RESUMEN
Small-angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN3) at pressures up to 300 MPa. Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performed using various concentrations of MbN3, show that the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa. The value of the second virial coefficient of the protein solution indicates that the interactions between the molecules are always strongly repulsive even if their magnitude decreases with increasing pressure. Taking advantage of the pressure-induced contrast variation, these experiments allow the partial specific volume of MbN3 to be determined as a function of pressure. Its value decreases by 5.4% between atmospheric pressure and 300 MPa. In this pressure range the isothermal compressibility of hydrated MbN3 is found to be almost constant. Its value is (1.6 +/- 0.1) 10-4 MPa-1.
Asunto(s)
Metamioglobina/análogos & derivados , Metamioglobina/química , Animales , Fuerza Compresiva , Caballos , Presión Hidrostática , Técnicas In Vitro , Miocardio/química , Neutrones , Conformación Proteica , Dispersión de Radiación , SolucionesRESUMEN
A resonance Raman band involving significantly the iron(III)-histidine stretching (upsilonFe-His) character is identified for metmyoglobin (metMb) through isotope sensitivity of its low-frequency resonance Raman bands, but the identification was not successful for methemoglobin (metHb) and its isolated alpha and beta subunits. A band at 218 cm-1 of natural abundance metMb exhibited a low-frequency shift for 15N-His-labeled metMb (-1.4 cm-1 shift), while the strong porphyrin bands at 248 and 271 cm-1 did not shift significantly. The frequency of the 218-cm-1 band of metMb decreased by 1.6 cm-1 in D2O, probably due to Ndelta-deuteration of the proximal His, in a similar manner to that of the upsilonFe-His band of deoxyMb in D2O. This 218-cm-1 band shifted slightly to a lower frequency in H2(18)O, whereas it did little upon 54Fe isotopic substitution (<0.3 cm-1), presumably because of the six-coordinate structure. The lack of the 54Fe-isotope shift shows that the 218-cm-1 band is specific to metMb and not due to the deoxy species. The intensity of this band decreased for hydroxymetMb and was indiscernible for cyanometMb. For metHb and its alpha and beta subunits, however, the frequencies of the band around 220 cm-1 were not D2O sensitive. These results suggest an assignment of the band around 220 cm-1 to a pyrrole tilting mode, which significantly contains the Fe-His stretching character for metMb but scarcely for metHb and its subunits. The differences in the isotope sensitivity of this band in different proteins are considered to reflect the heme distortion from the planarity and the Fe-His geometry specific to individual proteins.
Asunto(s)
Monóxido de Carbono/química , Histidina/química , Hierro/química , Metamioglobina/análogos & derivados , Metamioglobina/química , Mioglobina/análogos & derivados , Espectrometría Raman/métodos , Animales , Marcaje Isotópico/métodos , Metahemoglobina/química , Mioglobina/química , Subunidades de Proteína , Proteínas Recombinantes/química , BallenasRESUMEN
The 1H and 13C chemical shifts for the heme methyls of low-spin, ferric sperm whale cyanometmyoglobin reconstituted with a variety of centrosymmetric and pseudocentrosymmetric hemins have been recorded and analyzed to shed light on the nature of heme-protein contacts, other than that of the axial His, that modulate the rhombic perturbation to the heme's in-plane electronic asymmetry. The very similar 1H dipolar shifts for heme pocket residues in all complexes yield essentially the same magnetic axes as in wild type, and the resultant dipolar shifts allow the direct determination of the heme methyl proton and 13C contact shifts in all complexes. It is demonstrated that, even when the magnetic axes and anisotropies are known, the intrinsic uncertainties in the orientational parameters lead to a sufficiently large uncertainty in dipolar shift that the methyl proton contact shifts are inherently significantly less reliable indicators of the unpaired electron spin distribution than the methyl 13C contact shifts. The pattern of the noninversion symmetry in 13C contact shifts in the centro- or pseudocentrosymmetric hemes is shown to correlate with the positions of aromatic rings of Phe43(CD1) and His97(FG3) parallel to, and in contact with, the heme. These results indicate that such pi-pi interactions significantly perturb the in-plane asymmetry of the heme pi spin distribution and cannot be ignored in a quantitative interpretation of the heme methyl 13C contact shifts in terms of the axial His orientation in b-type hemoproteins.
