Hemoglobin (Hb) is an essential component of the circulatory system of vertebrates. Its chief physiological function is to transport oxygen from the lungs to the tissues. For reviews of the structure-function relationship in hemoglobin, see Dickerson and Geiss, 1 Ho, 2 and Ho and Lukin. 3 Human normal adult hemoglobin (Hb A) is one of the most studied proteins and has served as a model or paradigm for multimeric allosteric proteins. Hemoglobin is a useful system for testing a basic premise of structural biology, which holds that the functional properties of a protein can be explained in terms of its structure and dynamic behavior on the atomic scale. Since the first crystal structures of Hb A were determined by Perutz and colleagues in the 1960s, extensive efforts have been devoted to elucidating the relationship of hemoglobin’s structure with its physiologically important properties, including the cooperative binding of oxygen, and the control of oxygen affinity by pH (the Bohr effect) and allosteric effectors such as 2, 3-bisphosphoglycerate (2, 3-BPG). Despite these efforts, the detailed structural basis of these properties is not fully understood, and some aspects remain controversial. Much of our current understanding of Hb A has been obtained with reference to X-ray crystal structures. However, since the protein performs its physiological functions in the solution state, it is useful to investigate its structure-function relationships in solution, using techniques including infrared, resonance Raman, and nuclear magnetic resonance (NMR) spectroscopies.

One-dimensional (1D) 1H NMR spectroscopy has been used extensively to study the behavior of Hb in solution by following the response of assigned 1H resonances to varied conditions, and to modifications of the protein. 2, 4, 5 This technique is limited, however, by the broadening of resonance lines observed for large proteins such as Hb, which results in a degenerate 1H NMR spectrum, where only a few resonances are well-resolved. The spectral resolution, and consequently the amount of structural and dynamic information obtainable, can be greatly enhanced by the application of multidimensional, multinuclear NMR to isotopically labeled protein samples. Labeling of Hb A with 2H, 15N, and 13C has become feasible through the development of a bacterial expression system for this protein. 6, 7 Expression of hemoglobin in Escherichia coli also makes it possible to produce any desired mutant recombinant hemoglobin (rHb), by site-directed mutagenesis. Much insight into the structure-function relationships of Hb has been gained by investigating the properties of rHbs where amino acid substitutions have been made at key residues. 8-14 This article will review recent progress in understanding the structure-function relationships of hemoglobin in solution, emphasizing results obtained by NMR spectroscopy.