ROLE OF THE HEME PROPIONATES IN THE INTERACTION OF HEME WITH APOMYOGLOBIN AND APOCYTOCHROME B(5)

The heme propionate groups of both myoglobin (Mb) and cytochrome b(5) form hydrogen bonds with nearby surface amino acids residues that are believed to stabilize the heme-protein complex. To evaluate the magnit ude of this stabilization, the kinetics of heme dissociation from vari ants of horse heart Mb and cytochrome b(5) in which these hydrogen bon ding interactions have been systematically eliminated were studied by the method of Hargrove and colleagues (1994), and their thermal stabil ity was assessed. Elimination of each hydrogen bond was found to decre ase the thermal stability of the proteins and increase the rate consta nt for heme dissociation in a progressive fashion. For the Mb derivati ves, H-1-NMR studies indicate that the elimination of individual hydro gen bonds also affects the rate at which the heme orientational equili brium is achieved, In both types of kinetics experiment, the effects o f decreasing the number of potential hydrogen bonding interactions are found to be cumulative, Despite their kinetic effects, elimination of these hydrogen bonding interactions had no influence on the initial d istribution of heme orientational isomers immediately following recons titution or on the equilibrium constant of heme orientational disorder . The interactions between the heme propionates and nearby protein res idues play a partial role in the stabilization of the heme-protein com plex and are a major factor in the kinetic ''trapping'' of the minor h eme orientation. Comparisons of the various rate constants determined for the mechanism of heme binding and reorientation suggests that the intramolecular reorientation mechanism is slightly favored over the in termolecular mechanism.