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

Human hemoglobin produced in the Escherichia coli coexpression system of He rnan et al. [(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous protein whose properties closely approximate thos e of normal hemoglobin A. Both of the alpha and beta chains of this hemoglo bin contain a valine-methionine substitution at position 1 in order to acco mmodate 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 alpha and beta globins and their reass embly in the presence of hemin. The kinetics of CO combination and the ther modynamics of O-2 binding and cooperativity of the reassembled alpha V1M-be ta V1M hemoglobin closely approximate those of HbA. The alpha globin obtain ed from the E. coli expressed hemoglobin was also combined with normal huma n beta chains and hemin to form the alpha V1M variant. The alpha+M variant of HbA, in which the normal N-terminal valine of the alpha chains is preced ed by a methionine residue, was prepared by the same procedure, The kinetic s of the reactions of CO with the alpha V1M and alpha+M variants are simila r to those for HbA. The equilibria of oxygen binding to alpha V1M and HbA a re similar whereas alpha+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of alpha V1M and alpha+M offer an explana tion for the latter affinity difference, Although the structures of alpha V 1M and HbA, which have been determined by X-ray crystallography, are virtua lly indistinguishable except at the N-terminal residues, that of alpha+M in dicates the displacement of a solvent molecule, possibly a chloride ion, fr om arginine 141 alpha. Such an alteration in an anion binding site could re sult in increased oxygen affinity.