Nitrous oxide (N2O), a driver of global warming and climate change, has reached unprecedented concentrations in Earth’s atmosphere1. Current N2O sources outpace N2O sinks, emphasizing the need for comprehensive understanding of processes that consume N2O. Microbes that express the enzyme N2O reductase (N2OR) convert N2O to climate change-neutral dinitrogen (N2). Known N2ORs belong to the canonical clade I and clade II NosZ reductases and are considered key enzymes for N2O reduction2–4. Here we report a previously unrecognized protein family with a role in N2O reduction, clade III lactonase-type N2OR (L-N2OR), which diverges in sequence from canonical NosZ but conserves three-dimensional protein structural features. Integrated physiological, metagenomic, proteomic and structural modelling studies demonstrate that L-N2ORs catalyse N2O reduction. L-N2OR genes occur in several phyla, predominantly in uncultured taxa with broad geographic distribution. Our findings expand the known diversity of N2ORs and implicate previously unrecognized taxa (for example, Nitrospinota) in N2O consumption. The expansion of N2OR diversity and the identification of a novel type of catalyst for N2O reduction advances the understanding of N2O sinks, has implications for greenhouse gas emission and climate change modelling, and expands opportunities for innovative biotechnologies aimed at curbing N2O emissions5,6.