Asunto(s)
Hemina/análogos & derivados , Metamioglobina/análogos & derivados , Metamioglobina/química , Animales , Isótopos de Carbono , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Protones , BallenasRESUMEN
Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.
Asunto(s)
Cisteína/genética , Mutagénesis Sitio-Dirigida , Mioglobina/química , Mioglobina/genética , Óxido Nítrico/metabolismo , Ácido Trifluoroacético/metabolismo , Animales , Monóxido de Carbono/metabolismo , Ditionita/farmacología , Flúor/metabolismo , Glicina/genética , Histidina/genética , Ligandos , Metamioglobina/análogos & derivados , Metamioglobina/genética , Metamioglobina/metabolismo , Mioglobina/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica/genética , Protones , Compuestos de Sulfhidrilo/metabolismo , BallenasRESUMEN
We report experimental observation and numerical simulation of a two-dimensional multiplet effect in the heteronuclear correlation spectrum of a paramagnetic protein that depends on molecular geometry. This effect arises as a consequence of cross-correlated relaxation involving the Curie spin relaxation and internuclear dipolar relaxation mechanisms. It also manifests itself in resolution and sensitivity improvement in transverse relaxation optimised spectroscopy (TROSY) kind of experiments. Characteristic multiplet patterns in heteronuclear coupled two-dimensional NMR spectra encode directional information for the heteronuclear bond with respect to the paramagnetic center. These patterns, which are simulated here using Redfield's relaxation theory, can be used to obtain a new type of geometry restriction for structure determination and refinement of paramagnetic macromolecular systems.
Asunto(s)
Metamioglobina/análogos & derivados , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Animales , Magnetismo , Metamioglobina/química , Proteínas Recombinantes de Fusión/química , Sensibilidad y EspecificidadRESUMEN
Myoglobin was reconstituted with the ferric complex of corrphycene, a novel porphyrin isomer with a rearranged tetrapyrrole array, to investigate the influence of porphyrin deformation on the equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states in the azide derivative. The azide affinity, 2.5 x 10(4) M(-1), was 1 order of magnitude lower than the corresponding values of a reference myoglobin containing an electron-deficient diformylheme similar to the corrphycene. Analysis of the visible absorption spectrum over a range of 0-40 degrees C reveals that the population of high-spin iron is 76-82% at room temperature for azide metmyoglobin complexed with ferric corrphycene. The unusual predominance of the high-spin state was verified from the infrared spectrum of coordinating azide, where the high-spin peak at 2046 cm(-1) is 4-fold larger in intensity than the 2023 cm(-1) low-spin band. Electron paramagnetic resonance at 15 K further indicated that the iron-histidine bond is cleaved to form a five-coordinate derivative in some fraction of the myoglobin. The remarkable high-spin bias of the spin equilibrium at room temperature and cleavage of the iron-histidine bond at 15 K could be explained in terms of the contracted and trapezoidal metallo core that weakens the iron-histidine bond of azide metmyoglobin bearing corrphycene.
Asunto(s)
Compuestos Ferrosos/química , Metaloporfirinas/química , Metamioglobina/análogos & derivados , Metamioglobina/química , Electroquímica , Estructura Molecular , Oxidación-Reducción , Espectrofotometría Atómica , Espectrofotometría Infrarroja , Relación Estructura-ActividadRESUMEN
The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution (1)H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small approximately 2 degrees rotation of the axial His in rWT relative to true WT and a approximately 3 degrees rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.
Asunto(s)
Aplysia , Hemo/metabolismo , Metamioglobina/análogos & derivados , Acetilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Hidrógeno , Enlace de Hidrógeno , Metamioglobina/química , Metamioglobina/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , BallenasRESUMEN
In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. (1)H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.
Asunto(s)
Mioglobina/genética , Mioglobina/metabolismo , Alanina/genética , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/genética , Glicina/genética , Histidina/genética , Enlace de Hidrógeno , Imidazoles/química , Ligandos , Sustancias Macromoleculares , Metamioglobina/análogos & derivados , Metamioglobina/química , Metamioglobina/metabolismo , Modelos Químicos , Mutagénesis Insercional , Mioglobina/química , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Resonancia Magnética Nuclear Biomolecular , Protones , Serina/genética , Treonina/genética , BallenasRESUMEN
Asn and Gln with an amide group at gamma- and delta-positions, respectively, were substituted for distal His-E7 of bovine myoglobin to establish a system where hydrogen bonding interaction between the distal residue and bound-ligand can be altered by changing donor-acceptor distance. Two mutant myoglobins showed nearly identical (1)H-NMR spectral pattern for resolved heme peripheral side-chain and amino acid proton signals and similar two-dimensional NMR connectivities irrespective of cyanide-bound and -unbound states, indicating that the heme electronic structure and the molecular structure of the active site are not affected by a difference in one methylene group at the E7 position. Chemical exchange rate of Asn-E7 N(delta)H proton in met-cyano myoglobin is larger than that of Gln-E7 N(epsilon)H proton by at least two orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the differential donor-acceptor distance between the two mutants. Thus a comparative study between the two proteins provides an ideal system to delineate a relationship between the stabilization of bound-ligand by the hydrogen bond and myoglobin's ligand affinity. The Asn-mutant showed a faster dissociation of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Similarly, oxygen dissociation is faster in the Asn-mutant than in the Gln-mutant by approximately 100-fold. Association of cyanide anion to the mutant met-myoglobin was accelerated by changing Gln to Asn by a 4-fold. Likewise, oxygen binding was accelerated by approximately 2-fold by the above substitution. The present findings confirm that hydrogen bonding with the distal residue is a dominant factor for determining the ligand dissociation rate, whereas steric hindrance exerted by the distal residue is a primary determinant for the ligand association.
Asunto(s)
Amidas/química , Mioglobina/química , Animales , Asparagina/química , Secuencia de Bases , Sitios de Unión , Bovinos , Cianuros/metabolismo , Glicina/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Metamioglobina/análogos & derivados , Metamioglobina/química , Datos de Secuencia Molecular , Mutación , Mioglobina/genética , Mioglobina/metabolismo , Oxígeno/metabolismo , ProtonesRESUMEN
An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.
Asunto(s)
Metamioglobina/análogos & derivados , Animales , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Metamioglobina/química , Metamioglobina/genética , Metamioglobina/metabolismo , Mutagénesis Sitio-Dirigida , Mioglobina/química , Mioglobina/genética , Mioglobina/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Cloruro de Sodio , Electricidad Estática , Urea , BallenasRESUMEN
A series of proximal side mutants of sperm whale metmyoglobin (metMb) that involves residues which provide hydrogen bonds to the axial His and heme have been prepared, and the CO binding and solution molecular and electronic structure has been investigated by 1H NMR. These include Ser92(F7), whose O gamma serves as a hydrogen-bond acceptor to the axial His ring NdeltaH and whose O gamma H serves as hydrogen-bond donor to the 7-propionate carboxylate, and His97(FG3) whose ring provides the other hydrogen-bond donor to the 7-propionate carboxylate. 2D NMR data on the S92A-metMbCN, S92P-metMbCN and H97F-metMbCN show that the distal structure is completely conserved and that proximal side structural changes are highly localized. For the S92A-metMbCN, altered dipolar contacts to the F-helix backbone show that the axial His imidazole has rotated clockwise by approximately 10 degrees relative to a stationary heme, while in H97F-metMbCN, the altered heme-E helix backbone contacts reveal that the heme has rotated counterclockwise by approximately 3 degrees relative to a conserved axial His. The pattern of axial His rotation was qualitatively predicted by energy minimization calculations. The assignments and conserved structural elements allow the determination of a set of magnetic axes whose major magnetic axis is unchanged with respect to WT and confirms that local distal, and not proximal, interactions control the orientation of the major magnetic axis and, by inference, the degree and direction of tilt of the Fe-CN from the heme normal. The rhombic magnetic axes in S92A-metMbCN are rotated approximately 10 degrees in the opposite direction from the established approximately 10 degrees rotation for the axial His ring as expected. It is shown, moreover, that the pairwise alpha-, gamma-meso vs beta-, delta-meso-H hyperfine shift differences are well predicted by the change in the location of the rhombic magnetic axes. Carbon monoxide ligation rates experience minor but systematic perturbation for the S92A substitutions which confirms an influence (albeit very small) for axial His orientation on ligand affinity.
