Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c

Katherine A Gentry,Lucy Waskell,Mukesh Mahajan,Carlo Barnaba,G M Anantharamaiah,Ayyalusamy Ramamoorthy,Sang-Choul Im,Elke Prade,Meng Zhang,Satoshi Nagao

doi:10.1038/s41598-017-08130-7

Katherine A Gentry, Lucy Waskell + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-08130-7

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Journal: Scientific Reports	Publication Date: Aug 10, 2017
Citations: 15	License type: open-access

Affiliation: University of Michigan–Ann Arbor

Abstract

Cytochrome b5 (cytb5) 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 cytb5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb5 also uses a distal region. Using restraints generated from both cytb5 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.

Highlights

It has puzzled investigators how cytb[5] is able to enhance, reduce, or exert no effect on cytochrome P450 (cytP450) metabolism depending on the substrate and the isoform of cytP450 involved[6,7,8,9,10]
As recent studies have identified, cyt c can undergo structural changes in the presence of cardiolipin[35, 36], membrane mimetics used in these experiments were cardiolipin-free, such that cyt c would not interact with the membrane environment
We investigated the interaction between full-length rabbit cytb[5] and full-length cyt c in different membrane mimetic environments, including lipid-free, isotropic bicelles, and lipid nanodiscs, utilizing solution NMR techniques

Summary

Introduction

It has puzzled investigators how cytb[5] is able to enhance, reduce, or exert no effect on cytP450 metabolism depending on the substrate and the isoform of cytP450 involved[6,7,8,9,10]. We investigated the interaction between full-length rabbit cytb[5] and full-length cyt c in different membrane mimetic environments, including lipid-free, isotropic bicelles, and lipid nanodiscs, utilizing solution NMR techniques. Three different cytb[5] samples were prepared in this study: cytb[5] in buffer (membrane-free cytb5); cytb[5] in isotropic bicelles; and cytb[5] in lipid nanodiscs.

Results

Conclusion