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 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.

1H NMR Investigation of the Distal Hydrogen Bonding Network and Ligand Tilt in the Cyanomet Complex of Oxygen-avidAscaris suum Hemoglobin

“…The temperature dependence of the heme methyl shifts reveals that the 1-CH 3 , 5-CH 3 exhibit positive slopes that are steeper than Curie (T Ϫ1 ) behavior, whereas the 3-CH 3 , 5-CH 3 exhibit slopes that are negative or exhibits antiCurie behavior. This effect is expected on the basis of thermal population of the excited orbital state, where the lone spin on the iron becomes delocalized into pyrroles B and D (60,(67)(68)(69). Lastly, the magnetic axes reported above allow the determination of ␦ dip for the axial His, which, in turn, provides ␦ con for each of the positions, as shown in Table I.…”

Solution 1H NMR of the Active Site of Substrate-bound, Cyanide-inhibited Human Heme Oxygenase

“…It is reasonable to expect that using the ␦ dip values as constraints, a more quantitative description of the active site is attainable once NOESY rise curves have allowed quantitative estimates of internuclear distances. The inefficiency in detecting TOCSY peaks is surprising, because the helical segments of both isoelectronic but bigger 44-kDa HRP-CN (44) and 65-kDa metHbCN (45) have yielded the necessary fingerprint cross-peaks. The difficulty with hHOhemin-CN is likely due to line broadening from the dynamic averaging of more than one unique structure in solution (see "Dynamic Properties of the Pocket").…”

Heme Ligation and Conformational Plasticity in the Isolatedc Domain of Cytochrome cd 1 Nitrite Reductase

“…In DEFT-NOESY and DEFT-TOCSY experiments (27,28), the first 90°pulse in the ordinary NOESY and TOCSY pulse sequences was replaced with the modified DEFT sequence (90°--180°--90°) (29) H NMR data were processed using the XWINNMR package. One-dimensional and twodimensional 1 H NMR data sets were apodized using an exponential decay in the direct dimension and using a 30 -40°shifted sine-bellsquared window function in the indirect dimension.…”