Cytochrome P-450 spin state and leakiness of the monooxygenase pathway

Abstract

1. The monooxygenase and oxidase activities of liver microsomes from phenobarbital (PB)-treated rabbits were investigated for their dependence on the high spin shift (Δα) of the ferric cytochrome P-450 induced by a series of benzphetamine analogues.

2. The spin shift activity of the substrate determines, via the first electron transfer kinetics, the steady-state level of the reaction intermediate oxycytochrome P-450. Correlation of the amount or oxycytochrome P-450 with Δα can be experimentally proved.

3. The spin-state-dependent formation of oxycytochrome P-450 regulates quantitatively the rates of NADPH oxidation and substrate N-demethylation. Both activities correlate with Δα. Oxycytochrome P-450 is substrate-stabilized towards decay with the formation of O₂⁻ which, upon dismutation, gives rise to H₂O₂.

4. The ratio of N-demethylase to NADPH oxidase activity (coupling ratio) also increases with the spin shift, Δα. Concomitantly, the proportion of NADPH accounted for by H₂O₂ and H₂O formation via two- and four-electron reduction of dioxygen decreases. This indicates that the substrate-induced structural changes in the enzyme active centre which give rise to spin transition may likewise modify the coupling properties.

5. Perfluorinated compounds, which fail to undergo monooxygenation, fall in line with the benzphetamine derivatives with respect to the dependence of NADPH oxidation rate and steady-state oxycytochrome P-450 level on Δα. The increased oxidase activity results mostly in H₂O formation.

6. The leakiness of the PB-induccd monooxygenase pathway in the biotransformation of oxygen in the presence of the benzphetamines and perfluorinated compounds does not result in marked increases in H₂O₂ formation. Therefore, the increase of NADPH oxidase activity by these substrates does not significantly enhance H₂O₂-mediated oxygen tissue toxicity.