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 approximately 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 mini-globin with cobalt protoprophyrin 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.
De Sanctis
G
G
Department of M.C.A. Biology, University of Camerino, Italy.
Falcioni
G
G
Grelloni
F
F
Desideri
A
A
Polizio
F
F
Giardina
B
B
Ascoli
F
F
Brunori
M
M
D000818:Animals; D003035:Cobalt / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006736:Horses; D007700:Kinetics; D008024:Ligands; D009206:Myocardium / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry*; D010782:Photolysis; D011524:Protoporphyrins / Q000737:chemistry; D013053:Spectrophotometry
D008024:Ligands; D009211:Myoglobin; D010446:Peptide Fragments; D011524:Protoporphyrins; D003035:Cobalt; C007095:cobaltiprotoporphyrin; D010100:Oxygen