We are carrying out extended X-ray absorption fine structure (EXAFS) studies of mixed metal hybrid hemoglobins at the National Synchrotron Light Source. Mixed metal hybrid hemoglobins are hemoglobin tetramers in which one or more of the four heme irons has been replaced by another metal and serve as mimics of intermediates of oxygen binding (see Section II). The proposed studies aim at obtaining a complete documentation of the metal-ligand bond lengths of each of the four metal centers in every intermediate of oxygen binding. Concurrent with the proposed EXAFS investigations, electron-spin echo envelope modulation (ESEEM) (an advanced EPR technique) and ultraviolet resonance Raman (UV RR) studies are being carried out at the Albert Einstein College of Medicine. The ESEEM studies examine the electronic structure of a metal-dioxygen bond and complement the bond length measurements achieved with EXAFS spectroscopy. The UV RR studies examine the tryptophan residues at the subunit interfaces of the mixed metal hybrid hemoglobins as a means of identifying the quaternary state of the tetramers. The EXAFS studies thus represent a crucial component of a detailed investigation Hof how the quaternary state of the hemoglobin tetramer affects the Hstructure of each of the four metals and consequently, its function, Hi.e., cooperative oxygen binding. Advancement in understanding the Hstructural-function relationship of human hemoglobin is essential for Hthe design of blood substitutes, and may also provide a model for the Hstructural basis of allostery in biological macromolecules in general.
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