MINI-MYOGLOBIN - ELECTRON-PARAMAGNETIC RESONANCE AND REVERSIBLE OXYGENATION OF THE COBALT DERIVATIVE

Mini-myoglobin, obtained by limited proteolysis of horse heart myoglobin (residues 32 to 139), represents a good model for testing the correlation between an exon and a protein domain. We have shown that ligand binding kinetics, spectral and folding features of mini-myoglobin are very similar to those of native myoglobin. In order to develop further the analysis of the structure-function relationship in this mini-protein, mini-globin was reconstituted with the heme moiety in which iron is replaced by cobalt. The Soret absorption spectra of oxy and deoxy cobaltous mini-myoglobin are very similar to those of cobaltous myoglobin derivatives; in addition, Co-mini-myoglobin binds oxygen reversibly with an n value ~ 1 and a p50 value of 45 to 50 mm Hg (the same as Co-myoglobin). Oxy Co-mini-myoglobin shows a well-resolved electron paramagnetic resonance (e.p.r.) spectrum typical of an oxygenated hemoprotein, while the spectrum of the deoxy derivative, although similar to that of deoxy Co-myoglobin, displays a lower resolution of the complex hyperfine structure. Moreover, photodissociation experiments on oxy Co-mini-myoglobin allow e.p.r. detection of an intermediate state, already observed in most hemoproteins and diagnostic for the interaction of bound oxygen with the distal histidine residue. Thus, reconstitution of miniglobin with cobalt protoporphyrin IX has provided, for the first time, a stable oxygenated complex that reflects a correct folding of the protein surrounding the heme pocket and possesses the functional behaviour typical of a hemoprotein. © 1991.