Substitution of the heme binding module in hemoglobin alpha- and beta-subunits - Implication for different regulation mechanisms of the heme proximalstructure between hemoglobin and myoglobin

In our previous work, we demonstrated that the replacement of the "heme bin ding module," a segment from F1 to G5 site, in myoglobin with that of hemog lobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J . Mel. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the beta alpha(HBM)- subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin a-subunit. Based on the gel chromatograp hy, the beta alpha(HBM)-subunit was preferentially associated with the alph a-subunit to form a heterotetramer, alpha(2)[beta alpha(HBM)(2)], just as i s native beta-subunit. Deoxy-alpha(2)[beta alpha(HBM)(2)] tetramer exhibite d the hyperfine-shifted NMR resonance from the proximal histidyl NdeltaH pr oton and the resonance Raman band from the Fe-His vibrational mode at the s ame positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and c yanomet alpha(2)[beta alpha(HBM)(2)] tetramer were quite similar to those o f native hemoglobin. Consequently, the heme environmental structure of the beta alpha(HBM)-subunit in tetrameric alpha(2)[beta alpha(HBM)(2)] was simi lar to that of the beta-subunit in native tetrameric Hb A, and the structur al conversion by the module substitution was not clear in the hemoglobin su bunits, The contrastive structural effects of the module substitution on my oglobin and hemoglobin subunits strongly suggest different regulation mecha nisms 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 hemoglo bin appears to be closely coupled to the subunit interactions.