Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b and Cytochrome c.

Cytochrome b (cytb ) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb -cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb in the presence and absence of membrane. Notably, in the presence of membrane, cytb only engaged cyt c at its lower and upper clefts while the membrane-free cytb also uses a distal region. Using restraints generated from both cytb and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.