1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl α-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole π bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

Assignment of heme resonances and determination of the electronic structures of high- and low-spin nitrophorin 2 by 1H and 13C NMR spectroscopy: an explanation of the order of heme methyl resonances in high-spin ferriheme proteins

The (1)H NMR resonances of the heme substituents of the low-spin Fe(III) form of nitrophorin 2, as its complexes with N-methylimidazole (NP2-NMeIm) and imidazole (NP2-ImH), have been assigned by a combination of (1)H homonuclear two-dimensional NMR techniques and (1)H-(13)C HMQC. Complete assignment of the proton and partial assignment of the (13)C resonances of the heme of these complexes has been achieved. Due to favorable rates of ligand exchange, it was also possible to assign part of the (1)H resonances of the high-spin heme via saturation transfer between high- and low-spin protein forms in a partially liganded NP2-NMeIm sample; additional resonances (vinyl and propionate) were assigned by NOESY techniques. The order of heme methyl resonances in the high-spin form of the protein over the temperature range of 10-37 degrees C is 8 = 5 > 1 > 3; the NMeIm complex has 5 > 1 > 3 > 8 as the order of heme methyl resonances at <30 degrees C, while above that temperature, the order is 5 > 3 > 1 > 8, due to crossover of the closely spaced 3- and 1-methyl resonances of the low-spin complex at higher temperatures. This crossover defines the nodal plane of the heme orbital used for spin delocalization as being oriented 162 +/- 2 degrees clockwise from the heme N(II)-Fe-N(IV) axis for the heme in the B orientation. For the NP2-ImH complex, the order of heme methyl resonances is 3 > 5 > 1 > 8, which defines the orientation of the nodal plane of the heme orbital used for spin delocalization as being oriented approximately 150-155 degrees clockwise from the heme N(II)-Fe-N(IV) axis. In both low-spin complexes, the results are most consistent with the exogenous planar ligand controlling the orientation of the nodal plane of the heme orbital. In the high-spin form of NP2, the proximal histidine plane is shown to be oriented 135 degrees clockwise from the heme N(II)-Fe-N(IV) axis, again for the B heme orientation. A correlation between the order of heme methyl resonances in the high-spin form of NP2 and several other ferriheme proteins and an apparent 90 degrees shift in the nodal plane of the orbital involved in spin delocalization from that expected on the basis of the orientation of the axial histidine imidazole nodal plane have been explained in terms of bonding interactions between Fe(III), the axial histidine imidazole nitrogen, and the porphyrin pi orbitals of the high-spin protein.     

Assignment of heme and distal amino acid resonances in the 1H-NMR spectra of the carbon monoxide and oxygen complexes of sperm whale myoglobin

Assignments of resonances of the heme and distal amino acid protons in spectra of the CO and O2 complexes of sperm whale myoglobin are reported. These resonances provide information on the conformation of the heme pocket. For oxymyoglobin, the assignments of the heme meso protons disagree with those proposed previously on the basis of partial deuteration experiments. Rapid ring flips about the C beta-C gamma bond are detected for Phe-CD1. Recent claims for two conformational substates of valine-E11 in carbonmonoxymyoglobin (Bradbury, J.H. and Carver, J.A. (1984) Biochemistry 23, 4905-4913) are shown to be in error. The pK of His-97 (FG3) in carbonmonoxymyoglobin has been determined (pK = 5.9). This residue appears to influence many spectroscopic properties of myoglobin. The distal His-E7 in carbonmonoxymyoglobin has pK less than 5.0. Differences in the heme pocket conformation in the CO complexes of myoglobin and leghemoglobin are discussed. These differences may be influential in O2 and CO association reactions.