Site E14 in Hemoglobins and Myoglobins: A Key Residue That Affects Hemin Loss and Lipid Oxidation Capacity

Abstract

Fish hemoglobins (Hbs) frequently contain glycine at site E14 while mammalian Hbs contain larger residues (e.g., alanine and serine). These differences were examined by creating structural variants at E14 using recombinant bovine myoglobin (Mb) as a model heme protein that contains alanine at E14. The Ala(E14)Gly mutation increased k(ox) and hemin loss 3-fold and 45-fold, respectively. Glycine at E14 creates a channel for solvent to enter the heme crevice, which enhances autoxidation and hemin loss rates. Hydration of the proximal heme pocket facilitates hemin loss because protonation of the proximal histidine weakens the linkage of the imidazole group to the iron atom of the hemin moiety. Ala(E14)Gly promoted lipid oxidation in washed fish muscle more rapidly during iced storage compared to wild type Mb at pH 5.7. This suggested that the rapid hemin loss from Ala(E14)Gly accelerated lipid oxidation. Ala(E14)Ser and Ala(E14)Val had little effect on k(ox) but somewhat accelerated net hemin loss. These studies suggest that enhanced access of solvent to the heme crevice of many fish Hbs at site E14 facilitates rapid hemin loss and moderately accelerates autoxidation. This likely is part of the reason fish Hbs promote lipid oxidation much more effectively compared to mammalian Hbs.