Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al. [(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the α and β chains of this hemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functional homogeneity of the E. coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component α and β globins and their reassembly in the presence of hemin. The kinetics of CO combination and the thermodynamics of O₂ binding and cooperativity of the reassembled αV1M-βV1M hemoglobin closely approximate those of HbA. The α globin obtained from the E. coli expressed hemoglobin was also combined with normal human β chains and hemin to form the αV1M variant. The α+M variant of HbA, in which the normal N- terminal valine of the α chains is preceded by a methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with the αV1M and α+M variants are similar to those for HbA. The equilibria of oxygen binding to αV1M and HbA are similar whereas α+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of αV1M and α+M offer an explanation for the latter affinity difference. Although the structures of αV1M and HbA, which have been determined by X- ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of α+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141α. Such an alteration in an anion binding site could result in increased oxygen affinity.