Approaches to the Solution NMR Characterization of Active Sites for 65 kDa Tetrameric Hemoglobins in the Paramagnetic Cyanomet State

Abstract

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 3J(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 positio...