Abstract
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.
Highlights
The structural disorder in the ␣(HBM)-subunit was reduced by the association with the ␣-subunit, and its heme environmental structure was almost identical to that of the -subunit bound to the ␣-subunit
In the results we could not confirm that the heme binding module corresponds to a structural unit regulating the heme proximal structure and heme electric state in globin proteins
In tetrameric Hb A, the subunit interactions appear to play a role in the regulation of the heme environmental structure
Summary
Construction of Expression Vector—To construct the gene of the ␣(HBM)-subunit, KpnI (GGTACC) and SacI (GAGCTC) sites were introduced at the boundaries of the heme binding module in the ␣- and -subunits, both, by polymerase chain reaction (Fig. 1), accompanied with the mutation Arg(G6) to Glu. The small KpnI-SacI fragment of the ␣-subunit amplified by polymerase chain reaction was ligated with the digested T7 expression vector encoding the -subunit. We confirmed the correct expression of the desired subunits by fast atom bombardment mass spectroscopy (data not shown) [22], and no additional mutations were detected. Tetramer-dimer dissociation constants of the samples were determined by the concentration dependence of the elution volume in the column over the range from 0.5 to 800. The dependence of the centroid elution volume Ve versus protein concentration (CT), Equation 1, allows us to determine the dimer-tetramer equilibrium constants for the samples,. The volume of the NMR sample was 600 l, and the protein concentration was approximately 1 mM on the heme basis. Sample volume was 6 ml, and concentration was 100 M on the heme basis. The hemoglobin reductase system [29] was added to the sample before each measurement to reduce oxidized subunits. The oxygenation data were acquired by use of a microcomputer (model PC-98XA, Nippon Electric Co., Tokyo), which was interfaced to the oxygenation apparatus [32]
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