title: Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance.
abstract: The interaction between bovine cytochrome b(5) (cyt b(5)) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b(5) backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b(5) with cyt c is in fast exchange. An equilibrium association constant of (6 +/- 3) x 10(4) M(-1) was obtained with a lower limit of 180 s(-1) for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, (15)N relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b(5)-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b(5) surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. These data support the models proposed by Salemme and Mauk [(1976) J. Mol. Biol. 102, 563-568; (1993) Biochemistry 32, 6613-6623].
authors.affiliation: Department of Anesthesiology, University of Michigan, VA Medical Center, 2215 Fuller Road, Ann Arbor, Michigan 48105, USA.
authors.forename: Weiping
authors.initials: W
authors.lastname: Shao
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.forename: Erik R P
authors.initials: ER
authors.lastname: Zuiderweg
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
mesh_terms: D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes / Q000737:chemistry; D002417:Cattle; D017124:Conserved Sequence; D015786:Cytochromes b5 / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D006736:Horses; D008956:Models, Chemical; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D045304:Cytochromes c; D015786:Cytochromes b5