Structural and kinetic characterization of myoglobins from eurythermal and stenothermal fish species

Teleost myoglobin (Mb) proteins from four fish species inhabiting different temperature environments were used to investigate the relationship between protein function and thermal stability. Mb was isolated from yellowfin tuna (homeothermal warm), mackerel (eurythermal warm), and the Antarctic teleost Notothenia coriiceps (stenothermal cold). Zebrafish (stenothermal tropical) myoglobin was expressed from cloned cDNA. These proteins differed in oxygen affinity, as measured by O2 dissociation rates and P50 values, and thermal stability as measured by autooxidation rates. Mackerel Mb had the highest P50 value at 25 degrees C (3.7 mmHg), corresponding to the lowest O2 affinity, followed by zebrafish (1.0 mmHg), yellowfin tuna (1.0 mmHg), and N. coriiceps (0.6 mmHg). Oxygen dissociation rates and Arrhenius plots were similar between all teleost species in this study, with the exception of mackerel myoglobin, which was two-fold faster at all temperatures tested. Myoglobin from the Antarctic teleost had the highest autooxidation rate (0.44 h(-1)), followed by mackerel (0.26 h(-1)), zebrafish (0.22 h(-1)), and yellowfin tuna (0.088 h(-1)). Primary structural analysis revealed residue differences distributed throughout the polypeptide sequences, making it difficult to identify, which, if any, residues contribute to structural flexibility. However, analysis of molecular dynamics trajectories indicates that Mb from the eurythermal mackerel is predicted to be the most flexible protein within the D loop and FG turn. At the same time, it has the lowest O2 affinity and the highest O2 dissociation rates when compared to myoglobins from teleosts that appear to be less flexible in our dynamics simulations.     

Comparative Studies of the Indoleamine Dioxygenase-like Myoglobin from the Abalone Sulculus diversicolor

The abalone Sulculus diversicolor contains abundant myoglobin in its buccal mass. The myoglobin is homodimeric and the molecular mass of the constituent polypeptide chain is 41,000 Da. The amino acid sequence and gene structure are highly homologous with those of a vertebrate tryptophan-degrading enzyme, indoleamine dioxygenase (IDO). Thus Sulculus myoglobin evolved from an IDO gene, and represents a typical case of functional convergence. The oxygen equilibrium properties of Sulculus myoglobin were examined and compared with those of myoglobins from other sources. It binds oxygen reversibly, and the P₅₀ was determined to be 3.8 mmHg at 20°C and pH 7.4, showing that the oxygen affinity of Sulculus myoglobin is significantly lower than those of usual 16 kDa myoglobins. It also displays no cooperativity (n_max: 1.02–1.06) and no alkaline Bohr effect between pH 7.0 and 7.9. The cDNA-derived amino acid sequences of vertebrate IDOs, molluscan IDO-like myoglobins and a homolog in the yeast Saccharomyces were aligned, and several amino acid residues were proposed as candidates for key residues to control the function of IDO or myoglobin.     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates

Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates. These hemoglobins appear to be the result of gene duplication and fusion. Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids. These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form. Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined. The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate. On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively). The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin. These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen.     

Molecular mechanism for ligand stabilization in the mollusc myoglobin possessing the distal Val residue.

Myoglobin extracted from the triturative stomach of Dolabella auricularia, a common mollusc found on the Japanese coast, possesses naturally occurring substitution at the distal E7 position (Val-E7) and its oxygen affinity is only slightly lower than those of the common mammalian myoglobins possessing the usual His-E7. 1H nuclear magnetic resonance studies of Dolabella met-cyano myoglobin have revealed that a guanidino NH proton of Arg-E10 is hydrogen-bonded to the Fe-bound CN-. The role of Arg-E10 as a hydrogen-bond donor for Fe-bound ligand in the present myoglobin appears to be responsible for its relatively high ligand affinity.     

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.     

The primary structure of myoglobin from pacific green sea turtle (Chelonia mydas caranigra)

The amino acid sequence of the main component myoglobin from skeletal muscle of Pacific green sea turtle (Chelonia mydas caranigra) has been determined. The globin is 153 residues in length and has a free amino-terminus. The heme-binding and internal residues are as found in mammalian myoglobins. Ten substitutions are observed between this myoglobin and that from map turtle. About 38, 52, 47 and 86 substitutions are noted in comparison with the myoglobins of other reptiles, mammals, birds and fish, respectively. The inferred pattern of structural stabilization and conservation of two loci are typical of tetrapod myoglobin.     

