FT-IR DIFFERENCE SPECTROSCOPY OF HEMOGLOBINS-A AND KEMPSEY - EVIDENCETHAT A KEY QUATERNARY INTERACTION INDUCES PROTONATION OF ASP-BETA-99

Fourier transform infrared difference spectra are reported for the CO adduct of human hemoglobin versus deoxyHb, in H2O and D2O. In addition to the well-known CO stretching and S-H(D) stretching bands, the diff erence spectra reveal numerous bands in the 1200-1700 cm(-1) region, a number of which are assigned. Several amide modes are identified via their frequencies and D2O sensitivities. Bands arising from histidine protonation have also been found via comparison of the difference spec tra at different pH(D) values, with the aid of aqueous histidine spect ra. Of particular interest is the observation of a negative band at 16 97 cm(-1), which is assigned to the C=O stretch of carboxylic acid. Th is carboxylic acid is tentatively identified as the side chain of Asp beta 99, because it is missing in the difference spectrum of Hb Kempse y, a mutant in which Asp beta 99 is replaced by Asn. Asp beta 99 forms a critical contact with Tyr alpha 42 across the alpha(1) beta(2) inte rface in deoxyHb, which is broken upon ligation. Protonation of Asp be ta 99 in deoxyHb is consistent with UV resonance Raman evidence that T yr alpha 42 is the acceptor rather than the donor of the quaternary H- bond.