Structure-function relations in hemoglobin as determined by x-ray absorption spectroscopy.

Abstract

Conclusions concerning the structure around the Fe atom in oxy- and carbonmonoxyhemoglobin were obtained by fluorescent X-ray absorption studies. The bis-imidazole heme complex was used as a model system of known structure. The ligated forms of Hb, and cytochrome c at high pH, gave spectra which were very similar to the bis-imidazole complex, where the average Fe-N bond distance is 1.98 .ANG.. By comparison it was possible to determine that the average Fe-N bond distances were 1.99 .ANG. in oxyhemoglobin, 1.98 .ANG. in carbonmonoxyhemoglobin, and 1.98 .ANG. in cytochrome c at pH > 10.5, with an experimental accuracy of .+-. 0.01 .ANG.. An experimental comparison between oxy- and deoxyhemoglobin A showed much larger spectral changes than among the ligated forms. A comparison was made between the low O2 affinity form of deoxy HbA and the high affinity form of deoxy Hb Kempsey (.alpha.2.beta.299 Asp .fwdarw. Asn). All the spectral features coincided, allowing the conclusion that the average Fe-ligand bond differences must be .ltoreq. 0.02 .ANG.. Since the strain energy is proportional to the square of this displacement, the strain energy at the Fe is .ltoreq. 4 .times. 10-3 eV. This is negligible compared to the difference of binding energy of the high and low affinity forms, which is 0.15 eV, showing that the energies responsible for the increase of O2 affinity are not localized at the heme.