The dimeric and co-operative myoglobin ofNassa mutabilis. A peculiar case

The myoglobin present in the radular muscle of the Prosobranchia sea snailNassa mutabilis is a peculiar case among myoglobins. It is a dimer showing co-operative oxygen binding equilibrium curves with pO_{2 1/2}=4.7 mmHg, invariant with pH, and n=1.6. Although the globin is composed of 147 amino acid residues, corresponding to a molecular mass of 15760 D, gel filtration chromatography of the native myoglobin indicate Mr=26000±2000 D. Similarly, acrylamide electrophoretic analyses in SDS and velocity sedimentation indicate a molecular mass of about 13000 D for the denatured globin. The molecule is highly unstable and forms slowly a chromogen when aged or immediately upon oxidation to the ferric state. The visible region of the absorption spectrum of the O₂ or CO liganded myoglobin derivatives indicate an altered heme environment. Circular dichroism analyses confirm this indication showing negative Cotton effects in all regions of the heme absorption spectra of the MbO₂ and MbCO derivatives. Interestingly, the CD spectrum of the oxidised met-form shows a positive band almost symmetrical with respect to that of the MbO₂ derivative. This is similar to what reported for the monomeric hemoglobin ofGlycera dibranchiata for which a reversed heme orientation was proposed. Detailed resonance Raman spectroscopic studies have permitted a more direct investigation of the interactions between the heme and the protein. The proximal Fe-Im bond shows a stretching mode frequency down shifted by 5 cm^–1 with respect to the corresponding band of horse heart myoglobin, in good correlation with the much higher instability ofNassa m. myoglobin and its much lower oxygen affinity. The unusual bond instability finds additional support in a kinetic study in which the myoblogin is mixed with CO in buffered solutions at different pH values. This approach gives evidence that the Fe-Im bond is broken upon lowering the pH, with a pK of 4.0±0.2, the highest among those of deoxy hemoproteins. The rupture of the proximal bond appears to occur with a proton-linked transition showing n=1.8±0.1, again indicating cooperativity between the two subunits. The vinyl and propionate heme substituents show resonance Raman spectroscopic bands indicating different modes of interaction with their environment with respect to other myoglobins. Most interestingly, the vinyl stretching mode frequency, typically a single band, appears split in two bands inNassa m. myoglobin. This splitting is evident in all the investigated derivatives of the myoglobin, indicating that vinyl 2 and 4 are not equivalent in this molecule. A similar splitting has been found so far only inChironomus t.t. hemoglobin.