Characterization and Calculation of a Cytochrome <i>c</i>−Cytochrome <i>b</i>₅ Complex Using NMR Data^†

Abstract

To identify the binding site for bovine cytochrome b5 (cyt b5) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with 2H- and 15N-enriched cyt c and unlabeled cyt b5. In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b5 concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A Ka of (4 ± 3) × 105 M-1 was obtained with a lower limit of 855 s-1 for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b5 heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c−cyt b5 complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein−protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces