A Comparative NMR Study of the Polypeptide Backbone Dynamics of Hemoglobin in the Deoxy and Carbonmonoxy Forms

摘要

Model-free based NMR dynamics studies have been undertaken for polypeptide backbone amide N-H bond vectors for both the deoxy- and carbonmonoxy forms of chain-specific, isotopically (¹⁵N and ²H) labeled tetrameric hemoglobin (Hb), using ¹⁵N-relaxation parameters [longitudinal relaxation rate (R1), transverse relaxation rate (R2), and heteronuclear nuclear Overhauser effect (NOE)] measured at two temperatures (29 and 34 °C) and two magnetic field strengths (11.7 and 14.1 Tesla). In both deoxy- and carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N-H bonds of most amino acid residues are rigid on the fast time scale (ns-ps), except for the loop regions and certain helix-helix connections. Although rigid in deoxy-Hb A, β146His has been found to be free from restriction of its backbone motions in the CO form, presumably due to the rupture of its hydrogen-bond/salt-bridge network. We now have direct dynamics evidence for this structural transition of Hb in solution. While remarkably flexible in the deoxy state, α31Arg and β123Thr, neighbors in the intra-dimer (α1β1) interface, exhibit stiffening upon CO binding. These findings imply a role for α31Arg and β123Thr in the intra-dimer communication, but contradict the results from X-ray crystallography. We have also found that there is considerable flexibility in the intra-dimer (α1β1) interface (i.e., B, G, and H helices, and the GH corner), and possible involvement of several amino acid residues (e.g., α31Arg, β3Leu, β41Phe, β123Thr, and β146His) in the allosteric pathway. Several amino acid residues at the intra-dimer interfaces, such as β109Val, appear to be involved in possible conformational exchange processes. The dynamic picture derived from the present study provides new insights into the traditional description of the stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographic results.