Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: the effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

Biochim Biophys Acta. 2010 Mar;1797(3):378-90. doi: 10.1016/j.bbabio.2009.12.008. Epub 2009 Dec 21.


We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55+/-6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism*
  • Boron Compounds
  • Cell Membrane / metabolism*
  • Cytochrome P-450 CYP3A / chemistry*
  • Cytochrome P-450 CYP3A / genetics
  • Cytochrome P-450 CYP3A / metabolism*
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Electron Transport
  • Flavins / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Humans
  • Kinetics
  • Mutation / genetics
  • NADP / metabolism
  • NADPH-Ferrihemoprotein Reductase / chemistry*
  • NADPH-Ferrihemoprotein Reductase / metabolism*
  • Nanotechnology
  • Oxidation-Reduction
  • Proteolipids / metabolism
  • Substrate Specificity


  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Bacterial Proteins
  • Boron Compounds
  • Flavins
  • Fluorescent Dyes
  • Proteolipids
  • proteoliposomes
  • NADP
  • Cytochrome P-450 Enzyme System
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • NADPH-Ferrihemoprotein Reductase
  • flavocytochrome P450 BM3 monoxygenases