Asunto(s)
Metamioglobina/análogos & derivados , Mutagénesis Sitio-Dirigida , Animales , Sitios de Unión/genética , Monóxido de Carbono/metabolismo , Transferencia de Energía , Hemo/química , Histidina/química , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Masculino , Metamioglobina/química , Metamioglobina/genética , Metamioglobina/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Protones , Espermatozoides , BallenasRESUMEN
Residual dipolar couplings observed in NMR spectra at very high magnetic fields have been measured to a high degree of accuracy for the paramagnetic protein cyanometmyoglobin. Deviations of these measurements from predictions based on available crystallographic and solution structures are largely systematic and well correlated within a given helix of this highly alpha-helical protein. These observations can be explained by invoking collective motion and small displacements of representative helices from their reported average positions in the solid state. Thus, the measurements appear to be capable of providing important insights into slower, collective protein motions, which are likely to be important for function, and which have been difficult to study using established experimental techniques.
Asunto(s)
Metamioglobina/análogos & derivados , Espectroscopía de Resonancia Magnética/métodos , Metamioglobina/química , Modelos Químicos , Modelos Moleculares , Movimiento (Física)RESUMEN
Very precise measurements of 1JNH couplings have been made for approximately 40% of the amide sites in cyanometmyoglobin using two different experimental approaches. The first approach is a previously described frequency-based method in which the couplings are observed as splittings in the frequency-domain spectrum. The second is a new approach, along the lines of quantitative J-correlation spectroscopy, in which the coupling is encoded in the resonance intensity. Measurements obtained from the two experimental techniques are in agreement to a high degree of precision (s.d. of 0.17 Hz) and residual deviations appear to be largely random. The new method offers substantial time savings when resonances are widely dispersed in the frequency domain and may offer improved precision in these instances.
Asunto(s)
Amidas/química , Espectroscopía de Resonancia Magnética , Metamioglobina/análogos & derivados , Metamioglobina/química , Isótopos de Nitrógeno , Conformación ProteicaRESUMEN
We report the Soret absorption spectra (500-350 nm) of the cyanomet derivatives of human hemoglobin and horse myoglobin, in the temperature range 300-20 K and in two different solvents (65% v/v glycerol-water or 65% v/v ethylene glycol-water). In order to obtain information on stereodynamic properties of active site of the two hemeproteins, we perform an analysis of the band profiles within the framework of electron-vibrations coupling. This approach enables us to single out the various contributions to the spectral bandwidth, such as those arising from non-radiative decay of the excited electronic state (homogeneous broadening) and from the coupling of the electronic transition i) with high frequency modes (that determines the vibronic structure of the band) and ii) with a "bath" of low frequency modes (that is responsible for the temperature dependence of the experimental spectra). We discuss the relevant parameters and their temperature dependence and compare them with the ones already reported for other derivatives of the same hemeproteins in the same solvents. In particular, non-harmonic contributions to soft modes are found, for cyanomet derivatives, to be larger than those observed for liganded carbonmonoxy but smaller than those observed for unliganded deoxy derivatives. The reported data enable us to obtain information on the dependence of stereodynamic properties of the heme pocket upon iron oxidation state, dimensions of the exogenous ligand and composition of the external matrix.
Asunto(s)
Metahemoglobina/análogos & derivados , Metamioglobina/análogos & derivados , Animales , Sitios de Unión , Frío , Caballos , Humanos , Hierro/química , Metahemoglobina/química , Metamioglobina/química , Músculo Esquelético/química , Oxidación-Reducción , Solventes , Espectrofotometría/métodosRESUMEN
The paramagnetic shifts of 13C nuclei positioned alpha to the haems in the A and B forms of rat cytochrome b5 and in metcyanomyoglobin have been analysed in terms of molecular orbitals based on D4h symmetry with a rhombic perturbation. The contribution to the 13C shifts from pseudocontact interactions is calculated from parameters obtained for a metal-centred dipolar shift tensor by fitting 1H shifts. The effect of electron delocalisation onto the vinyl groups of these haems b is separated with reference to the shifts of the vinyl beta carbons. In each case, it was found that the orientation of the magnetic axes in the plane of the haem is rotated away from the iron-nitrogen vectors in the opposite sense to the rotation of the rhombic perturbation and the molecular orbitals. The orientation of the orbitals is closely aligned with the normal to the single His ligand in metcyanomyoglobin, and with the average of the two normals in the bis-His cytochrome b5. It is concluded that the in-plane anisotropy of haems b is dominated by the orientation of the axial ligands in a similar manner to that in haems c and that the approximations used are weakened, but not invalidated, by the presence of partially conjugated vinyl groups.
