Global analysis of protein-protein interactions reveals multiple CYP2E1-reductase complexes

Biochemistry. 2007 Sep 4;46(35):10192-201. doi: 10.1021/bi7003476. Epub 2007 Aug 9.

Abstract

Although a single binary functional complex between cytochrome P450 (P450 or CYP for a specific isoform) and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain the experimentally observed catalytic activity of recombinant CYP2E1 in dependence on the total concentration of the added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450.CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450.CPR, the quaternary complex (P450)2.(CPR)2, and the homodimer (P450)2. The second plausible model additionally involves a weakly bound ternary complex (P450)2.CPR. Importantly, only the binary complex P450.CPR seems catalytically active in either of the two most probable mechanisms.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Computer Simulation
  • Cytochrome P-450 CYP2B1
  • Cytochrome P-450 CYP2E1 / metabolism*
  • Glutamine / chemistry
  • Lysine / chemistry
  • Mathematical Computing
  • Microsomes, Liver / metabolism
  • Models, Biological*
  • Models, Chemical
  • Multienzyme Complexes / metabolism*
  • Mutation
  • NADPH-Ferrihemoprotein Reductase / metabolism*
  • Nitrophenols / metabolism
  • Oxidation-Reduction
  • Protein Binding
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism*
  • Thermodynamics*
  • Titrimetry*

Substances

  • Multienzyme Complexes
  • Nitrophenols
  • Recombinant Proteins
  • Glutamine
  • Cytochrome P-450 CYP2E1
  • Cytochrome P-450 CYP2B1
  • NADPH-Ferrihemoprotein Reductase
  • Lysine