A further investigation of the cytochrome b5–cytochrome c complex

The interaction of reduced rabbit cytochrome b5 with reduced yeast iso-1 cytochrome c has been studied through the analysis of 1H–15N HSQC spectra, of 15N longitudinal (R1) and transverse (R2) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b5 has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b5.