Solution ~1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated α-chain from human hemoglobin A

摘要

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated α-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional ~1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the α-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (～70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the α-chain in terms of the crystal coordinates of the α-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated α-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted ～10°from the heme normal so that the Fe-CN unit is tilted toward the β-meso-H in a fashion remarkably similar tot he Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet α-chain and that this approach should be similarly effective in HbA.