Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

Dual nature of the distal histidine residue in the autoxidation reaction of myoglobin and hemoglobin comparison of the H64 mutants.

The oxygenated form of myoglobin or hemoglobin is oxidized easily to the ferric met-form with generation of the superoxide anion. To make clear the possible role(s) of the distal histidine (H64) residue in the reaction, we have carried out detailed pH-dependence studies of the autoxidation rate, using some typical H64 mutants of sperm whale myoglobin, over the wide range of pH 5-12 in 0.1 M buffer at 25 degrees C. Each mutation caused a dramatic increase in the autoxidation rate with the trend H64V >/= H64G >/= H64L > H64Q > H64 (wild-type) at pH 7.0, whereas each mutant protein showed a characteristic pH-profile which is essentially different from that of the wild-type or native sperm whale MbO2. In particular, all the mutants have lost the acid-catalyzed process that can play a dominant role in the autoxidation reaction of most mammalian myoglobins or hemoglobins. Kinetic analyses of various types of pH-profiles lead us to conclude that the distal histidine residue can play a dual role in the nucleophilic displacement of O2- from MbO2 or HbO2 in protic, aqueous solution. One is in a proton-relay mechanism via its imidazole ring, and the other is in the maximum protection of the FeO2 center against a water molecule or an hydroxyl ion that can enter the heme pocket from the surrounding solvent.     

Aplysia oxymyoglobin with an unusual stability property

Native oxymyoglobin was isolated directly from the radular muscle of Aplysia kurodai with complete separation from metmyoglobin on a DEAE-cellulose column. It was examined for its spectral and stability properties. The spectrum of Aplysia MbO2 , which lacks the distal histidine, is very similar to those of mammalian oxymyoglobins , the alpha-peak being higher than the beta-peak and the absorbance ratio being 1.03. Its stability, however, is quite different from those of the mammalian oxymyoglobins , and Aplysia MbO2 is found to be extremely susceptible to autoxidation. Its rate is one-hundred times higher at pH 9.0, and its pH dependence is unusual and much less steep, when compared with sperm whale MbO2 as reference.     

Stability properties of oxymyoglobin from chicken gizzard smooth muscle

1. Oxymyoglobin (MbO2) was isolated directly from the smooth muscle of chicken gizzard and was examined for its spectral and stability properties. 2. When compared with sperm whale MbO2 as a reference, chicken gizzard MbO2 was found to be much more susceptible to autoxidation. Its pH-dependence was therefore analyzed in terms of an "acid-catalyzed three-state model". 3. The complete amino acid sequence of the myoglobin was also determined. Its hydropathy profile revealed that the region corresponding to the distal side of the heme iron appears to be less hydrophobic.     

Shark myoglobins. II. Isolation, characterization and amino acid sequence of myoglobin from Galeorhinus japonicus

Native oxymyoglobin (MbO2) was isolated from red muscle of G. japonicus by chromatographic separation from metmyoglobin (metMb) on DEAE-cellulose and the amino acid sequence of the major chain was determined with the aid of sequence homology with that of G. australis. It was shown to differ in amino acid sequence from that of G. australis by 10 replacements, to be acetylated at the amino terminus and to contain glutamine at the distal (E7) residue. It was also shown to have a spectrum very similar to that of mammalian MbO2. However, the pH-dependence for the autoxidation of MbO2 was seen to be quite different from that of sperm whale (Physeter catodon) MbO2. Although the sequence homology between sperm whale and G. japonicus myoglobins is about 40%, their hydropathy profiles were very similar, indicating that they have a similar geometry in their globin folding.     

Histidine and not tyrosine is required for the peroxide-induced formation of haem to protein cross-linked myoglobin

Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 - 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr(103) --> Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome.     

New transient species of sperm whale myoglobin in photodissociation of dioxygen from oxymyoglobin

We carried out the flash photolysis of oxy complexes of sperm whale myoglobin, cobalt-substituted sperm whale myoglobin, and Aplysia myoglobin. When the optical absorption spectral changes associated with the O2 rebinding were monitored on the nanosecond to millisecond time scale, we found that the transient spectra of the O2 photoproduct of sperm whale myoglobin were significantly different from the static spectra of deoxy form. This was sharply contrasted with the observations that the spectra of the CO photoproduct of sperm whale myoglobin and of the O2 photoproducts of cobalt-substituted sperm whale myoglobin and Aplysia myoglobin are identical to the corresponding spectra of their deoxy forms. These results led us to suggest the presence of a fairly stable transient species in the O2 photodissociation from the oxy complex of sperm whale myoglobin, which has a protein structure different from the deoxy form. We denoted the O2 photo-product to be Mb*. In the time-resolved resonance Raman measurements, the nu Fe-His mode of Mb* gave the same value as that of the deoxy form, indicating that the difference in the optical absorption spectra is possibly due to the structural difference at the heme distal side rather than those of the proximal side. The structure of Mb* is discussed in relation to the dynamic motion of myoglobin in the O2 entry to or exit from the heme pocket. Comparing the structural characteristics of several myoglobins employed, we suggested that the formation of Mb* relates to the following two factors: a hydrogen bonding of O2 with the distal histidine, and the movement of iron upon the ligation of O2.     

