Disruption of the heme iron-proximal histidine bond requires unfolding of deoxymyoglobin.

The unfolding behavior of 10 different distal heme pocket mutants of sperm whale deoxymyoglobin (deoxyMb) has been investigated. The effects of distal histidine (His 64) replacement were the primary focus; however, mutations at Leu 29, Val 68, and Ile 107 were also examined. Formation of the spectroscopically distinguishable heme intermediate (I') of deoxyMb was tracked as a function of pH and guanidinium chloride (GdmCl) concentration. The appearance of this intermediate signals cleavage of the iron-proximal histidine (His 93) bond. The key observations are as follows. (1) None of the distal heme pocket mutations investigated alter the nature of the heme intermediates that are formed under low pH unfolding conditions. (2) Unfolding of deoxyMb at high concentrations of GdmCl proceeds through the same heme intermediates that occur under low pH conditions. (3) The rate of the iron-histidine bond cleavage in an acidic medium is dramatically slowed when large hydrophobic residues (Leu and Phe) replace the distal histidine, whereas there is little correlation between the polarity of the residue at position 64 and the rate of denaturation by GdmCl. (4) However, apolar residues at position 64 enhance significantly the equilibrium resistance of deoxyMb to iron-histidine bond cleavage under both low pH and high GdmCl unfolding conditions. There is a direct correlation between the equilibrium pH and GdmCl values for maximum intermediate formation and the stabilities of the corresponding apoproteins. Collectively, these observations suggest that substantial unfolding of deoxyMb is required for Fe(II)-His 93 bond cleavage. Unlike the situation for aquometMb, heme loss from deoxyMb is not driven by protonation of the proximal histidine ligand. Instead, the process is mediated by more global unfolding of the protein that leads to solvation of the prosthetic group.