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 cyto chrome b(5) and horse heart cytochrome c was investigated by C-13 NMR spectroscopy and X-ray crystallography. To achieve these aims, C-13-la beled heme OM cytochrome b(5) was expressed in Escherichia coli as pre viously described [Rivera M., Walker, F. A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propiona te carbons in ferricytochrome b(5) was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [C-13]heme-la beled OM cytochrome b(5) with horse heart cytochrome c were carried ou t in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b(5). The results from these titrati ons clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b(5) participates in the electrostatic stab ilization of the complex between OM cytochrome b(5) and horse heart cy tochrome c, Similar experiments carried out monitoring C-13 resonances arising from several other heme substituents demonstrated that the st oichiometry of the complex is 1:1. A conditional binding constant, K w hich equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained f or the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crys tal structure of rat liver OM cytochrome bs solved to 2.7 Angstrom res olution shows that the structures of bovine liver microsomal cytochrom e b(5) and rat liver OM cytochrome b(5) are almost identical when comp ared at medium resolution, The similarity between the two structures, combined with the findings that only the heme propionate located on th e exposed heme edge of OM cytochrome bs participates in the electrosta tic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytoch rome b(5) and cytochrome c, clearly indicates that the site of interac tion on OM cytochrome b(5) is almost identical to the one elucidated f or microsomal cytochrome b(5). It is therefore possible to conclude th at the large body of information gathered by many investigators for th e 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 inde ed biological as well as pedagogical validity.