A NOVEL LOW-OXYGEN AFFINITY RECOMBINANT HEMOGLOBIN (ALPHA-96VAL -] TRP) - SWITCHING QUATERNARY STRUCTURE WITHOUT CHANGING THE LIGATION STATE

Using our Escherichia coli expression plasmid (pHE2) in which syntheti c human alpha and beta-globin genes are coexpressed with the E. coli m ethionine aminopeptidase gene under the control of separate tac promot ers, we have constructed a new artificial hemoglobin in which the vali ne residue at position 96 of the alpha chain, located in the alpha(1) beta(2) subunit interface, has been replaced by a tryptophan residue u sing site-directed mutagenesis. We have determined the oxygen-binding properties of this recombinant hemoglobin, r Hb (alpha 96Val-->Trp), a nd have used proton nuclear magnetic resonance spectroscopy to investi gate its tertiary structure around the heme group and the quaternary s tructure in the alpha(1) beta(2) subunit interface. This artificial he moglobin shows a low oxygen affinity, but high cooperativity in oxygen binding, and exhibits no unusual subunit dissociation when ligated. M olecular dynamics simulations suggest that the unique oxygen-binding p roperty of r Hb (alpha 96Val-->Trp) may be due to an extra hydrogen bo nd 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) be ta(2) subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure i n the alpha(1) beta(2) subunit interface compared to those of human no rmal adult hemoglobin. Another unique feature of this artificial hemog lobin is that the ligated form, e.g. carbonmonoxy form, of this hemogl obin in the oxy-quaternary structure can be converted to the deoxy-lik e quaternary structure by the addition of an allosteric effector, inos itol hexaphosphate, as well as by lowering the temperature in the abse nce of inositol hexaphosphate, without changing its ligation state. Th us, this recombinant hemoglobin can be used to gain new insights regar ding the nature of subunit interactions in the alpha(1) beta(2) interf ace and the molecular basis for the allosteric mechanism of hemoglobin .