STRUCTURAL FACTORS GOVERNING HEMIN DISSOCIATION FROM METMYOGLOBIN

Rates of hemin dissociation from similar to 100 different metmyoglobin mutants were measured to determine which amino acid residues are impo rtant for retaining the prosthetic group, Most of the amino acids exam ined are within 4 Angstrom of the porphyrin ring, but replacements of a number of noncontact residues were also made. Mutations of His(93)(F 8) and Leu(89)(F4) can result in >100-fold increases in the rate of he min loss at pH 5 and 7. Some replacements of the contact residues His( 64)(E7), Val(68)(E11), His(97)(FG3), Ile(99)(FG5), Thr(39)(C4), and Ty r(103)(G4) cause >10-fold changes in the rate of hemin dissociation. S ubstitutions of the noncontact residues Leu(29)(B10), Phe(46)(CD4), an d Gly(65)(E8) can also increase the rate of hemin loss >10-fold. The k ey structural factors stabilizing bound hemin in myoglobin are (1) hyd rophobic interactions between apolar residues in the heme pocket and t he porphyrin ring, (2) the covalent bond between His(93)(F8) and the F e3+ atom, and (3) hydrogen bonding between distal residues and coordin ated water. Specific electrostatic interactions between the heme propi onates and amino acids at the surface of the protein appear to be less important. Loss of these polar interactions can be compensated by inc reasing the apolar character of either the heme group by esterificatio n of the propionates or replacement of charged surface residues with l arge apolar side chains [e.g., replacing His(97)(FG3) with Phe].