Mechanism-based inactivation of cytochrome P450 2B1 by 8-methoxypsoralen and several other furanocoumarins

Biochemistry. 1998 Sep 22;37(38):13184-93. doi: 10.1021/bi981198r.


Of several furanocoumarins [5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 5-hydroxypsoralen (5-OH-P), 8-hydroxypsoralen (8-OH-P), 4',5'-dihydro-8-MOP (DH-8-MOP), and psoralen (P)] tested as mechanism-based inactivators (MBIs) of purified reconstituted cytochrome P450 (P450) 2B1, 8-MOP was found to be the most potent (KI, kinact, and partition ratio of 2.9 microM, 0.34 min-1, and 1.3, respectively). The inactivation was not prevented by reactive oxygen species scavengers or nucleophilic trapping agents and proceeded with a decrease in P450 spectral content. Liquid chromatography (LC) separation of the reconstituted enzyme mixture, followed by liquid scintillation counting, indicated that [14C]-8-MOP binding was specific to the apoprotein of P450 2B1 with a binding stoichiometry of 0.7:1. The major metabolites formed by P450 2B1 from the furanocoumarins that were MBIs were characterized by LC electrospray ionization tandem mass spectrometry (ESI-MS/MS) as dihydro diols. Results from H218O incorporation experiments supported initial oxidation of 8-MOP and P to an epoxide which can react with some nucleophilic active site residue and inactivate the enzyme or partition to a dihydro diol metabolite by hydrolytic ring opening. On the other hand, 5-MOP was converted to an epoxide or gamma-keto enal intermediate prior to inactivation or dihydro diol formation. Comparison of the ESI mass spectra of P450 2B1 and furanocoumarin exposed P450 2B1, indicated a mass difference consistent with the covalent addition of a furanoepoxide to P450 2B1.

Publication types

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

MeSH terms

  • 5-Methoxypsoralen
  • Animals
  • Binding Sites
  • Chromatography, Liquid
  • Coumarins / metabolism
  • Coumarins / pharmacology*
  • Cytochrome P-450 CYP2B1 / antagonists & inhibitors*
  • Cytochrome P-450 CYP2B1 / metabolism*
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Ficusin / metabolism
  • Male
  • Mass Spectrometry
  • Methoxsalen / analogs & derivatives
  • Methoxsalen / metabolism
  • Methoxsalen / pharmacology*
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • NADP / physiology
  • Rats
  • Rats, Sprague-Dawley


  • Coumarins
  • Enzyme Inhibitors
  • 5-Methoxypsoralen
  • NADP
  • Cytochrome P-450 CYP2B1
  • Ficusin
  • Methoxsalen