Functional role of the distal valine (E11) residue of α subunits in human haemoglobin

Abstract

We have expressed human α-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor X a. The apo globin has been refolded and reconstituted with haem and native β-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the α subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al.. Here, we present the oxygen equilibrium curves of three E11α mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of α-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of β subunits. The crystal structure of the valine E11 α → isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of α-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.