C-13 NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b(5) heme propionates in the electrostatic binding to cytochrome c

The role played by the outer mitochondrial membrane (OM) cytochrome b(5) heme propionate groups in the electrostatic binding between OM cytochrome b(5) and horse heart cytochrome c was investigated by C-13 NMR spectroscopy and X-ray crystallography. To achieve these aims, C-13-labeled heme OM cytochrome b(5) 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 b(5) was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [C-13]heme-labeled OM cytochrome b(5) with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b(5). The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b(5) participates in the electrostatic stabilization of the complex between OM cytochrome b(5) and horse heart cytochrome c, Similar experiments carried out monitoring C-13 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 bs solved to 2.7 Angstrom resolution shows that the structures of bovine liver microsomal cytochrome b(5) and rat liver OM cytochrome b(5) 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 bs 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 b(5) and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b(5) is almost identical to the one elucidated for microsomal cytochrome b(5). It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b(5) 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.