NMR Studies of the Association of Cytochrome b 5 with Cytochrome c

• Published in: Biochemistry (Easton) 2000-11, Vol.39 (46), p.14025-14039
• Main Authors: Hom, Kellie, Ma, Qi-Feng, Wolfe, Gary, Zhang, Hong, Storch, Elizabeth M, Daggett, Valerie, Basus, Vladimir J, Waskell, Lucy
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001129o

In an effort to gain greater insight into the molecular mechanism of the electron-transfer reactions of cytochrome b 5, the bovine cytochrome b 5−horse cytochrome c complex has been investigated by high-resolution multidimensional NMR spectroscopy using 13C, 15N-labeled cytochrome b 5 expressed from a synthetic gene. Chemical shifts of the backbone 15N, 1H, and 13C resonances for 81 of the 82 residues of [U-90% 13C,U-90% 15N]-ferrous cytochrome b 5 in a 1:1 complex with ferrous cytochrome c were compared with those of ferrous cytochrome b 5 in the absence of cytochrome c. A total of 51% of these residues showed small, but significant, changes in chemical shifts (the largest shifts were 0.1 ppm for the amide 1H, 1.15 for 13Cα, 1.03 ppm for the amide 15N, and 0.15 ppm for the 1Hα resonances). Some of the residues exhibiting chemical shift changes are located in a region that has been implicated as the binding surface to cyt c [Salemme, F. R. (1976) J. Mol. Biol. 10, 563−568]. Surprisingly, many of the residues with changes are not located on this surface. Instead, they are located within and around a cleft observed to form in a molecular dynamics study of cytochrome b 5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682−9693]. The rim of this cleft can readily accommodate cytochrome c. Molecular dynamics simulations of the Salemme and cleft complexes were performed for 2 ns and both complexes were stable.