An Investigation of the Distal Histidyl H-Bonds in Oxyhemoglobin: Effects of Temperature, pH, and Inositol Hexaphosphate

Abstract

Ligand binding to hemoglobin (Hb) is a dynamic process involving many tertiary and quaternary structural changes. It has been inferred that the O2 binding to Hb is stabilized by the H-bonds between the O2 ligands and the side-chains of two distal histidyl residues, α58His and β63His. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO2 A) in solution. Here, the NMR spectra of uniformly 15N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH, temperature, and inositol hexaphosphate (IHP). We have found that these H-bonds can be used as markers to reflect structural changes in the distal heme pocket. At lower pH and/or higher temperature, the side-chains of the distal histidines appear to be more mobile and the exchange with water molecules in the distal heme pockets is faster, which is consistent with the changes in the O2 affinity of Hb as a function of pH and temperature. Our NMR results have also confirmed that the H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. These H-bonds are sensitive to the mutation in the distal heme pockets, but only affected indirectly by the mutations in the quaternary interfaces and IHP. The recent findings are consistent with our previous NMR results showing that IHP exerts its effects on both the tertiary and quaternary structures of Hb A. (Supported by NIH grant R01GM084614)