Fourier Transform Infrared Evidence against Asp β99 Protonation in Hemoglobin: Nature of the Tyr α42−Asp β99 Quaternary H-Bond

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Abstract
The Tyr α42−Asp β99 intersubunit H-bond stabilizes the T quaternary structure in hemoglobin (Hb) tetramers. We had proposed that Tyr α42 acts as an acceptor in this H-bond, because the tyrosine Y8a/8b and Y7a‘ UVRR (ultraviolet resonance Raman) bands shift in directions opposite to those expected if tyrosine is an H-bond donor. If Asp β99 is the H-bond donor, then it must be protonated in the T state, and would be a previously unrecognized contributor to the Bohr effect. This implication was strengthened by the discovery that an R-minus-T difference FTIR (Fourier transform infrared) band at 1693 cm-1, which might be a signal from protonated carboxylate, is missing in Hb Kempsey, a mutant in which Asp β99 is replaced by Asn. However, we now find that this FTIR signal is insensitive to 13C-labeling of the aspartate residues in Hb, and cannot arise from protonated Asp β99. There are no other difference signals in the 1700 cm-1 region at a sensitivity of one COOH group. We conclude that Asp β99 is not protonated, and that the anomalous UVRR shifts must arise from compensating polarization of the Tyr α42 OH. Candidates for this compensation are the H-bond donated by the Asp β94 backbone NH, and the nearby positive charge of Arg β40.