Hemoglobin and myoglobin stabilization by heme-fluoride complexes

Abstract

Under physiological conditions, hemoglobin (Hb) and myoglobin (Mb) are subject to autoxidation, leading to the formation of ferric heme proteins with weakened globular structures and resulting in the loss of the heme cofactor. To gain further insights into the role of the heme pocket in the stabilization of the heme-containing proteins (holo-Mb and holo-Hb), the relative stabilities of heme-bound fluoride complexes (Mb-F and Hb-F) were examined under denaturing conditions. The electronic spectra of the heme proteins were followed upon addition of guanidine hydrochloride (GuHCl) and under denaturation-induced acidification. In the presence of ~ 2 M GuHCl, the heme-bound fluoride complexes are 27 – 46% more stable than metaquo Mb and Hb and about 50% more stable under acidic conditions. Mb and Hb denaturation equilibria at 25 and 40 °C were analyzed by using two-state and three-state models, respectively. Under GuHCl-induced and acid-induced denaturations, Mb-F and Hb-F behave as a two-state process. In addition, the affinity for fluoride under these denaturing conditions were determined. The dissociation constants (Kd) for fluoride binding in Hb and Mb under 1.5 M GuHCl are slightly greater than the measured Kd without GuHCl, which we attribute to the presence of molten-globular holo protein intermediates that are not favorable for fluoride binding. This study shows that the measured fluoride affinities in the presence of GuHCl at various temperatures and the effects of pH on the Kd can provide additional thermodynamic properties of Mb and Hb denaturation processes at low pH.