Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide

Abstract

Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-cis(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene with consumption of O2 and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O2 utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O2 was detected as hydrogen peroxide. The rate of benzene-dependent O2 consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O2 consumption by NDO. Detailed experiments showed that the total amount of O2 consumed and the rate of benzene-induced O2 consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISP(NAP)) of NDO. Kinetic studies showed that ISP(NAP) was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISP(NAP) gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISP(NAP) containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene- dependent O2 consumption and as an inactivator of ISP(NAP). It is proposed that the irreversible inactivation of ISP(NAP) occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (·OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [14C]benzene was used as the substrate, cis- benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O2 consumption and NADH oxidation.