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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96 of the alpha chain, located in the alpha 1 beta 2 subunit interface, has been
replaced by a tryptophan residue using site-directed mutagenesis.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.
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