Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A.
The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-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 alpha-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 (approximately 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 alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-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 approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the 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 alpha-chain and that this approach should be similarly effective in HbA.
Tran
Anh-Tuyet T
AT
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
Kolczak
Urszula
U
La Mar
Gerd N
GN
D001665:Binding Sites; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006441:Hemoglobin A / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy
D006418:Heme; D006441:Hemoglobin A