FOURIER-TRANSFORM INFRARED EVIDENCE AGAINST ASP BETA-99 PROTONATION IN HEMOGLOBIN - NATURE OF THE TYR ALPHA-42 ASP BETA-99 QUATERNARY H-BOND

The Tyr alpha 42-Asp beta 99 intersubunit H-bond stabilizes the T quat ernary structure in hemoglobin (Hb) tetramers. We had proposed that Ty r alpha 42 acts as an acceptor in this H-bond, because the tyrosine Y8 a/8b and Y7a' UVRR (ultraviolet resonance Raman) bands shift in direct ions opposite to those expected if tyrosine is an H-bond donor. If Asp beta 99 is the H-bond donor, then it must be protonated in the T stat e, and would be a previously unrecognized contributor to the Bohr effe ct. This implication was strengthened by the discovery that an R-minus -T difference FTIR (Fourier transform infrared) band at 1693 cm(-1) wh ich might be a signal from protonated carboxylate, is missing in Hb Ke mpsey, a mutant in which Asp beta 99 is replaced by Asn. However, we n ow find that this FTIR signal is insensitive to C-13-labeling of the a spartate residues in Hb, and cannot arise from protonated Asp beta 99. There are no other difference signals in the 1700 cm(-1) region at a sensitivity of one COOH group. We conclude that Asp beta 99 is not pro tonated, and that the anomalous UVRR shifts must arise from compensati ng polarization of the Tyr alpha 42 OH. Candidates for this compensati on are the H-bond donated by the Asp beta 94 backbone NH, and the near by positive charge of Arg beta 40.