Horse heart mini- and full length-myoglobin: pH effects on CO binding

Abstract

Mini-myoglobin (mini-HH-Mb) is a proteolytic fragment of horse heart myoglobin (HH-Mb) comprising residues 32–139, grossly corresponding to the central exon of the HH-Mb gene, which encodes residues 31–105. Unlike HH-Mb, which displays a single exponential for both CO association and CO dissociation kinetics, mini-HH-Mb shows a biphasic kinetic behavior for both processes, indicating the presence of at least two distinct conformations which are in a very slow (or no) equilibrium with each other. Between pH 2 and 12, CO association to both species of mini-HH-Mb shows two proton-linked transitions, one in the neutral-alkaline pH range (not observed for HH-Mb) and a second one in the acidic region displaying a pK_a of 2.9 like that observed in HH-Mb (pK_a = 2.7). Kinetics of CO dissociation from both species of mini-HH-Mb-CO was investigated between pH 5.5 and 10.5 only, since outside this pH range the slow CO dissociation kinetics are affected by protein denaturation, which shows up after few seconds. The CO dissociation rate shows a bell-shaped pH dependence for both conformations, while ligand dissociation from HH-Mb-CO is pH-independent. These features find a structural basis on molecular modelling, displaying a higher flexibility of both the proximal and distal side of the heme pocket in mini-HH-Mb, envisaging multiple conformations with different reactivity. This indicates that mini-HH-Mb differs from HH-Mb, suggesting a significant structural-functional role for the N- and C-terminal regions in O₂ supply to highly demanding tissues, like the retina, with implications for improving retinal blood flow in ocular pathologies.