Modulating Distal Cavities in the α and β Subunits of Human HbA Reveals the Primary Ligand Migration Pathway

Abstract

The free volume in the active site of human HbA plays a crucial role in governing the bimolecular rates of O2, CO, and NO binding, the fraction of geminate ligand recombination, and the rate of NO dioxygenation by the oxygenated complex. We have decreased the size of the distal pocket by mutating Leu(B10), Val(E11) and Leu(G8) to Phe and Trp and of other more internal cavities by filling them with Xe at high gas pressures. Increasing the size of the B10 side chain reduces bimolecular rates of ligand binding nearly 5,000-fold and inhibits CO geminate recombination due to both reduction of the capture volume in the distal pocket and direct steric hindrance of Fe-ligand bond formation. Phe and Trp(E11) mutations also cause a decrease in distal pocket volume but, at the same time, increase access to the Fe atom due to the loss of the γ2 CH3 group of the native Val(E11) side chain. The net result of these E11 substitutions is a dramatic increase in geminate recombination because dissociated CO is sequestered close to the Fe atom and can rapidly rebind without steric resistance. However, the bimolecular rate constants for ligand binding to the Phe and Trp(E11) mutants are decreased 5–30-fold, due to a smaller capture volume. Geminate and bimolecular kinetic parameters for Phe and Trp(G8) mutants are similar to those for the native HbA subunits because the aromatic rings at this position cause little change in distal pocket volume and because ligands do not move past this position into the globin interior of wild-type HbA subunits. The latter conclusion is verified by the observation that Xe binding to the α and β Hb subunits has little effect on either geminate or bimolecular ligand rebinding. All of these experimental results argue strongly against alternative ligand migration pathways that involve movements through the protein interior in HbA. Instead, ligands appear to enter through the His(E7) gate and are captured directly in the distal cavity.

How Do Heme-Protein Sensors Exclude Oxygen? Lessons Learned from Cytochrome c′,Nostoc puntiformeHeme Nitric Oxide/Oxygen-Binding Domain, and Soluble Guanylyl Cyclase

“…This effect is best seen for the Val68 to Ile mutation in Mb, where the Cd-methyl group lies directly over the iron atom and decreases the affinity of Mb for all three gases by *5-fold. Similar nonselective effects are seen in Mb and Hb when Leu at the B10 helical position is increased in size to Trp and causes a *25-fold decrease in all three ligand affinities (12,86,93).…”

Development of Recombinant Hemoglobin-Based Oxygen Carriers

“…3B) (15,35,38,101), which cause the incoming ligand to reflect back out of the pocket before the His(E7) gate closes. Insertion of large aromatic side chains at the B10 position are much more effective at reducing k¢ NOD in a-subunits than mutations at the E11 and G8 positions, whereas the opposite is true in bsubunits (13,38). The largest reductions in the rate of NO scavenging in b-subunits have been achieved with Trp replacements at the E11 position (13,38,99).…”

“…The distal histidine (E7) acts like the thumb of a baseball glove, rotating outward to allow ligand entry, which is then captured in the internal pocket surrounded by Leu(B10), Phe(CD1), Val(E11), and Leu (G8) (Fig. 3B) (13)(14)(15)131). Both NOD and bimolecular ligand binding can be inhibited by filling the back of the pocket with large Phe or Trp replacements at these positions ( Fig.…”

Free hemoglobin increases von Willebrand factor–mediated platelet adhesion in vitro: implications for circulatory devices

“…13,16 Human collagen type III was purchased from Advanced BioMatrix, human fibrinogen from Calbiochem, and extracellular matrix (ECM) from Sigma-Aldrich. Anti-GPIba antibody 6D1 was a gift from Dr Barry Coller (The Rockefeller University, New York, NY).…”