A solution 1H 2D NMR investigation has been carried out on low-spin, human adult cyanomet hemoglobin (HbA) to elucidate molecular, electronic, and magnetic properties of the heme cavities in the hetero-tetrameric globin. It is shown that, in spite of its size, 65 kDa, and paramagnetism (S = 1/2), appropriately tailored 2D experiments to suppress rotating frame dipolar correlation allow detection of the scalar connectivities needed to identify heme and heme pocket residue spin topology, as well as the backbone 3 J(α-N) necessary to assign residues sequence-specifically. NOESY rise curves clearly differentiate between primary and secondary NOEs and afford the sensitivity for providing interproton distance estimates. The combined NMR strategies provide the complete assignment of the heme, a key portion of the F-helix, the F-G turn, and residues in contact with ligands. Unambiguous subunit differentiation for all signals was achievable independently, either sequence-specifically via the Alaα vs Cysβ at position F9 or by conserved heme C-helix contacts for Tyrα vs Pheβ at position C7. The dipolar shifts for the assigned heme pocket residues provide the orientation of the anisotropic paramagnetic susceptibility tensor in the molecular framework, showing that the major axis in each subunit is tilted from the heme normal by ∼11° in a direction consistent with a bound cyanide exhibiting a tilt from the heme normal similar to that as observed for CO in the HbACO crystal. Numerous identified residues have been implicated in the mechanism of cooperativity. Analysis of the dipolar contacts between the heme vinyl and/or propionate groups with the adjacent heme methyls and neighboring protein residues identifies different 6-propionate mobility in the two subunits and 4-vinyl orientations with out-of-plane orientations to opposite sides of the heme in the two subunits. It is concluded that homonuclear 2D NMR is capable of providing unique information on functionally relevant structural and dynamic properties of HbA in the cyanomet form in spite of its size and paramagnetism; in fact, the paramagnetism facilitates the structural studies by significantly improving spectral resolution for the heme cavity.
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