NMR studies of the association of cytochrome b(5) with cytochrome c

In an effort to gain greater insight into the molecular mechanism of the el ectron-transfer reactions of cytochrome b(5), the bovine cytochrome b(5)-ho rse cytochrome c complex has been investigated by high-resolution multidime nsional NMR spectroscopy using C-13,N-15-labeled cytochrome b(5) expressed from a synthetic gene. Chemical shifts of the backbone N-15, H-1, and C-13 resonances for 81 of the 82 residues of [U-90% C-13,U-90% N-15]-ferrous cyt ochrome 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 s hifts (the largest shifts were 0.1 ppm for the amide H-1, 1.15 for C-13(alp ha), 1.03 pp, for the amide N-15, and 0.15 ppm for the H-1(alpha) resonance s). 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 wi thin and around a cleft observed to form in a molecular dynamics study of c ytochrome b(5) [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682 -9693]. The rim of this cleft can readily accommodate cytochrome c. Molecul ar dynamics simulations of the Salemme and cleft complexes were performed f or 2 ns and both complexes were stable.