a, SznF contains a large cavity (grey surface, 1.9 Å probe radius) in the middle of its central helical bundle domain (orange). Additionally, most of the secondary structures in this domain contain loop disruptions and disordered regions, suggesting considerable refolding upon binding or release of the l-NMA substrate and/or assembly of the iron-based cofactor. b, The central domain of SznF is similar in topology to haem oxygenase (HO), compared here to HO-2 from Synechocystis sp. PCC 6803 (PDB: 1WOW)40. SznF contains an open pocket near the haem-binding site in HO-2 but lacks conserved cofactor ligation and hydrogen-bonding motifs (d). c, SznF instead more closely resembles a C. trachomatis dinuclear iron protein in this structural superfamily (CADD)21 implicated in the biosynthesis of para-aminobenzoic acid41. SznF conserves all of the metal-binding residues but fails to stably incorporate iron in this domain in the current preparations. All three systems share a propensity for distorted secondary-structure motifs that perhaps enable complex formation with large and polar substrates for oxidative transformations. d, A structure-based sequence alignment42 of six HO-like enzymes in selected regions relevant to substrate–cofactor interaction and catalysis. SznF conserves all six histidine and carboxylate residues used to coordinate a dinuclear iron cluster in the active form of fatty acid oxidative decarboxylase UndA (PDB: 4WWZ)16 and in the uncharacterized CADD protein (PDB: 1RCW)21. Before our discovery and characterization of SznF, UndA was the only HO-like enzyme with a defined substrate and activity. The published crystal structure of UndA contains only a single iron ion and a mechanism was initially proposed using a mononuclear cofactor (located in site 1)16. However, recent spectroscopic studies20 show that this enzyme uses a dinuclear non-haem iron cofactor and corresponding alternative reaction pathway. So far, the dinuclear form of UndA has remained refractory to crystallographic characterization owing to a propensity for disorder in the helix containing the site 2 metal ion ligands. As in SznF, mutagenesis of any of the six predicted ligands to the recently characterized dinuclear site in UndA completely abolishes activity20. As a consequence, we propose that all HO-like non-haem-iron proteins (including SznF) assemble a multinuclear cofactor but require a second protein or other factor to stabilize the active form in high yield and at high concentration. e, Comparative views of the cofactor site and/or substrate binding site in (left to right, top to bottom) SznF, C. trachomatis CADD, Pseudomonas fluorescens UndA, Klebsiella pneumoniae pyrroloquinoline quinone (PQQ) synthase PqqC (PDB: 1OTW)43, Synechocystis sp. PCC 6803 haem oxygenase (HO) 2 (PDB: 1WOW)40, and Bacillus subtilis thiamin synthase TenA (PDB: 1YAK)44. Substrates, products and selected side chains are shown in stick format. Iron ions and water molecules are shown as orange and red spheres, respectively. f, Additional mutation of the predicted iron-binding residues in the SznF central bundle helix domain abolished N-oxygenation activity. Assay mixtures contained 1 mM l-NMA, 80 μM SznF or variant, 20 μM PMS and 5 mM NADH and were incubated at room temperature for 1 h. The EIC traces were generated with a 5 p.p.m. window using the [M − H]− masses.