Insights into the Solution Structure of Human Deoxyhemoglobin in the Absence and Presence of an Allosteric Effector

Abstract

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15 N-1 H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). In order to have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1 H-15 N Jcouplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed and these changes are largely within the α 1 β 1 (or α 2 β 2 ) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.Human normal adult hemoglobin (Hb A), a heterotetrameric protein that transports oxygen from the lungs to tissues, has served as an excellent model for investigating the structurefunction relationship in multimeric, allosteric proteins. Hb A consists of four subunits, i.e., two identical α-chains of 141 amino acid residues each and two identical β-chains of 146 amino acid residues each; each subunit contains a heme group leading to a complex with a molecular weight of ~ 64.5 kDa. The heme iron atom undergoes a spin-state change in going from a highspin paramagnetic Fe +2 (S=2) in the deoxy or unligated form to a diamagnetic Fe +2 (S=0) in the ligated form (with O 2 or CO). The oxygenation of Hb A is regulated by interactions between the O 2 -binding sites (homotropic interactions) and interactions between individual amino acid residues in the protein molecule and various solutes (heterotropic interactions). Heterotropic effectors include hydrogen ions, chloride ions, carbon dioxide, inorganic phosphate ions, and organic phosphate ions [e.g., 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP)] and are known to modulate the oxygen affinity of hemoglobin. For reviews on the NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript structure-function relationship on hemoglobin, see (1-3). Allosteric effectors, 2,3-BPG and IHP, exert a significant effect on the oxygen-binding pr...

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

“…The experimentally determined RDC values can be well reproduced by calculations based on either of the X-ray structures for isolated α- or β-chain or that of an isolated αβ dimer. However, a larger χ 2 value is obtained when based on the whole tetramer (39, 57). For HbCO A, a better fitting is obtained by a rotation of one αβ dimer with respect to the other (57).…”

Effector-Induced Structural Fluctuation Regulates the Ligand Affinity of an Allosteric Protein: Binding of Inositol Hexaphosphate Has Distinct Dynamic Consequences for the T and R States of Hemoglobin

“…Gray and Gibson (24) observed a kinetic difference between the R-and ·-chains of Hb induced by IHP binding. Recently, a study from our laboratory based on RDC measurements has shown that IHP causes changes mainly in the tertiary structure of deoxy-Hb and mostly in the ·-subunit (11). These observations show that in general, IHP can affect the two types of subunits of Hb A differently.…”

New Look at Hemoglobin Allostery

“…71-72 The calculated RDCs 72 were obtained from eight crystal structures of deoxy-Hb A, 4HHB, 1HGA, 1KD2, 1RQ3, 1XXT, 1BZ0, 2DN2, and 1YHR. The scatter in the eight correlation plots between the observed and calculated RDCs is larger than the experimental precision.…”

“…(iii) Allosteric effectors, e.g., IHP, alter both the tertiary and quaternary structure of deoxy- and CO-Hb as well as the dynamics of the Hb molecule, thus affecting its ligand affinity. 70-72,74 (iv) RDC measurements of Hb A in different aqueous liquid crystalline media and at several magnetic field strengths have shown that in solution, the quaternary conformations for both the CO- and deoxy-forms of Hb A are different from their crystal structures and both exist as dynamic ensembles of various structures. 69-72 It is tempting to speculate that the various T- and R-type structures reported by crystallographers (Table 1) may be the so-called “pre-existing protein conformations” and represent some of the conformational ensembles detected by our NMR measurements.…”