Evidence from infrared and 13C NMR spectra for discrete rapidly interconverting conformers at the carbon monoxide binding sites of hemoglobins A and Zurich.

Fully liganded hemoglobin carbonyls in solution exhibit infrared and 13C NMR spectra that indicate the normal presence of discrete, rapidly interconverting conformers at the carbon monoxide binding sites. The CO ligands bound to Hb A give infrared bands with vCO = 1968 and 1951 cm-1. With Hb Zurich [beta 63 His leads to Arg], vCO values of 1968, 1958, and 1951 cm-1 are observed. Changing either the temperature (3-32 degrees C) or the pH (4 to 11.9) produces reversible redistributions of individual infrared band intensities without a significant change in the total integrated intensity for all bands; only small shifts in frequency occur. In 13C NMR spectra for 13C16O ligands only one resonance is observed for each type of subunit: alpha A and alpha Zh at approximately 206.4 ppm, beta A at approximately 206.0 ppm, beta Zh at approximately 205.5 ppm, each from tetramethylsilane. Since the multiple conformers observed in infrared spectra are not evident in the 13C NMR spectra, the interconversions among the conformers are considered to be too rapid for the individual conformers to be detected by NMR spectroscopy. Estimated differences in enthalpy and in entropy between the separate conformers range from 1.5 to 5.2 kcal/mol and 6.6 to 11.5 entropy units, respectively. The structural differences between carbonyl conformers are sufficiently great that iron (II)--CO bonding and the reactivity of the individual conformers are expected to vary significantly. These findings demonstrate a dynamic aspect of structure due to the flexibility of the protein at the ligand binding site that is important in any consideration of structure-function or structure-property relationships.