The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation.
Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives. Little enzyme destruction is observed when the 4-alkyl group is of low radical stability (methyl, phenyl) and good destruction, but no isolable heme adducts when the 4-substituent is of very high radical stability (isopropyl, benzyl). Spin-trapping studies have established that the 4-ethyl group in DDEP is lost as a radical as a result of oxidation by cytochrome P-450. Of three commonly used spin traps, only alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone was found suitable for such studies. The other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and alpha-phenyl N-tert-butylnitrone, were found to be ineffective, the latter because it strongly inhibits cytochrome P-450. Hydrogen peroxide formed in situ can support a part of the cytochrome P-450-catalyzed ethyl radical formation and DDEP-dependent self-inactivation. The results provide persuasive evidence that oxidation of the nitrogen in DDEP by cytochrome P-450 proceeds in one-electron steps. Cytochrome P-450 may thus function, at least with certain substrates, as a one-electron oxidant.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003997:Dicarbethoxydihydrocollidine / Q000031:analogs & derivatives; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013113:Spin Labels; D013379:Substrate Specificity
D013113:Spin Labels; D003997:Dicarbethoxydihydrocollidine; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital