An investigation of the distal histidyl hydrogen bonds in oxyhemoglobin: effects of temperature, pH, and inositol hexaphosphate.

Hemoglobin (Hb) is a tetrameric, heme-containing protein consisting of two α- and two β-chains and is the carrier of oxygen from lungs to tissues in vertebrates. Oxygen binding to Hb is reversible and cooperative (1). The ratio of the equilibrium-binding constants for CO and O2 is approximately 20,000 for free heme, and is lowered to about 250 in the case of Hb A (2). Thus, the relative affinity for O2 versus CO is greatly increased in Hb, due to having its hemes embedded in the heme pockets of the protein molecule and interacting with their environment. It is currently accepted that O2 bound to Hb is stabilized by the hydrogen bonds between oxygen ligands and the distal histidyl residues. Early X-ray crystal structural studies at moderate resolution of 2.1 A (3) suggested the existence of a H-bond in the α-heme pocket between the bound oxygen and the distal histidine E7 (αHis58), and possibly a very weak one in the β-heme pocket to the E7 (βHis63) histidine. High-resolution (1.25 A) X-ray crystal structures (4) gave further support to this idea. The existence of such an H-bond has been inferred from the crystal structures (4) to explain the stronger binding of O2 to Hb and Mb, relative to the free heme. Although reasonable, this suggestion has remained controversial due to a lack of direct experimental evidence, especially for Hb in solution, under physiological conditions (5–8). Our previous NMR study (9) has provided the first direct evidence for the presence of the distal histidyl H-bonds in HbO2 A in solution. Here, we investigate the stability of these H-bonds as affected by pH, temperature, and an organic phosphate, inositol hexaphosphate (IHP). We have also examined the change to these H-bonds caused by mutations at the subunit interfaces (α1β1 and α1β2) and at the distal heme pockets. Uniformly 15N-labeled recombinant human normal adult hemoglobin A (rHb A) and five recombinant mutant rHbs were prepared and the NMR spectra were recorded on Bruker AVANCE DRX-600 MHz spectrometers. The changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the affinity of Hb for O2 and are clearly shown by our NMR experiments. We have found that the H-bond markers, namely the cross peaks for α58His (1He2, 15Ne2) and β63His (1He2, 15Ne2) are significantly affected by pH and temperature. At lower pH and/or higher temperature, the side-chains of the distal histidines appear to be more mobile and the proton exchange with water molecules in the distal heme pocket is faster, causing these markers to lose intensity and even to disappear. Our present findings have confirmed that the H-bond in the β-chain is weaker than that in the α-chain as suggested by the crystallographic studies (3–4). The allosteric effector, inositol hexaphosphate (IHP), binds to Hb and decreases its affinity for O2. However, the H-bond markers, the cross-peaks for α58His (1He2, 15Ne2) and β63His (1He2, 15Ne2), do not change significantly upon the addition of IHP at pH 8.0 and pH 7.0, although these peaks become weaker, suggesting that the effect of IHP might not be strong enough to break these H-bonds under those conditions.