Interaction of ligands with cytochrome P-450. On the 442 nm spectral species generated during the oxidative metabolism of pyridine

Abstract

When added to aerobic rabbit liver microsomal fractions fortified with an NADPH-generating system, pyridine initially produces a type II difference spectrum such as is observed with other aromatic amines. There is a time-dependent conversion of this perturbation into a new spectral species characterized by an absorbance maximum at 442 nm and a minor peak at 389 nm. Experiments with inhibitors of the cytochrome P-450-dependent electron-transport chain suggest that these species originate from binding to the haemoprotein of metabolic intermediate(s) derived from the amine substrate. Analysis of the incubation mixtures by t.l.c., high-pressure liquid chromatography, u.v.- and mass-spectrometry reveals the presence of a single metabolite arising from cytochrome P-450-catalysed oxidation of the heteroaromatic tertiary amine, which was identified as pyridine N-oxide, obviously accounting for adduct formation. This view is supported by comparative studies on the spectral changes generated by exogenous amine oxide with NADPH-reduced cytochrome P-450. Moreover, dithiothreitol, a potent N-oxidase inhibitor, strongly suppresses development of the 442 nm and 389 nm complexes. The ability of forming low-spin adducts with ferrous cytochrome P-450 absorbing around 440 nm appears to be an inherent property of different types of N-oxides. Considering the dipole character of the N+-O- function, a co-ordinate iron-oxygen bond is proposed to be formed in these complexes.

Some aspects of the role of cytochrome P‐450 isozymes in the n‐oxidative transformation of secondary and tertiary amine compounds

“…The lack of correlation between the Hammett a ; values and the rates of N-oxide formation from a series of para-substituted N,N-di-methylanilines has been considered to hint at the intermediacy of Fe/heteroatom oxide complexes, the formation or decomposition of which may be rate limiting (59). This concept is compatible with spectral work demonstrating the production of metabolic-intermediate complexes inherently linked to the P-450-catalyzed N-oxygenation of N,N-dimethylaniline (35) or pyridine (37). In view of the relatively strong dipole character of the "-0function, a coordinate Fe-0 bond has been proposed to be formed in these Fe(III)/ N-oxide adducts, which are destroyed by the presence of sodium dithionite (35,37).…”

“…This concept is compatible with spectral work demonstrating the production of metabolic-intermediate complexes inherently linked to the P-450-catalyzed N-oxygenation of N,N-dimethylaniline (35) or pyridine (37). In view of the relatively strong dipole character of the "-0function, a coordinate Fe-0 bond has been proposed to be formed in these Fe(III)/ N-oxide adducts, which are destroyed by the presence of sodium dithionite (35,37).…”

N‐Oxidative Transformations ofCNGroups as Means of Toxification and Detoxification

“…In many instances, oximes 13,22,23 , nitrones 24,25,36 and N-oxides 9,18,29 isolated by the chromatographic procedures decribed above have been subjected to mass-spectral analysis to verify their chemical nature. The majority of oximes derived from phenylethylamines produced spectra with fairly abundant molecular ions.…”

“…In experiments with subcellular tissue fractions, microsomal preparations have been detected to be abundant in N-oxygenating activity giving rise to the formation of oximes 1,5,29,51 , nitrones 3,6,26,53 and heteroaromatic N-oxides 15,16,28,65 ; these reactions invariably required the presence of oxygen and NADPH as the electron donor. Using 18 O, Parli and coworkers were able to demonstrate that the oxygen atom inserted into 2,4,6-trimethylacetophenone imine to yield the corresponding oxime derived from molecular oxygen and not from water 1 .…”

“…Electron transfer from this species (b) gives a cation/Fe 3C DO pair that generates an Fe(III) Noxide adduct (c). Indeed, N-oxidative turnover of pyridine by hepatic microsomes from phenobarbital-pretreated rabbits is associated with the gradual formation of a 442-nmabsorbing metabolic-intermediate spectral complex having the oxygen atom of the polar N O function coordinated to the heme iron 29 . This adduct decays to release stable Noxide and ferric pigment (d).…”