Structural Factors Governing Hemin Dissociation from Metmyoglobin

Abstract

Rates of hemin dissociation from ∼100 different metmyoglobin mutants were measured to determine which amino acid residues are important for retaining the prosthetic group. Most of the amino acids examined are within 4 Å of the porphyrin ring, but replacements of a number of noncontact residues were also made. Mutations of His⁹³(F8) and Leu⁸⁹(F4) can result in >100-fold increases in the rate of hemin loss at pH 5 and 7. Some replacements of the contact residues His⁶⁴(E7), Val⁶⁸(E11), His⁹⁷(FG3), Ile⁹⁹(FG5), Thr³⁹(C4), and Tyr¹⁰³(G4) cause >10-fold changes in the rate of hemin dissociation. Substitutions of the noncontact residues Leu²⁹(B10), Phe⁴⁶(CD4), and Gly⁶⁵(E8) can also increase the rate of hemin loss >10-fold. The key structural factors stabilizing bound hemin in myoglobin are (1) hydrophobic interactions between apolar residues in the heme pocket and the porphyrin ring, (2) the covalent bond between His⁹³(F8) and the Fe³⁺ atom, and (3) hydrogen bonding between distal residues and coordinated water. Specific electrostatic interactions between the heme propionates and amino acids at the surface of the protein appear to be less important. Loss of these polar interactions can be compensated by increasing the apolar character of either the heme group by esterification of the propionates or replacement of charged surface residues with large apolar side chains [e.g., replacing His⁹⁷(FG3) with Phe].