Asunto(s)
Citocromos b5/química , Hemo/química , Metamioglobina/análogos & derivados , Animales , Carbono/química , Bovinos , Electroquímica , Histidina/química , Ligandos , Espectroscopía de Resonancia Magnética , Metamioglobina/química , Estructura Molecular , Ratas , Proteínas Recombinantes/química , BallenasRESUMEN
The measurement of dipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported. The splittings appear as small field-dependent perturbations of normal scalar couplings. Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein. Implications as an additional source of information for protein structure determination in solution are discussed.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Metamioglobina/análogos & derivados , Mioglobina/química , Estructura Secundaria de Proteína , Proteínas/química , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli , Hemo/análisis , Hidrógeno , Metamioglobina/química , Modelos Moleculares , Datos de Secuencia Molecular , Mioglobina/biosíntesis , Mioglobina/aislamiento & purificación , Isótopos de Nitrógeno , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Programas Informáticos , SolucionesRESUMEN
The crystal structure of Asian elephant cyano-metmyoglobin which has a glutamine instead of the usual distal site histidine has been determined to high resolution. In addition to this replacement, the substitution of a conserved leucine residue in position 29(B10) at the distal side by a phenylalanine was unambiguously identified based on the available electron density. The suspicion, that there were errors in the original sequence which has caused some confusion, is thus confirmed. Comparison with other myoglobin structures in various ligated forms reveals an essentially unchanged tertiary structure in elephant myoglobin despite the two amino acid substitutions in the heme pocket. Our current structural model shows that the N epsilon 2 atom of Gln64(E7) has moved with respect to the corresponding nitrogen position of His64(E7) in the CO complex of sperm whale myoglobin. The newly assigned residue Phe29(B10) penetrates into the distal side of the heme pocket approaching the ligand within van der Waals distance and causing a much more crowded heme pocket compared to other myoglobins. Kinetic properties of Asian elephant myoglobin, wild type, and recombinant sperm whale myoglobins are discussed in relation to the structural consequences of the two amino acid substitutions H64Q and L29F.
Asunto(s)
Metamioglobina/análogos & derivados , Fenilalanina , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Asia , Sitios de Unión , Cromatografía por Intercambio Iónico , Cristalización , Cristalografía por Rayos X/métodos , Elefantes , Variación Genética , Histidina , Enlace de Hidrógeno , Ligandos , Metamioglobina/química , Metamioglobina/aislamiento & purificación , Metamioglobina/metabolismo , Modelos Moleculares , Músculo Esquelético/metabolismo , Mioglobina/química , Mioglobina/aislamiento & purificación , Programas Informáticos , BallenasRESUMEN
The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.
Asunto(s)
Mioglobina/química , Mioglobina/metabolismo , Mutación Puntual , Conformación Proteica , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X/métodos , Elefantes , Espectroscopía de Resonancia Magnética/métodos , Matemática , Metamioglobina/análogos & derivados , Metamioglobina/química , Modelos Moleculares , Modelos Teóricos , Mioglobina/genética , Ingeniería de Proteínas , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría Raman/métodos , BallenasRESUMEN
The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.
Asunto(s)
Hemo/química , Metamioglobina/análogos & derivados , Metamioglobina/química , Conformación Proteica , Tortugas/metabolismo , Animales , Azidas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cianuros/metabolismo , Mamíferos/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Reptiles/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Agua/químicaRESUMEN
The characterization of the dynamics of conformational changes that accompany the ligand binding and dissociation reactions of myoglobin may provide insights into the events that control the physiological function of this oxygen storage protein. The cyanometmyoglobin system was chosen for this study because cyanide binds to the metmyoglobin state and dissociates upon reduction. The potential step spectroelectrochemical method was used here because the rate of cyanometmyoglobin reduction at an electrode can be controlled so that the rate of dissociation of the reduced cyanomyoglobin can then be followed.