Yeast flavohemoglobin from Candida norvegensis. Its structural,spectral, and stability properties

Flavohemoglobin was isolated directly from the yeast Candida norvegensis and studied on its structural, spectral, and stability properties. In Candida flavohemoglobin, the 155 N-terminal residues make a heme-containing domain, while the remaining 234 C-terminal residues serve as a FAD-containing reductase domain. A pair of His-95 and Gln-63 was assigned to the proximal and distal residues, respectively. In purification procedure FAD was partially dissociated on a Butyl-Toyopearl column, so that FAD-lacking flavohemoglobin was also obtainable. In this ferric species, the Soret and charge-transfer bands were all characteristic of a penta-coordinate form. Compared with the recombinant heme domain expressed in Escherichia coli, we have measured the autoxidation rate over a wide pH range. The resulting pH dependence curves were then analyzed in terms of a nucleophilic displacement mechanism. As a result, the heme domain was found to be extremely susceptible to autoxidation, its rate being more than 100 times higher than that of sperm whale MbO2. However, this inherently high oxidation rate was dramatically suppressed in Candida flavohemoglobin to an extent almost comparable to the stability of mammalian myoglobins. These new findings lead us to conclude that Candida flavohemoglobin, differently from bacterial flavohemoglobins, can serve as an oxygen storage protein in aerobic conditions.     

Identification of the titrating group in the heme cavity of myoglobin. Evidence for the heme-protein pi-pi interaction

The pH dependence of the proton NMR chemical shifts of met-cyano and deoxy forms of native and reconstituted myoglobins reflects a structural transition in the heme pocket modulated by a single proton with pK 5.1-5.6. Comparison of this pH dependence of sperm whale and elephant myoglobin and that of the former protein reconstituted with esterified hemin eliminates both the distal histidine as well as the heme propionates as the titrating residue. Reconstitution of sperm whale met-cyano myoglobin with hemin modified at the 2,4-positions leads to a systematic variation in the pK for the structural transition, thus indicating the presence of a coupling between the titrating group and the heme pi system. The results are consistent with histidine FG3 (His-FG3) being the titrating group, and a donor-acceptor pi-pi interaction between its imidazole and the heme is proposed.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

Rearrangement of the distal pocket accompanying E7 His----Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket

Two-dimensional 1H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO2). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C zeta H proton of the Phe CD4 was found to move toward the iron of the heme by approximately 4 A relative to the position of sperm whale MbCO, requiring minimally a 3-A movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His----Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.     

Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix.     

Myoglobin and mitochondria: kinetics of oxymyoglobin deoxygenation in mitochondria suspension

The kinetics of whale MbO2 deoxygenation was studied spectrophotometrically in the presence of breathing rat mitochondria under conditions when mitochondria were separated from the protein solution by a semipermeable film capable to transfer only low-molecular-weight compounds and directly in the solution of MbO2 with mitochondria (incubation medium: 15-35 mM succinate, 150 mM sucrose, 100 mM KCl, 0.5 mM EGTA, 5 mM KH2PO4, 10 mM MOPS, pH 7.4). It was shown that the splitting of O2 from MbO2 at physiological pO2 is possible only if it directly contacts mitohondria. The deoxygenation rate does not depend on the protein concentration (zero order on [MbO2] as opposite to the first order reaction in the absence of mitochondria) and completely coincides with the rate of oxygen consumption by mitochondria under the same conditions, as indicated by the polarographic data. The dependence of the MbO2 deoxygenation rate on the concentration of mitochondria and the protein, and on the total charge of the MbO2 molecule was studied using horse MbO2 (pI 7.1), sperm whale MbO2 (pI 8.3), its zinc complex, Zn-MbO2 (pI > 8.3), and the sperm whale MbO2 derivative carboxymethylated at His residues, CM-MbO2 (pI 5.2). The mechanism of MbO2 deoxygenation in the cell obviously actuates its interplay with the mitochondrial membrane. As a result, the affinity of Mb to oxygen decreases several times, which corresponds to a shift of the Mb dissociation curve to higher pO2 values.     

On the difference in stability between horse and sperm whale myoglobins

The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both apo and holo forms, and was independent of pH from 5 to 8 and of the presence of sodium chloride. We also show that the substitution of sperm whale myoglobin residues Ala15 and Ala74 to Gly, the residues found at positions 15 and 74 in horse myoglobin, decreased the stability by 1.0 kcal/mol, indicating that helix propensity is an important component of the explanation for the difference in stability between the two proteins.     

