Structure of the Fe-heme in the homodimeric hemoglobin from Scapharca inaequivalvis and in the T721 mutant: an X-ray absorption spectroscopic study at low temperature

The Fe site structure in the recombinant wildtype and T72I mutant of the co operative homodimeric hemoglobin (HbI) of the mollusc Scapharca ca inaequiv alvis has been investigated by measuring the Fe K-edge Xray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy deriva tives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is q uite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in na tive HbI-CO it seems intermediate between that of HbA-CO and sperm whale Mb CO. The XANES spectra of oxy and deoxy derivatives are similar to the homol ogous spectra of human HbA, except for T72I HbI, for which the absorption e dge is blue-shifted (about +1 eV) towards the spectrum of the oxy form. XAN ES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) a nd mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Feheme structures of the three photoproducts are similar; howeve r, while in the case of HbA* and HbI* the data indicate incomplete structur al relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxatio n in T72I HbI* is almost completely towards the proposed "high affinity" Fe -heme structure of T72I HbI. This evidence suggests that minor tertiary res traints affect the Fe-heme dynamics of T72I HbI, corresponding to a reducti on of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.