title: 13C NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c.
abstract: The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.
authors.affiliation: Department of Chemistry, Oklahoma State University, Stillwater 74078, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Rodríguez-Marañón
authors.forename: F
authors.initials: F
authors.lastname: Qiu
authors.forename: R E
authors.initials: RE
authors.lastname: Stark
authors.forename: S P
authors.initials: SP
authors.lastname: White
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.forename: S I
authors.initials: SI
authors.lastname: Foundling
authors.forename: V
authors.initials: V
authors.lastname: Rodríguez
authors.forename: C L
authors.initials: CL
authors.lastname: Schilling
authors.forename: R A
authors.initials: RA
authors.lastname: Bunce
authors.forename: M
authors.initials: M
authors.lastname: Rivera
mesh_terms: D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes; D002417:Cattle; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D009994:Osmolar Concentration; D017433:Protein Structure, Secondary; D051381:Rats; D055672:Static Electricity
chemical_list: D002247:Carbon Isotopes; D003574:Cytochrome c Group; D006418:Heme; D015786:Cytochromes b5