Substitution of the heme binding module in hemoglobin α- and β- subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin

In our previous work, we demonstrated that the replacement of the 'heme binding module,' a segment from F1 to G5 site, in myoglobin with that of hemoglobin α-subunit converted the heme proximal structure of myoglobin into the α-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the βα(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin β-subunit was replaced by that of hemoglobin α-subunit. Based on the gel chromatography, the βα(HBM)- subunit was preferentially associated with the α-subunit to form a heterotetramer, α₂[βα(HBM)₂], just as is native β-subunit. Deoxy- α₂[βα(HBM)₂] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(δ)H proton and the resonance Raman band from the Fe- His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet α₂[βα(HBM)₂] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the βα(HBM)-subunit in tetrameric α₂[βα(HBM)₂] was similar to that of the β-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.