Abstract
somal cytochrome P-450 (P-450~~) with aromatic hydroperoxides and peracids two transient spectral intermediates are produced reversibly: Complex C, which is directly involved in the hydroxylation pathway, and Complex D, which is formed in a nonproductive side reaction. In the present study the broad substrate specificity of P-450~~ was exploited to examine the effect of structural changes on the reaction Toluene + cumene hydroperoxide -j benzyl alcohol + cumyl alcohol Determination of Complexes C and D by stopped flow spectrophotometry and measurement of the initial rate of benzyl alcohol formation in the presence of 3-acetylpyridine adenine dinucleotide and alcohol dehydrogenase indicated that a number of m- and p-substituted toluenes and cumene hydroperoxides could be used in place of the parent compounds. The rate-limiting decay of Complex C was found to be dependent on the structure of both the peroxy compound and the hydroxylatable substrate. These results are compatible with a homolytic mechanism of oxygen-oxygen bond cleavage but not with the heterolytic formation of a common iron-oxo intermediate from the various peroxides.
Highlights
The cytochrome exhibits a very broad specificity withrespect to the peroxy compound in Reaction 2; alkyl where RH represents a variety of hydroxylatable sub- hydroperoxides [2,3,4], peroxy acids (21, sodium periodate [5], strates and XOOH a variety of peroxy compounds sodium chlorite [2,5],iodosobenzene [6,7], anidodosobenzene which serve as oxygen donors, and neither molecular oxygen nor an external electron donor is required
We have previously shown that in the reaction of the phenobarbital-inducible isozyme of rabbitliver microsomal cytochrome P-450 ( P - 4 5 0 ~w~ i)th aromatic hydroperoxides and peracids two transient spectrailntermediates are produced reversibly: Complex C, which is directly involved in the hydroxylation pathway, and Complex D, which is formed in a nonproductive side diacetate [7] have all been shown to participate in this reaction
The occurrenceof P-450-catalyzed substrate hydroxylation reactions involving either molecular oxygen or peroxy compounds as oxygen donors raises the possibility that the two pathways may have one or more intermediates in common
Summary
Vol 256, No., Issue of December 10, pp. 12127-18133, 1981 Printed in U.S.A. EVALUATION OF HOMOLYTIC AND HETEROLYTIC MECHANISMS OF OXYGEN-OXYGEN BOND CLEAVAGE DURING SUBSTRATE HYDROXYLATION BY PEROXIDES*. Materials-Cumene hydroperoxide was purchased from K and K Laboratories and purified by published procedures [19],andp-methyland p-fluoro-CHP were synthesized as described previously [8].Substituted toluenes were obtained from Aldrich; dilauroylglyceryl-3phosphorylcholine was from Serdary Research Laboratories, and horse liver alcohol dehydrogenase and 3-AcPyAD (grade 1) were from Sigma. The concentration of Complex C was calculated for each experimental condition by use of the solutions to the set of differential equations describing the mechanism, and the initial rate of benzyl alcohol formation was measured spectrophotometrically by an assay in which the production of benzyl alcohol was coupled via alcohol dehydrogenase tothe reduction of 3-AcPyAD. The value of n, which is a measure of the relative effect of the substituent upon the lipophilicity of the compound, is defined as log PX- log PH,
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