title: One- and two-dimensional nuclear Overhauser effect studies of the electronic/molecular structure of the heme cavity of ferricytochrome b5.
abstract: A nuclear Overhauser effect, NOE, study of solubilized native bovine ferricytochrome b5 has provided the complete assignment of the heme resonances as well as those of the majority of the amino acid side-chains making contact with the prosthetic group. The resonances which could not be identified are those from positions very close to the iron (less than 5 A) for which paramagnetic relaxation is sufficiently strong to significantly decrease the NOEs. The observed 1H-1H dipolar contacts generally confirm a solution structure unchanged from that described in single crystals, except for the detailed orientation of the heme side-chains. The 2-vinyl group is found in both the cis and trans in-plane orientation as opposed to exclusively cis in the crystal, and the 7-propionate group is rotated by 30 degrees in solution towards the 6-propionate group. Identification of resonances for the individual axial histidine residues indicates non-equivalent interaction with the heme iron, and the patterns of meso-H, pyrrole substituent and amino acid dipolar shifts allow the location of the principal magnetic axes in the protein coordinate system. This identifies His-39 as the dominant influence in determining the electronic ground state that orients the molecular orbital for facile electron transfer via the exposed heme edge. The complete two-dimensional NOESY map for ferricytochrome b5 is presented that yields all the cross peaks expected on the basis of the one-dimensional NOE studies, and indicates that such two-dimensional methods should have profitable extension to strongly hyperfine-shifted resonances in paramagnetic proteins.
authors.affiliation: Department of Chemistry, University of California, Davis 95616.
authors.forename: S J
authors.initials: SJ
authors.lastname: McLachlan
authors.forename: G N
authors.initials: GN
authors.lastname: La Mar
authors.forename: K B
authors.initials: KB
authors.lastname: Lee
mesh_terms: D000818:Animals; D002417:Cattle; D003573:Cytochrome b Group; D015786:Cytochromes b5; D006418:Heme; D008099:Liver / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011758:Pyrroles
chemical_list: D003573:Cytochrome b Group; D011758:Pyrroles; D006418:Heme; D015786:Cytochromes b5