loading . . . Microbial N2O reduction in sulfidic waters: Implications for Proterozoic oceans Throughout Earth’s history, shifts in ocean redox influenced the bioavailability of trace metals, shaping the activity of microorganisms. In Proterozoic oceans, the precipitation of copper (Cu) with sulfide was hypothesized to limit the bioavailability of Cu. This limitation may have suppressed microbial reduction of nitrous oxide (N2O), due to the Cu dependency of nitrous oxide reductase (Nos). It is thought that without this critical microbial sink, Proterozoic oceans were a significant net source of N2O. Here, we revisit this paradigm in light of recently derived ∼20-fold lower estimates for sulfide in Proterozoic seawater and an empirical evaluation of the potential for microbial N2O reduction under sulfidic conditions. Leveraging publicly available environmental metatranscriptomes, we infer active N2O reduction from the detection of nosZ transcripts in multiple marine and lacustrine systems in which sulfide and Cu concentrations are analogous to those of the Proterozoic. In controlled culture experiments, we demonstrate that the purple non-sulfur bacterium Rhodopseudomonas palustris can reduce N2O at sulfide concentrations up to 100 µM, well above levels predicted for Proterozoic oceans. Based on trace metal speciation modeling, we suggest that Cu remains bioavailable under Proterozoic-like conditions as a dissolved CuHS complex. Using phylogenetics, we infer that early N2O reducers were probably anoxygenic phototrophs and performed N2O reduction as dark metabolism. Collectively, these observations suggest microbial N2O reduction occurs under euxinic conditions, implying that Proterozoic marine N2O emissions were substantially lower than previously proposed. Our conclusions inform our understanding of the microbial ecology in sulfidic waters, the early climate, and the search for extraterrestrial life. ### Competing Interest Statement The authors have declared no competing interest. National Aeronautics and Space Administration, https://ror.org/027ka1x80, 80NSSC17K0296 https://www.biorxiv.org/content/10.1101/2025.06.28.662143v1