The sequence-immunology correlation revisited: Data for cetacean myoglobins and mammalian lysozymes

Quantitative microcomplement fixation tests employing rabbit antisera were done to compare immunologically 13 cetacean myoglobins and 15 mammalian lysozymes c of known amino acid sequence. In both cases there was a strong correlation between immunological distance (y) and percent sequence difference (x), as had been found for several other globular proteins. For myoglobin the relationship could be described by y = 10.5x and for lysozyme by y = 8.5x. The coefficients in both of these equations are appreciably higher than the values of 5.1–6.9 reported for three other vertebrate globular proteins (bird lysozyme c, mammalian ribonuclease, and mammalian serum albumin), and they imply that rabbit antisera to mammalian myoglobins and lysozymes are more sensitive to evolutionary substitutions. A strong inverse correlation (r = -0.95) was found when the slope of the line relating y to x for these five data sets was plotted against the percent sequence difference between the rabbit's own protein and the proteins immunized with. Specifically, the cetacean myoglobins on average differ in amino acid sequence from rabbit myoglobin by less than 13% and exhibit the steepest slope (10.5), while bird lysozyme sequences differ by nearly 40% from rabbit lysozyme and exhibit the shallowest slope (5.1).     

Oxygenation and EPR spectral properties of Aplysia myoglobins containing cobaltous porphyrins.

Cobalt myoglobins (Aplysia) have been reconstituted from apo-myoglobin (Aplysia) and proto-, meso-, and deutero-cobalt porphyrins. Each of them showed the 30--60 times lower oxygen affinity than those of the corresponding cobalt myoglobins (Sperm whale). Kinetic investigation of their oxygenation by the temperature-junp relaxation technique showed that the low oxygen affinity of cobalt myoglobin (Aplysia) is due to a large dissociation rate constant. the electron paramagnetic resonance (EPR) spectrum of oxy cobalt myoglobin (Aplysia) is affected by the replacement of H2O with D2O, suggesting a possible interaction between the bound oxygen and the neighboring hydrogen atom. A low temperature photodissociation study showed that the product of photolysis of oxy cobalt myoglobin (Aplysia) gives an EPR spectrum different from that of the deoxy-cobalt myoglobin (Aplysia) and from that of the photolysed form of oxy-cobalt myogloin (Sperm whale). These observations suggest that in oxy-cobalt myoglobin (Aplysia) the bound oxygen might interact with amino acid adjacent to it, but the interaction is weaker than that in oxy cobalt myoglobin (Sperm whale).     

Aplysia myoglobins with an unusual amino acid sequence

The complete amino acid sequence of the myoglobin from Aplysia juliana, a species distributed world-wide, has been determined and compared with the sequence of the myoglobin of Aplysia limacina, a Mediterranean species, and of Aplysia kurodai, a Japanese and Asian species. Unlike mammalian myoglobins, Aplysia myoglobins contain only a single histidine residue, lacking the distal one, the homology being 76% between A. juliana and A. limacina, 74% between A. juliana and A. kurodai, and 83% between A. limacina and A. kurodai. The hydropathy profiles of the Aplysia myoglobins are very similar, but completely different from that of sperm whale myoglobin, taken as the reference.     

Oxygen binding of myoglobins of diving animals]

When studying oxygen binding by myoglobins of diving animals it is shown that half-saturation of myoglobins obtained from the muscles of animals who can stop the external respiration for a long period of time occurs at oxygen strength of 0.62-0.67 mm Hg. Such indices of oxygenation of respiratory hemoprotein of muscles are characteristic of most mammals. Temperature being decreased from 37 to 15 degrees C, myoglobin affinity to oxygen considerably increases.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

