A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Microsomal cytochrome b 5 (cytb 5 ) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb 5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb 5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb 5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb 5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb 5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb 5 . The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb 5 . We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb 5 -cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. Background: cytb 5 modulates catalysis performed by cytsP450, in vivo and in vitro . Results: The structure of full-length cytb 5 was solved by NMR, and the cytP450-binding site on cytb 5 was identified by mutagenesis and NMR. Conclusion: A model of the cytb 5 -cytP450 complex is presented. Addition of a substrate strengthens the cytb 5 -cytP450 interaction. Significance: The cytb 5 -cytP450 complex structure will help unravel the mechanism by which cytb 5 regulates catalysis by cytP450.