Comparison of the dynamics of myoglobin in different crystal forms.

Crystals have been grown of "sperm whale" myoglobin produced in Escherichia coli from a synthetic gene and the structure has been solved to 1.9 A resolution. Because of a remaining initiator methionine, this protein crystallizes in a different space group from native sperm whale myoglobin. The three-dimensional structure of the synthetic protein is essentially identical to the native sperm whale protein. However, the crystallographic B-factors for parts of the molecule are quite different in the two crystal forms, and provide a measure of the effect of different packing constraints on the flexibility of the protein. The effect of the packing forces is to reduce the mobility of the protein in the regions of contact and thereby introduce differences in mobilities between the two crystal forms. Discrepancies between mobilities calculated from molecular dynamics simulations and crystallography can be reduced by considering the data from both crystal forms.     

X-ray crystal structure of a recombinant human myoglobin mutant at 2.8 A resolution

We have grown crystals in trigonal space group P3(2)21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the molecular replacement method to determine the X-ray crystal structure of the mutant at 2.8 A resolution. At the present stage of refinement, the crystallographic R-value for the model, with tightly restrained stereochemistry, is 0.158 for 5.0 to 2.8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.     

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.     

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.     

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.     

A primitive myoglobin from Tetrahymena pyriformis: its heme environment, autoxidizability, and genomic DNA structure

A myoglobin-like protein isolated from Tetrahymena pyriformis is composed of 121 amino acid residues. This is much smaller than sperm whale myoglobin by 32 residues, suggesting a distinct origin from the common globin gene. We have therefore examined this unique protein for its structural, spectral and stability properties. As a result, the rate of autoxidation of Tetrahymena oxymyoglobin (MbO(2)) was found to be almost comparable to that of sperm whale MbO(2) over a wide range of pH 4-12 in 0.1 M buffer at 25 degrees C. Moreover, both pH profiles exhibited the remarkable proton-assisted process, which can be performed in sperm whale myoglobin by the distal (E7) histidine as its catalytic residue. These kinetic observations are also in full accord with spectral examinations for the presence of a distal histidine in ciliated protozoa myoglobin. At the same time, we have isolated the globin genes both from T. pyriformis and Tetrahymena thermophila, and found that there is no intron in their genomic structures. This is in sharp contrast to previous reports on the homologous globin genes from Paramecium caudatum and Chlamydomonas eugametos. Rather, the Tetrahymena genes seemed to be related to the cyanobacterial globin gene from Nostoc commune. These contracted or truncated globins thus have a marked diversity in the cDNA, protein, and genomic structures.     

Comparative oxygen affinity of fish and mammalian myoglobins

Summary Myoglobins from rat, coho salmon (Oncorhynchus kisutch), buffalo sculpin (Enophrys bison) hearts, and yellowfin tuna (Thunnus albacares) red skeletal muscle were partially purified and their O₂ binding affinities determined. Commercially prepared sperm whale myoglobin was employed as an internal standard. Tested at 20°C, myoglobins from salmon and sculpin bound O₂ with lower affinity than myoglobins from the rat or sperm whale. Oxygen binding studies at 12°C and 37°C suggest that this difference is adaptive, permitting myoglobins from cold-adapted fish to function at physiologically relevant temperatures. Taken together, purification and O₂ binding data obtained in this study reveal a previously unrecognized diversity of myoglobin structure and function.     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

Complete amino acid sequence of the major component myoglobin of finback whale (Balaenoptera physalus)

The complete amino acid sequence of the major component myoglobin from finback whale, Balaenoptera physalus, was determined by the automated Edman degradation of several large peptides obtained by specific cleavages of the protein. Three easily separable peptides were obtained by cleaving with cyanogen bromide at the two methionine residues and one large peptide was isolated after cleavage with (2-p-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. More than 60% of the covalent structure was established by the sequential degradation of three of these peptides and the apomyoglobin. An additional 30% of the primary sequence was established with peptides obtained from tryptic digestion of both the apomyoglobin and the acetimidoapomyoglobin, and the final 10% of the sequence was completed after digestion of the two larger cyanogen bromide peptides with S. aureus strain V8 protease. This myoglobin differs from that of the sperm whale, Physeter catodon, at 15 positions, from that of the arctic minke whale, Balaenoptera acutorostrata, at 3 positions, and from that of the California gray whale, Eschrichtius gibbosus, at 4 positions. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

Fluorescence decay kinetics of the tryptophyl residues of myoglobin: effect of heme ligation and evidence for discrete lifetime components

The fluorescence decay kinetics of the tryptophyl residues of sperm whale and yellowfin tuna myoglobin have been determined by using time-correlated single photon counting, with picosecond resolution. Purification by HPLC techniques resulted in the isolation of samples that exclusively displayed picosecond decay kinetics. Lifetimes of 24.4 ps for Trp14 and 122.0 ps for Trp7 were found for oxy sperm whale myoglobin (pH 7), which agree with theoretical predictions [Hochstrasser, R. M., & Negus, D. K. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4399-4403]. The effects of ligand binding and pH on the decay kinetics were investigated, and the results were shown to be consistent with the known crystal structures. Data for the met form of sperm whale myoglobin were analyzed both in terms of a sum of discrete exponential components and as a continuous gamma distribution of exponential decays. The results were not found to support the existence of multiple, structurally distinct conformation states in myoglobin.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.     

Structural and functional aspects of the heart ventricle myoglobin of bluefin tuna

The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna. The alpha-helix content of tuna myoglobin has been found considerably lower than that of mammalian myoglobin. No correlation has been discovered between the conformational stability and alpha-helix content. Denaturation experiments have shown that the whole structure of tuna myoglobin results from the interaction of two structural units which represent the product of independent folding processes. The structure of tuna myoglobin has been found more open and disorganized than that of sperm whale. This result has been related to the low content of electrostatic interactions and explained in terms of evolutive adaptations.     

X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin at 2.0 A resolution. Stabilization of the fluoride ion by hydrogen bonding to Arg66 (E10)

The X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin has been solved and refined at 2.0 A resolution; the crystallographic R-factor is 13.6%. The fluoride ion binds to the sixth co-ordination position of the heme iron, 2.2 A from the metal. Binding of the negatively charged ligand on the distal side of the heme pocket of this myoglobin, which lacks the distal His, is associated with a network of hydrogen bonds that includes the fluoride ion, the residue Arg66 (E10), the heme propionate III, three ordered water molecules and backbone or side-chain atoms from the CD region. A comparison of fluoride and oxygen dissociation rate constants of A. limacina myoglobin, sperm whale (Physeter catodon) myoglobin and Glycera dibranchiata monomeric hemoglobin, suggests that the conformational readjustment of Arg66 (E10) in A. limacina myoglobin may represent the molecular basis for ligand stabilization, in the absence of a hydrogen-bond donor residue at the distal E7 position.     

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.     

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.