A model of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data

J Biol Chem. 2013 Jul 26;288(30):22080-95. doi: 10.1074/jbc.M112.448225. Epub 2013 May 24.


Microsomal cytochrome b5 (cytb5) 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 cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 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 cytb5 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 cytb5 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 cytb5. 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 cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.

Keywords: Cytochrome P450; Enzyme Catalysis; Membrane Proteins; NMR; Structural Biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arginine / chemistry
  • Arginine / genetics
  • Arginine / metabolism
  • Binding Sites / genetics
  • Biocatalysis
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Cytochromes b5 / chemistry*
  • Cytochromes b5 / genetics
  • Cytochromes b5 / metabolism
  • Electron Transport / genetics
  • Heme / analogs & derivatives
  • Heme / chemistry
  • Heme / metabolism
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular*
  • Molecular Sequence Data
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Mutagenesis, Site-Directed
  • Mutation
  • Oxidation-Reduction
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Rabbits
  • Sequence Homology, Amino Acid
  • Substrate Specificity


  • Multiprotein Complexes
  • heme propionate-7
  • Heme
  • Cytochromes b5
  • Cytochrome P-450 Enzyme System
  • Arginine

Associated data

  • PDB/2M33