Evidence of met-form myoglobin from Theliostyla albicilla radular muscle

Gastropod mollusc myoglobins provide interesting clues to the evolution of this family of proteins. In addition to conventional monomeric myoglobins, this group also has dimeric and unusual indoleamine dioxygenase-like myoglobins. We isolated myoglobin from the radular muscle of living gastropod mollusc Theliostyla albicilla. The myoglobin appeared to be present in an oxidized met-form, a physiologically inactive form that is not capable of binding oxygen. Under the same extraction conditions, myoglobins mainly of the physiologically active oxy-form have been isolated from other molluscs. The complete amino acid sequence of 157 residues of Theliostyla myoglobin shows that it has a long N-terminal extension of seven residues and contains three functional key residues: CD1-Phe, E7-His, and F8-His. The metmyoglobin can easily be reduced to a ferrous state with Na(2)S(2)O(4). The autoxidation rate of the oxy-form was comparable to other molluscan myoglobins over a wide pH range, and Theliostyla myoglobin was shown to be stable as an oxygen-binding protein. Thus, the predominantly met-form of myoglobin in Theliostyla can be attributed to the incomplete functioning of the myoglobin reduction system in the radular muscle. Although the function of Theliostyla myoglobin is unclear, it may be a scavenger of H(2)O(2).     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

The myoglobin of rodents Proechimys guairae (casiragua) and Mus musculus (house mouse)

The amino acid sequences of the myoglobins of two rodents, the casiragua and the house mouse, have been determined. The myoglobin of casiragua differs from that of viscacha (another hystricomorph) at 6 positions. Mouse myoglobin differs from that of mole-rat (another myomorph) at 17 positions, whereas casiragua and mouse differ at 22 positions. Mouse myoglobin possesses several features unique among all known myoglobins (Gly 31, Cys 66, Thr 74 and Glu 113) and one substitution unique among known mammalian myoglobins (Glu 53).

Amino acid sequence Myoglobin Rodent Casiragua Mouse Phylogeny     

Crystal structures of modified myoglobins. II. Relation between oxygen affinity properties and structural changes around heme in myoglobins reconstituted with 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme

The crystal structures of sperm whale metmyoglobins reconstituted with three kinds of modified hemes, 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.216, 0.219, and 0.195, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The 2-vinyl-4-isopropyldeuteroheme was found to be in a reverse orientation, in which the heme plane is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other two hemes were the same as that of protoheme in native myoglobin. In the myoglobins with 2,4-diisopropyldeuteroheme and 2-vinyl-4-isopropyldeuteroheme, both of which have lower oxygen affinities than native myoglobin, the bulky isopropyl side chain pushes Phe 43 0.7 A toward His 64 (the distal histidine) in the former, and the whole E helix at most 1.5 A, including a 0.7 A shift of the His 64 imidazole ring, in the latter. The changes of the structures prevent His 64 from forming a hydrogen bond with the liganded oxygen molecule, so that these two modified myoglobins show low oxygen affinities. On the other hand, there is no such drastic displacement in myoglobin with 2-isopropyl-4-vinyldeuteroheme, which has a slightly higher oxygen affinity than native myoglobin.     

Comparison of the biochemical and molecular properties of myoglobins from three Biomphalaria species

Myoglobin is a globin with heme as prosthetic group whose main known biological role is to bind to O2 reversibly. On account of their large diversity, globins from mollusks have contributed to the study of this protein class. The cDNA of the myoglobins from Biomphalaria straminea and Biomphalaria tenagophila, which have a glutamine as distal residue (E7), were constructed and analyzed by bioinformatic tools. Native (not recombinant) myoglobins of these two Biomphalaria species were purified and their experimental molecular mass (about 16 kDa) and pI (about (8.0) were provided. Data analysis showed that these proteins are monomers with the signature for the classic myoglobin fold and they are blocked in amino terminus probably by an acetyl group. Values of the autoxidation rates showed that these myoglobins oxidized slowly. About the primary sequences of the myoglobins, they turned out to be satisfactory to group mollusks in phylogenetic class.     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Myoglobin in control and dystrophic chicken muscle

This study shows no differences in the character of myoglobin in muscles from control and genetically dystrophic chickens. Our data on cellulose chromatography, acrylamide gel electrophoresis, sedimentation velocities, spectra, isoelectric points, and amino acid analyses provide useful information on the first avian myoglobin to be added to the list of mammalian and fish myoglobins which have already been characterized.