Study of the regulation mechanism of ligand affinity of hemoglobin by protein engineering

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The oxygen affinity of hemoglobin is regulated in a range of 25,000-folds depending on the living environment of animal species. To know by what mechanism the regulation is realized from amino acid sequence differences in protein moiety, we synthesized eight artificial mutants of human hemoglobin with particular amino acids replaced by others by means of protein engineering based on site-directed mutagenesis and elucidated their characteristics such as oxygen binding properties, giving the following results.1. Proximal His mutants : Six mutants, in which the heme iron-bound invariable His-87alpha residue is replaced by either Leu, Val, Ile, Ala or Phe, or His-92beta is replaced by Tyr, showed such oxygen equilibrium properties as 36-fold variation of oxygen affinity, various decreases in the effect of IHP(inositol hexaphosphate), the Bohr effect and cooperativity, and increase in autooxidation of heme iron.2. Thr-38alpha mutants : Two mutants in which Thr-38alpha, participating in hydrogen bond formation between the alpha1-beta2 subunits, is replaced by Ser or Val were synthesized. The former showed almost normal oxygen binding properties whereas the latter showed somewhat altered properties (2.8-fold increase in oxygen affinity). The analysis by means of electronic, vibration and proton NMR Spectroscopy indicated that their high order structure is normal.3. The present experimental results indicate that the residues at the proximal side extensively participate in oxygen affinity regualtion whereas Thr-38alpha does not so much. They also indicate that a 36-fold regulation of oxygen affinity can be realized by the proximal residue. However, examination with other investigators' data shows that the affinity reguation by replacements of only the residues surrounding the heme group is far from the 25,000-fold variation, suggesting that some other mechanism must be invoked.