Juglone, a plant-derived 1,4-naphthoquinone, binds to hydroxylamine oxidoreductase and inhibits the electron transfer to cytochrome c 554

Abstract

ABSTRACT Nitrification is an essential biological process that converts ammonia into nitrate via nitrite. In the initial phase of nitrification, ammonia-oxidizing bacteria convert ammonia to hydroxylamine and subsequently to nitrite. The electrons generated in this process provide energy for cellular growth. In this study, we observed the inhibitory effects of plant-derived 1,4-naphthoquinones, including juglone, plumbagin, and 1,4-naphthoquinone, on nitrification in Nitrosomonas europaea , a typical nitrifying bacterium. These compounds disrupted the transfer of electrons produced during hydroxylamine oxidation to cytochrome c 554 , a physiological electron acceptor. In vitro electron transfer experiments confirmed the function of these three 1,4-naphthoquinones as electron acceptors in hydroxylamine oxidation. The results indicate that these compounds interfere with electron transfer to cytochrome c 554 during the ammonia oxidation process leading to nitrification inhibition. Remarkably, lawsone, an isomer of juglone, exhibited no effect on the activity of hydroxylamine oxidoreductase, implying a correlation between the redox potential of each 1,4-naphthoquinone and their ability to hinder canonical electron transfer. Furthermore, X-ray crystallographic analysis revealed that juglone resides in close proximity to the catalytic heme P460 in hydroxylamine oxidoreductase. The hydrophobic environment surrounding the binding cavity appears critical for the selectivity of 1,4-naphthoquinones. IMPORTANCE Nitrification, the microbial conversion of ammonia to nitrate via nitrite, plays a pivotal role in the global nitrogen cycle. However, the excessive use of ammonium-based fertilizers in agriculture has disrupted this cycle, leading to groundwater pollution and greenhouse gas emissions. In this study, we have demonstrated the inhibitory effects of plant-derived juglone and related 1,4-naphthoquinones on the nitrification process in Nitrosomonas europaea . Notably, the inhibition mechanism is elucidated in which 1,4-naphthoquinones interact with hydroxylamine oxidoreductase, disrupting the electron transfer to cytochrome c 554 , a physiological electron acceptor. These findings support the notion that phytochemicals can impede nitrification by interfering with the essential electron transfer process in ammonia oxidation. The findings presented in this article offer valuable insights for the development of strategies aimed at the management of nitrification, reduction of fertilizer utilization, and mitigation of greenhouse gas emissions.