Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 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...

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Bibliographic Details
Published in:Scientific reports 2017-08, Vol.7 (1), p.7793-15, Article 7793
Main Authors: Gentry, Katherine A, Prade, Elke, Barnaba, Carlo, Zhang, Meng, Mahajan, Mukesh, Im, Sang-Choul, Anantharamaiah, G M, Nagao, Satoshi, Waskell, Lucy, Ramamoorthy, Ayyalusamy
Format: Article
Language:English
Subjects:
Animals
Cytochrome
Cytochrome b
Cytochrome c
Cytochrome P450
Cytochromes b5 - chemistry
Cytochromes c - chemistry
Electron transfer
Electrons
Epitopes
Lipid Bilayers - chemistry
Membrane proteins
Molecular Dynamics Simulation
NMR
Nuclear magnetic resonance
Peptides
Protein Binding
Proteins
Rabbits
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-08130-7