Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.
The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.
Santucci
R
R
Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy.
Ascoli
F
F
La Mar
G N
GN
Pandey
R K
RK
Smith
K M
KM
D000818:Animals; D002942:Circular Dichroism; D006427:Hemin / Q000737:chemistry*; D006736:Horses; D008745:Methylation; D008786:Metmyoglobin / Q000031:analogs & derivatives* / Q000737:chemistry; D008958:Models, Molecular; D009206:Myocardium / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation
D009211:Myoglobin; C007177:cyanometmyoglobin; D008786:Metmyoglobin; D006427:Hemin