a, SDS–PAGE of purified SznF and SznF–SznG complex. The molecular weights of SznF and SznG are 54 kDa and 13 kDa, respectively. Ladder = Precision Plus Protein All Blue Standards (BioRad). b, Nitrite and nitric oxide were detected when Feii–SznF and l-NMA were incubated together. Nitrite was detected with the Griess reagent, and absorbance was measured at 548 nm39. Data are mean ± s.d. of three replicates. NO was trapped with MGD and analysed by EPR spectroscopy at room temperature33. Sodium 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) was used as a positive control for NO detection. Assays using [guanidino-15N2]l-NMA as a substrate revealed changes in hyperfine splitting by EPR spectroscopy, indicating that NO is derived from the terminal guanidine group of l-NMA. We propose that the NO detected is derived from the degradation of 3 or is generated as part of the N–N bond-formation step. c, Comparison of retention times and MS/MS fragmentation patterns of 1, 2, Fmoc-3 and 4 generated in SznF assay mixtures with the corresponding synthetic standards. NMR characterization and synthetic procedures are reported in Supplementary Information.