| Abstract
| - Model-free-based NMR dynamics studies have been undertaken for polypeptide backboneamide 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 relaxationrate (R1), transverse relaxation rate (R2), and heteronuclear nuclear Overhauser effect (NOE)] measuredat two temperatures (29 and 34 °C) and two magnetic field strengths (11.7 and 14.1 T). In both deoxyand carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N−H bonds of mostamino acid residues are rigid on the fast time scale (nanosecond to picosecond), except for the loopregions and certain helix−helix connections. Although rigid in deoxy-Hb A, β146His has been found tobe free from restriction of its backbone motions in the CO form, presumably due to the rupture of itshydrogen bond/salt bridge network. We now have direct dynamics evidence for this structural transitionof Hb in solution. While remarkably flexible in the deoxy state, α31Arg and β123Thr, neighbors in theintradimer (α1β1) interface, exhibit stiffening upon CO binding. These findings imply a role for α31Argand β123Thr in the intradimer communication but contradict the results from X-ray crystallography. Wehave also found that there is considerable flexibility in the intradimer (α1β1) interface (i.e., B, G, and Hhelices 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 intradimerinterfaces, such as β109Val, appear to be involved in possible conformational exchange processes. Thedynamic picture derived from the present study provides new insights into the traditional description ofthe stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographicresults.
|
