Using our Escherichia coli expression plasmid (pHE2) in which synthetic human alpha and beta-globin genes are coexpressed with the E. coli methionine aminopeptidase gene under the control of separate tac promoters, we have constructed a new artificial hemoglobin in which the valine residue at position 96 of the alpha chain, located in the alpha 1 beta 2 subunit interface, has been replaced by a tryptophan residue using site-directed mutagenesis.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 (alpha 96Val-->Trp), and have used proton nuclear magnetic resonance spectroscopy to investigate its tertiary structure around the heme group and the quaternary structure in the alpha 1 beta 2 subunit 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 (alpha 96Val-->Trp) may be due to an extra hydrogen bond between alpha 96Trp and beta 99Asp in the alpha 1 beta 2 subunit interface in the deoxy form. Despite the replacement of a small amino acid residue, valine, by a large tryptophan residue in the alpha 1 beta 2 subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 subunit 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 alpha 1 beta 2 interface and the molecular basis for the allosteric mechanism of hemoglobin.