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 (delta alpha) 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 delta alpha 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 delta alpha. 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, delta alpha. 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 delta alpha. The increased oxidase activity results mostly in H2O formation. 6. The leakiness of the PB-induced 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.

CYP2E1 and oxidative liver injury by alcohol

“…ROS have been implicated in many of the major diseases that plague mankind, including the toxicity of O 2 itself; hyperbaric O 2 ; ischemiareperfusion injury; cardiovascular diseases; atherosclerosis; carcinogenesis; diabetes; neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease; toxicity of heavy metals, e.g., iron; asbestos injury; radiation injury; vitamin deficiency; drug (e.g., redox cycling agents) toxicity; aging; inflammation; smoking toxicity; emphysema; and toxicity of acute and chronic ethanol treatment (11)(12)(13)(14)(15). ROS can be produced from many systems in cells including the mitochondrial respiratory chain (16); the cytochrome P450s (10,17); oxidative enzymes such as xanthine oxidase, aldehyde oxidase, cyclooxygenase, monoamine oxidase, the NADPH oxidase complex (18,19); autooxidation of heme proteins such as ferrohemoglobin or myoglobin or biochemicals such as catecholamines, quinones or tetrahydrobiopterins. In addition to these cellular sources of ROS, environmental sources of ROS include radiation, UV light, smoke and certain drugs which can redox cycle.…”

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…In general, cooperativity in substrate binding is revealed by a sigmoidal curve of the substrate-induced displacement of the spin equilibrium of the heme iron, which is known to be an important determinant of the catalytic efficiency and coupling of cytochromes P450 [50][51][52]. Understanding the structural relationship between the binding of each substrate and/or effector molecule and the spin equilibrium of the heme iron is essential in unveiling the mechanism of P450 cooperativity.…”

Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4: The Stoichiometry of the Enzyme−Substrate Complexes Probed by FRET and Job's Titration

“…Therefore, many recent efforts in the studies of P450 cooperativity are increasingly focused on the substrate binding stage of the catalytic cycle (16,17,(19)(20)(21)(22). In the case of Type-I substrates these interactions are known to modulate the spin equilibrium of the enzyme, which is thought to be an important determinant of its catalytic efficiency and coupling (23)(24)(25)(26)(27)(28). Homotropic cooperativity in substrate binding is revealed in sigmoidal dependencies of the substrate-induced spin transitions on the substrate concentration.…”