Ultraviolet resonance Raman spectroscopy of hemoglobin with 200 and 212 nm excitation: H-bonds of tyrosines and prolines

Resonance Raman spectra are reported for hemoglobin (Hb) with deep UV excitation at 212 and 200 nm. At 200 nm, strongly enhanced amide bands appear, but the spectra are dominated by tyrosine residues, and substantial enhancement is seen for the Y7a′ mode, a sensitive monitor of H-bond status. T–R difference spectra between deoxyHb and HbCO reveal a positive Y7a′ band at 1249 cm-1 and a negative band at 1263 cm-1. This result implies that acceptance by one or more Tyr residues, rather than donation by, an H-bond in the T state, and supports previous evidence that the T state H-bond partner of Tyr α42, the carboxylate side chain of Asp β99, is protonated in the T state. At 212 nm, the X–Pro amide II bands are clearly observed, and the position of the T–R difference band, 1464 cm-1, implies that one or more X–Pro carbonyls forms a stronger H-bond in the T state. This residue may be Pro α37, whose carbonyl interacts with a water molecule that in turn interacts with His β146 in the T state. The X–Pro difference signal may be an indicator of the His β146 protonation status, and therefore of an important contributor to the Bohr effect.