Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric β-chain from human adult hemoglobin

Abstract

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated β-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D 1H NMR in order to assess the extent of assignments allowed by 1H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the β-chain to the crystallographically characterized complexes that contains the β-chain. 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 104 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. These comprehensive 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 β-chain in terms of the crystal coordinates of the β-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated β-chain relative to that in the intact R-state 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 to the Fe-CO tilt in the β-subunit of HbCO. It is concluded that a set of “preliminary” magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet β-chain and that this approach should be similarly effective in HbA.