Substitution of the heme binding module in hemoglobin alpha- and beta-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 alpha-subunit converted the heme proximal structure of myoglobin into the alpha-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 betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)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 alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-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.
Inaba
K
K
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
Ishimori
K
K
Imai
K
K
Morishima
I
I
D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013059:Spectrum Analysis, Raman
D006454:Hemoglobins; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D010100:Oxygen