Cooperative mechanism in the homodimeric myoglobin from Nassa mutabilis

Oxygen binding and spectroscopic propel-ties of the homodimeric myoglobin (Mb) from the prosobranchia sea snail Nassa mutabilis have been investigated. Oxygen equilibrium curves are pH-independent and cooperative with P-50 = 5 +/- 1 mmHg and n approximate to 1.5. Circular dichroism spectra of the oxygenated and deoxygenated form of N. mutabilis Mb are superimposable between 190 and 250 nm, suggesting a mechanism for cooperative ligand binding that does not involve changes in the a-helical content of the whole protein. The oxygen dissociation process is biphasic and pH-dependent, with different pK(a) values (= 6.7 +/- 0.2 and 8.5 +/- 0.3) for the two phases. Moreover, the activation energy is essentially the same for both oxygen dissociation processes (E-a = 56.4 +/- 2.1 kJ/mol for the fast phase, and E-a = 53.8 +/- 1.9 kJ/mol for the slow phase), indicating that the rate difference for O-2 dissociation between the diliganded and the monoliganded species is mostly dependent on a variation of the activation entropy. Ferrous nitrosylated N. mutabilis Mb shows. at alkaline and neutral pH, axial and rhombic X-band EPR signals, respectively, which display below pH 6 a three-hyperfine pattern typical of five-coordination. The results presented here suggest that in N. mutabilis Mb the kinetic control of cooperativity operates through a mechanism never observed before in other hemoproteins, which requires a ligand-linked large enhancement for the value of the oxygen association process in a molecule not undergoing changes in quaternary structure.