A Novel Low Oxygen Affinity Recombinant Hemoglobin (α96Val→Trp): Switching Quaternary Structure Without Changing the Ligation State

Abstract

Using ourEscherichia coliexpression plasmid (pHE2) in which synthetic human α and β-globin genes are coexpressed with theE. colimethionine aminopeptidase gene under the control of separatetacpromoters, we have constructed a new artificial hemoglobin in which the valine residue at position 96 of the α chain, located in the α1β2subunit interface, has been replaced by a tryptophan residue using site-directed mutagenesis. We have determined the oxygen-binding properties of this recombinant hemoglobin, r Hb (α96Val→Trp), and have used proton nuclear magnetic resonance spectroscopy to investigate its tertiary structure around the heme group and the quaternary structure in the α1β2subunit interface. This artificial hemoglobin shows a low oxygen affinity, but high cooperativity in oxygen binding, and exhibits no unusual subunit dissociation when ligated. Molecular dynamics simulations suggest that the unique oxygen-binding property of r Hb (α96Val→Trp) may be due to an extra hydrogen bond between α96Trp and β99Asp in the α1β2subunit interface in the deoxy form. Despite the replacement of a small amino acid residue, valine, by a large tryptophan residue in the α1β2subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure in the α1β2subunit interface compared to those of human normal adult hemoglobin. Another unique feature of this artificial hemoglobin is that the ligated form, e.g. carbonmonoxy form, of this hemoglobin in the oxy-quaternary structure can be converted to the deoxy-like quaternary structure by the addition of an allosteric effector, inositol hexaphosphate, as well as by lowering the temperature in the absence of inositol hexaphosphate, without changing its ligation state. Thus, this recombinant hemoglobin can be used to gain new insights regarding the nature of subunit interactions in the α1β2interface and the molecular basis for the allosteric mechanism of hemoglobin.