Nitroreductases (NTRs) catalyze the reduction of a wide range of nitro-compounds and quinones using NAD(P)H. Although the physiological functions of these enzymes remain obscure, a tentative function of resistance to reactive oxygen species (ROS) via the detoxification of menadione has been proposed. This suggestion is based primarily on the transcriptional or translational induction of an NTR response to menadione rather than on convincing experimental evidence. We investigated the performance of a fungal NTR from Aspergillus nidulans (AnNTR) exposed to menadione to address the question of whether NTR is really an ROS defense enzyme. We confirmed that AnNTR was transcriptionally induced by external menadione. We observed that menadione treatment generated cytotoxic levels of O₂^•-, which requires well-known antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin to protect A. nidulans against menadione-derived ROS stress. However, AnNTR was counterproductive for ROS defense, since knocking out AnNTR decreased the intracellular O₂^•- levels, resulting in fungal viability higher than that of the wild type. This observation implies that AnNTR may accelerate the generation of O₂^•- from menadione. Our in vitro experiments indicated that AnNTR uses NADPH to reduce menadione in a single-electron reaction, and the subsequent semiquinone-quinone redox cycling resulted in O₂^•- generation. We demonstrated that A. nidulans nitroreductase should be an ROS generator, but not an ROS scavenger, in the presence of menadione. Our results clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous. IMPORTANCE Menadione is commonly used as an O₂^•- generator in studies of oxidative stress responses. However, the precise mechanism through which menadione mediates cellular O₂^•- generation, as well as the way in which cells respond, remains unclear. Elucidating these events will have important implications for the use of menadione in biological and medical studies. Our results show that the production of Aspergillus nidulans nitroreductase (AnNTR) was induced by menadione. However, the accumulated AnNTR did not protect cells but instead increased the cytotoxic effect of menadione through a single-electron reduction reaction. Our finding that nitroreductase is involved in the menadione-mediated O₂^•- generation pathway has clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous.

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