Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b(5) and Cytochrome c

Cytochrome b (5) (cytb (5)) 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 (5) and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb (5) - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb (5)-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb (5) in the presence and absence of membrane. Notably, in the presence of membrane, cytb (5) only engaged cyt c at its lower and upper clefts while the membrane-free cytb (5) also uses a distal region. Using restraints generated from both cytb (5) 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.