Hidrógeno , Hierro/química , Cinética , Oxidación-Reducción , Citrato de Sodio , Espectrometría de Fluorescencia , Terbio/química , Zinc/químicaRESUMEN
The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6-9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm-1 and shifts to 553 cm-1 in H218O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.
Asunto(s)
Hemo/química , Hemoglobinas/química , Histidina/química , Hidróxidos/química , Animales , Bivalvos , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Espectrometría RamanRESUMEN
The analytical and biotechnological applications of human hemoglobin immobilized covalently on CNBr-Sepharose 4B are reviewed. Hemoglobin is bound to the matrix as alphabeta dimers via either chain. The immobilized alphabeta dimers maintain the capacity to interact reversibly with soluble ones under conditions where the soluble protein is in self-association equilibrium. Under these conditions, therefore, immobilized dimers bind part of the soluble protein. In turn, the binding process can be used to assess the specific features of the equilibrium on solid-phase and to extract selectively hemoglobin from a variety of biological specimens of practical interest. A different application of immobilized alphabeta dimers concerns their use in the determination of the equilibrium and kinetic stability of the heme-globin linkage, a property that is directly correlated with the stability of the hemoglobin molecule. The advantages and limitations attendant the use of the immobilized protein relative to the soluble one are discussed.
Asunto(s)
Biotecnología/métodos , Técnicas de Química Analítica/métodos , Hemoglobinas/química , Dimerización , HumanosRESUMEN
The effect of the apolar mutation of the distal histidine (His69-->Val) has been studied in the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Absorption, circular dichroism, and resonance Raman spectroscopy point to a more symmetric heme structure of the deoxy derivative, which is indicative of an R-like conformation of the deoxy heme. Resonance Raman spectroscopy also brings out alterations in the geometry and interactions of the bound CO molecule. The iron-carbon stretching frequency is decreased by about 30 cm-1 with respect to the native protein, while the diatomic ligand stretching frequency is increased by about the same degree. Consistent with the structural changes, the ligand binding properties are significantly altered. In the mutant the overall rate and the affinity for CO binding are increased about 100-fold with respect to the native protein, and cooperativity is abolished. In addition, the amplitude and the rate of the geminate rebinding process increase significantly. This finding may be correlated to the longer average residence time of the photolyzed CO molecule within the heme pocket of the H69V mutant, as indicated by molecular dynamics simulations.
Asunto(s)
Sustitución de Aminoácidos/genética , Hemoglobinas/química , Histidina/genética , Mutagénesis Insercional , Conformación Proteica , Valina/genética , Animales , Bivalvos , Monóxido de Carbono/química , Dicroismo Circular , Dimerización , Hemo/química , Hemoglobinas/genética , Ligandos , Unión Proteica/genética , Espectrofotometría , Espectrometría RamanRESUMEN
We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400]. We determined by analytical ultracentrifugation the dimer-tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary alpha1 beta1 interface, and all of the others are at the sliding alpha1 beta2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the alphabeta dimer interface. The slope yields an interface stabilization free energy of -15 +/- 1.2 cal/mol upon burial of 1 A2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199-203].