Studies on the Mechanism of Physiological Function of Hemoglobin by Using Artificial Mutants

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Artificial hemoglobin mutants which contained single amino acid substitutions at particular sites were synthesized by site-directed mutagenesis using recombinant DNA and their oxygen binding function, light absorption spectra, proton nuclear magnetic resonance (NMR) spectra and resonance Raman scattering were measured to explore the roles of amino acid residues which are considered to be a key residue for the allosteric properties of hemoglobin.Four mutants were synthesized : Hb Y145betaF in which Phe substitutes for Tyr-145beta which is considered to induce a tertiary structure change in the beta subunit ; Hb W37betaF in which Phe substitutes for Trp-37beta which forms a hydrogen bond with Asp-94alpha at the alpha1-beta2 interface ; Hb H92betaV and Hb H92betaD in which the proximal His at 92beta is replaced by Val or Asp. The M13 Phase-E. coli system was used to introduce the mutations into human globin gene and to express the globin gene.The changes in oxygen binding functions (oxygen affinity, the Bohr effect, co-operativity, effect of inositol hexaphosphate) of Hb Y145betaF were moderate while those of Hb W37betaF were drastic. The tertiary and quaternary structure data acquired from UV light absorption, NMR, and resonance Raman spectra were nearly consistent with the functional data. It was noted that the important role of Tyr-145beta is that it has a side chain whose size is appropriate to fit to the tyrosine pocket rather than that it forms a hydrogen bond with Asp-94beta. The drastic functional changes in Hb W37betaF may partly be attributed to partial dissociation into alphabeta dimers.Hb H92betaV and Hb H92betaD showed drastic functional changes. The replacement of the proximal His by neutral or negatively charged residue caused disapearance of allosteric effects whereas the heme iron of the mutant chains were maintained in a ferrous state, different from the M-type hemoglobins.