Generation of OH radical during enzymatic reduction of 9,10-anthraquinone-2-sulphonate. Can semiquinone decompose hydrogen peroxide?

Spin-trapping experiments have shown that 9,10-anthraquinone-2-sulphonate (AQS) can be reduced enzymatically with NADPH-cytochrome P-450 reductase (FP) producing OH radicals. It has been revealed for the first time that the radical anion of AQS cannot decompose H2O2 as indicated by flash-photolysis data. From the experimental results obtained it follows that an enzyme system containing NADPH, AQS and FP produces both accumulation of H2O2 from molecular oxygen and reduction of Fe3+ to Fe2+ which decomposes H2O2 catalytically via a Fenton reaction.

[Substrate reduction and oxygen activation during microsomal metabolism of quinones]. / Vosstanovlenie substrata i aktivatsiia kisloroda pri mikrosomal'nom metabolizme khinonov.

2-Dimethylamino-3-chloro-1,4-naphthaquinone (DCNQ) was used to study oxygen and substrate activation in microsomal system. DCNQ was shown to be bound to microsomal cytochrome P-450 as a type I substrate; its N-demethylation was catalyzed by cytochrome P-450. Cytochrome P-450 and NADPH-cytochrome P-450 reductase are capable of DCNQ reduction to semi- and hydroquinones. The OH-radical formed in the presence of DCNQ, NADPH and reductase was detected, using a spin trap (5,5-dimethylpyrroline-N-oxide). The OH-radical formation was shown to be stimulated by the Fe-EDTA complex. Using the OH-radical scavengers (mannitol, N-butanol, alpha-naphthol) and the catalase inhibitor sodium azide, it was shown that the OH-radical participates in microsomal oxidation of DCNQ and aminopyrine. It was assumed that in the course of microsomal oxidation the reduced DCNQ is responsible for: i) stimulation of molecular oxygen reduction to H2O2; ii) reduction of Fe ions (Fe3+----Fe2+) which cause the decomposition of H2O2 in the Fenton reaction resulting in the formation of a strong oxidizing agent--a hydroxyl radical.

Microsomal oxidation of 2-dimethylamino-3-chloro-1,4-naphthoquinone. The possibility of substrate activation by cytochrome P-450.

2-Dimethylamino-3-chloro-1,4-naphthoquinone (DCNQ) is bound to microsomal cytochrome P-450 as a type I substrate (lambda max = 391 nm, lambda min = 420 nm). The Ks is 40.5 microM. In a rat-liver microsomal system, the N-demethylation of DCNQ produces formaldehyde (rate 225 pmol/min per mg of protein). Induction by phenobarbital increases the rate of formation, while addition of metyrapone and SKF-525A into the system decreases the rate by 52% and 35%, respectively. The microsomal N-demethylation of DCNQ is not inhibited by CO. Under full anaerobiosis, the microsomal oxidation of DCNQ again gives formaldehyde (rate 416 pmol/min per mg of protein). The anaerobic oxidation of DCNQ is inhibited by metyrapone and SKF-525A. The microsomal, chemical and electrochemical reduction of DCNQ to the corresponding semiquinones and hydroquinones have been studied. Non-enzymic DCNQ reduction is insufficient for the formation of formaldehyde. Under anaerobic conditions the microsomal DCNQ oxidation is assumed to occur via the intramolecular oxazole bond which is then hydrolysed, yielding formaldehyde. This may be a new example of substrate activation by cytochrome P-450.