Mapping the Binding Interface of the Cytochrome b[sub5]-Cytochrome c complex by Nuclear Magnetic Resonance.

The interaction between bovine cytochrome b5 (cyt b5) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b5 backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b5 with cyt c is in fast exchange. An equilibrium association constant of (6 ±3) × 10[SUP4] M[SUP-1] was obtained with lowera limit of 180 s[SUP-] for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, [SUP15]SN relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b5-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b5 surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. [ABSTRACT FROM AUTHOR]