Effects of ionic strength on the functional interactions between CYP2B4 and CYP1A2

Biochemistry. 2005 Feb 22;44(7):2632-41. doi: 10.1021/bi0477900.


The presence of one P450 can influence the catalytic characteristics of a second enzyme through the formation of heteromeric P450 complexes. Such a complex has been reported for mixed reconstituted systems containing NADPH-cytochrome P450 reductase, CYP2B4, and CYP1A2, where a dramatic inhibition of 7-pentoxyresorufin-O-dealkylation (PROD) was observed when compared to simple reconstituted systems containing reductase and a single P450 enzyme. The goal of the present study was to characterize this interaction by examining the potential of the CYP1A2-CYP2B4 complex to be formed by charge-pair interactions. With ionic interactions being sensitive to the surrounding ionic environment, monooxygenase activities were measured in both simple systems and mixed reconstituted systems as a function of ionic strength. PROD was found to be decreased at high ionic strength in both simple and mixed reconstituted systems, due to disruption of reductase-P450 complexes. Additionally, the inhibition of PROD in mixed reconstituted systems was relieved at high ionic strength, consistent with disruption of the CYP2B4-CYP1A2 complex. When ionic strength was measured as a function of CYP1A2 concentration, a shift to the right in the inflection point of the biphasic curve occurred at high ionic strength, consistent with a loss in CYP1A2 affinity for CYP2B4. When this analysis was applied to the same systems using a different substrate, 7-EFC, evidence for a high-affinity complex was not observed, demonstrating that the characteristics of the CYP1A2-CYP2B4 complex are influenced by the substrates present. These results support the role for a substrate specific electrostatic interaction between these P450 enzymes.

Publication types

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

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors
  • Aryl Hydrocarbon Hydroxylases / chemistry*
  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Coumarins / metabolism
  • Cytochrome P-450 CYP1A2 / chemistry*
  • Cytochrome P-450 CYP1A2 / metabolism*
  • Cytochrome P-450 CYP1A2 Inhibitors
  • Cytochrome P450 Family 2
  • Dealkylation
  • Enzyme Activation
  • Kinetics
  • Magnesium / chemistry
  • Models, Chemical
  • Models, Molecular
  • Multienzyme Complexes / antagonists & inhibitors
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / metabolism
  • NADPH-Ferrihemoprotein Reductase / antagonists & inhibitors
  • NADPH-Ferrihemoprotein Reductase / metabolism
  • Osmolar Concentration
  • Oxazines / metabolism
  • Rabbits
  • Static Electricity
  • Substrate Specificity


  • Coumarins
  • Cytochrome P-450 CYP1A2 Inhibitors
  • Multienzyme Complexes
  • Oxazines
  • 7-ethoxy-4-trifluoromethylcoumarin
  • pentoxyresorufin
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP1A2
  • Cytochrome P450 Family 2
  • cytochrome P-450 CYP2B4 (rabbit)
  • NADPH-Ferrihemoprotein Reductase
  • Magnesium