APPROACHES TO THE SOLUTION NMR CHARACTERIZATION OF ACTIVE-SITES FOR 65 KDA TETRAMERIC HEMOGLOBINS IN THE PARAMAGNETIC CYANOMET STATE

A solution H-1 2D NMR investigation has been carried out on low-spin, human adult cyanomet hemoglobin (HbA) to elucidate molecular, electron ic, and magnetic properties of the heme cavities in the hetero-tetrame ric globin. It is shown that, in spite of its size, 65 kDa, and parama gnetism (S = 1/2), appropriately tailored 2D experiments to suppress r otating frame dipolar correlation allow detection of the scalar connec tivities needed to identify heme and heme pocket residue spin topology , as well as the backbone (3)J(alpha-N) necessary to assign residues s equence-specifically. NOESY rise curves clearly differentiate between primary and secondary NOEs and afford the sensitivity for providing in terproton 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 diffe rentiation for all signals was achievable independently, either sequen ce-specifically via the Ala(alpha) vs Cys(beta) at position F9 or by c onserved heme C-helix contacts for Tyr(alpha) vs Phe(beta) 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 subuni t is tilted from the heme normal by similar to 11 degrees in a directi on consistent with a bound cyanide exhibiting a tilt from the heme nor mal similar to that as observed for CO in the HbACO crystal. Numerous identified residues have been implicated in the mechanism of cooperati vity. Analysis of the dipolar contacts between the heme vinyl and/or p ropionate groups with the adjacent heme methyls and neighboring protei n residues identifies different 6-propionate mobility in the two subun its and 4-vinyl orientations with out-of-plane orientations to opposit e sides of the heme in the two subunits. It is concluded that homonucl ear 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 fa cilitates the structural studies by significantly improving spectral r esolution for the heme cavity.