1H NMR study of labile proton exchange in the heme cavity as a probe for the potential ligand entry channel in myoglobin

The exchange rates of heme cavity histidine nitrogen-bound protons in horse and dog metcyanomyoglobins have been determined at 40 degrees C as a function of pH by 1H NMR spectroscopy. They were compared to the results reported for the sperm whale homologue [Cutnell, J. D., La Mar, G. N., & Kong, S. B. (1981) J. Am. Chem. Soc. 103, 3567-3572]. The rate profiles suggest that the exchange follows EX2-type kinetics, and the relative rate values favor a penetration model over a local unfolding model. It was found that the behavior of protons located on the proximal side of the heme is similar in the three proteins. The distal histidyl imidazole NH, however, shows a highly accelerated hydroxyl ion catalyzed rate in horse and dog myoglobins relative to that in sperm whale myoglobin. NMR spectral and relaxational characteristics of the assigned heme cavity protons indicate that the global geometry of the heme pocket is highly conserved in the ground-state structure of the three proteins. We propose a model that attributes the different distal histidine exchange behavior to the relative dynamic stability of the distal heme pocket in dog or horse myoglobin vs. sperm whale myoglobin. This model involves a dynamic equilibrium between a closed heme pocket as found in metaquomyoglobin [Takano, T. (1977) J. Mol. Biol. 110, 537-568] and an open pocket as found in phenylmetmyoglobin [Ringe, D., Petsko, G. A., Kerr, D. E., & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4].(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning, overexpression and characterization of micro-myoglobin, a minimal heme-binding fragment.

We report the cloning and expression of micro-myoglobin, a 78-amino-acid fragment containing residues 29-105 of sperm whale myoglobin, and spanning the region from mid-helix B to mid-helix G of the globin fold. In contrast to full-length myoglobin and to mini-myoglobin (residues 32-129), the micro-myoglobin apoprotein is almost unfolded. However, circular dichroism and absorption spectroscopy data indicate that this fragment is capable of folding into a functional heme-binding unit forming a complex with the prosthetic group with characteristics similar to native myoglobin. Therefore, this case represents a new example of cofactor-assisted folding. The experimental data suggest independence between myoglobin subdomains.     

The homonuclear Overhauser effect in H2O solution of low-spin hemeproteins

Proton-proton Overhauser effects were observed in ¹H₂O solutions of sperm whale metcyano myoglobin. Dipolar connectivities involving hyperfine-shifted exchangeable protons such as the proximal and distal histidine ring NH's allowed us to categorize signals as arising from residues located on one side of the heme plane or on the other. With these connectivities, as well as spin-lattice relaxation times, spectral assignments were reached that were used to derive structural and dynamic information about the heme environment. Thus, it was shown that the distal histidine residue does not titrate down to pH 4.1 and that the CH₂ of the proximal histidine side chain tumbles with the same correlation time as the protein. Some other applications and limitations are presented.     

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins.

Some insects have a globin exclusively in their fast-growing larval stage. This is the case in the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta-VII Hb) was of particular interest. When its ferric met-form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped-flow and rapid-scan techniques, and the observed first-order rate constants of k(out) = 25 s(-1) and kin = 128 s(-1) were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 m buffer at 25 degrees C. For O2 affinity, Ta-VII Hb showed a value of P50 = 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (deltaH+ = -0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta-VII HbO2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH-dependence curve was compared with those of another component Ta-V HbO2 and sperm whale MbO2, and described based on a nucleophilic displacement mechanism. In light of the O2 binding affinity, Bohr effect and considerable stability of the bound O2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O2 transport and storage as the major component in the larval hemolymph.     

Solution 1H nuclear magnetic resonance determination of hydrogen bonding of the E10 (66) Arg side-chain to the bound ligand in Aplysia cyano-met myoglobin.

A combined one-dimensional nuclear Overhauser effect, paramagnetic-induced relaxation and two-dimensional sequence-specific 1H n.m.r. assignment of the spectrum of portions of the distal pocket of Aplysia cyano metMyoglobin (metMbCN) has been carried out in order to establish the presence and identity of distal residues in the heme pocket. In the absence of the usual distal E7 His in Aplysia Mb (E7 Val), the sequence-specific assignment of the E7 and E10 residues, together with their hyperfine shift patterns, relaxivities and dipolar connectivities to each other and the remainder of the E helix, reveal that the E10 Arg is turned into the pocket and hydrogen bonds to the bound cyanide group. We have previously found a similar rearrangement of the E10 Arg in Aplysia fluoro metMyoglobin, and the stabilizing effect of this residue was proposed to be responsible for the slow rate of cyanide dissociation from rapidly reduced ferrous Aplysia myoglobin. Based on the similar distal E7 His hydrogen-bonding interaction to the bound ligand in the crystal of sperm whale MbO2 and in solution of its cyano met complex, we propose that the E10 Arg similarly hydrogen bonds to the bound O2 in Aplysia MbO2 and accounts for its strong ligand binding and slow dissociation rate.