X-ray Analysis of Fine Structure of Heme in Normal and Thalassemic HbE/F Hemoglobin

Thalassemic hemoglobin (Hb) differs from normal Hb in its structure and oxygen-binding capacity. To find out structural differences in their heme-iron sites, X-ray absorption fine structure (XAFS) was applied. Resulting data in the range of and obtained from frozen aqueous solutions (10 K) of normal and thalassemic HbE/F deoxy forms were refined using multiple-scattering (MS) analyses. To test the robustness of the refinements R starting models, constraints, restraints, and k ranges were varied; for normal relaxed R- and tense T-deoxy-Hb final XAFS R values were 13% and 17% and for thalassemic HbE/F relaxed R-deoxy-Hb R value was 19-21%. With normal R-deoxy-Hb, we obtained Fe-Np (pyrrole) and Fe-NÎ (imidazole) distances of 2.05 and 2.14 , whereas in thalassemic HbE/F R-deoxy-Hb, these values were 2.11 and 1.9 , respectively. In the T-state of normal deoxy-Hb, Fe-Np distance (2.08 was longer and Fe-NÎ (1.96 shorter than in the R-state; from thalassemic HbE/F T-deoxy-Hb we could not obtain sufficiently stable probes for XAFS measurement. In normal and HbE/F R-deoxy-Hb the Fe-His imidazole bond deviated from the z-axis by ~100, whereas it was on the z-axis in the T-state of normal Hb. Heme doming increased from normal R-deoxy via normal T-deoxy to thalassemic R-deoxy forms with simultaneously increasing dislocation of the central iron from the plane of the porphyrin. Oxygenation brings the iron back into the plane of the porphyrin ring.