Dielectric properties of hemoglobin and myoglobin. II. Dipole moment of sperm whale myoglobin

This paper is concerned with the molecular origin of the dipole moment of sperm whale myoglobin as it can be calculated from the dielectric dispersion at 1 Mcps on the basis of a mechanism of orientational polarization. It was possible to compare the dielectric increment of native myoglobin and its change during the reaction with bromo acetate with dipole moments calculated according to the known coordinates of the charged groups of the molecule. The agreement between the two shows that in myoglobin only the permanent dipole moment due to these charged groups is important, and that contributions from other possible sources remain within the limits of experimental error.     

The mouse myoglobin gene. Characterisation and sequence comparison with other mammalian myoglobin genes

Seal myoglobin (Mb) exons 1 and 3 were used as probes to isolate the functional mouse Mb gene. This gene has a very low level of Mb expression in skeletal muscle. Although it is shorter, the mouse Mb gene displays the common organisation found in human and seal Mb genes. In addition, we have defined blocks of conserved sequences in the 5' flanking region by comparison with other mammalian Mb genes. Moreover, about 10(3) bases upstream of the cap site we identified a repetitive B1 element directly associated with two overlapping open reading frames, containing a putative polyadenylation signal. A polypyrimidine-rich region has also been located upstream from the gene.     

Low pH myoglobin photoproducts.

Recently, there has been interest in determining the conditions under which the iron-histidine bond ruptures in myoglobin at low pH, so that the effect of proximal heme ligation can be studied. A 220-cm-1 Raman mode, assigned to iron-histidine stretching, is clearly visible after photolysis of aqueous MbCO samples below pH4 at room temperature (Sage et al. Biochemistry. 30:1237-1247). In contrast, Iben et al. (Biophys. J. 59:908-919) do not observe this mode upon photolysis of a pH3 MbCO sample in a glycerol/water glass at low temperature. In order to account for both the low temperature and the room temperature experiments, Iben et al. suggest a scheme involving an unusual protonation state of the proximal histidine. Here, we discuss some inconsistencies in their explanation of the room temperature results and offer instead a simple modification of an earlier model. In addition, circular dichroism data are presented that indicate partial unfolding of MbCO in aqueous solution below pH4, and raise questions about the claim of Iben et al. that MbCO remains folded in 75% glycerol at pH3.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

Fe-heme conformations in ferric myoglobin.

X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry.     

Structural dynamics of liganded myoglobin.

X-ray crystallography can reveal the magnitudes and principal directions of the mean-square displacements of every atom in a protein. This structural information is complementary to the temporal information obtainable by spectroscopic techniques such as nuclear magnetic resonance. Determination of the temperature dependence of the mean-square displacements makes it possible to separate large conformational motions from simple thermal vibrations. The contribution of crystal lattice disorder to the overall apparent displacement can be estimated by Mössbauer spectroscopy. This technique has been applied to high resolution x-ray diffraction data from sperm whale myoglobin in its Met iron and oxy cobalt forms. Both crystal structures display regions of large conformational motions, particularly at the chain termini and in the region of the proximal histidine. Overall, the mean-square displacement increases with increasing distance from the center of gravity of the molecule. Some regions of the heme pocket in oxy cobalt myoglobin are more rigid than the corresponding regions in Met myoglobin.     

Organization of the human myoglobin gene.

Cross-hybridization of the grey seal myoglobin gene to human DNA detected a single human myoglobin gene plus an extensive family of sequences apparently related to the central exon of this gene. The functional human gene is 10.4 kb long and has a haemoglobin-like three exon/two intron structure with long non-coding regions similar to its seal homologue. At least 300 bp of 5'-flanking region are closely homologous between the two genes, with the exception of a divergent purine-rich region 68-114 bp upstream of the cap site. A diverged tandem repetitive sequence based on (GGAT)165 is located 1100-1750 bp upstream from the gene; internal homology units within this sequence suggest sequence homogenization by gene microconversions. A second 33-bp tandem repeat element in the first intron is flanked by a 9-bp direct repeat, shares homology with other tandem repetitive elements in the human genome and may represent a novel form of transposable element.     

Dynamics of ligand binding to myoglobin.

Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.     

Binding of aromatic isonitriles to haemoglobin and myoglobin.

A series of aromatic isonitriles were synthesized and their binding to sheep haemoglobin and horse heart myoglobin was investigated. The disubstituted ligands 2,6-dimethylphenylisonitrile and 2,6-diethylphenylisonitrile were found to bind to horse-heart myoglobin with affinities ranging from 500 to 5000 times greater than that of ethylisonitrile (4.6 x 10(-6) M) which has been the tightest binding isonitrile ligand for myoglobin thus far reported. The tight binding was not found to vary significantly with pH or temperature. An explanation for the unexpectedly high affinity is offered in terms of the electronic structure of aromatic isonitriles.     

Structure and dynamics of the water around myoglobin.

The interplay between simulations at various levels of hydration and experimental observables has led to a picture of the role of solvent in thermodynamics and dynamics of protein systems. One of the most studied protein-solvent systems is myoglobin, which serves as a paradigm for the development of structure-function relationships in many biophysical studies. We review here some aspects of the solvation of myoglobin and the resulting implications. In particular, recent theoretical and simulation studies unify much of the diverse set of experimental results on water near proteins.     

The covalent structure of dog myoglobin.

The primary structure of the myoglobin of the domestic dog (German shepherd) was studied. Tryptic and thermolytic peptides were compared with the sequence of other known myoglobins; the stepwise automatic Edman's degradation of the whole globin and also the chymotryptic digestion of the median fragment obtained by CNBr cleavage completed this sequence. Comparison of the established dog myoglobin structure with those from other carnivora shows 16 differences versus badger, 20 versus harbour seal and 15 versus California sea lion.     

Camel myoglobin

1. Crystalline myoglobin was prepared from camel heart muscle. 2. A method was developed for the isolation of myoglobin that employs molecular-sieve chromatography. 3. Analytical chromatography of the camel myoglobin on a molecular-sieve column and on two types of ion-exchange columns gave in each case a single elution band, which accounted for better than 98% recovery and showed that the product was free from haemoglobin. 4. The iron content on a dry weight basis was 0.308%. This value corresponds to a molecular weight of 18100. 5. The spectra of acidic ferrimyoglobin, basic ferrimyoglobin and ferrimyoglobin cyanide were measured. 6. The pK(a) of the dissociation of the haem-bound water molecule in acidic ferrimyoglobin was 8.53 at 25 degrees . 7. Conclusions are drawn about the charge on the surface of the camel ferrimyoglobin molecule as compared with horse and sperm-whale ferrimyoglobins.