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The facial 2-His-1-carboxylate (Asp/Glu) motif has emerged as the structural paradigm for metal binding in the alpha-ketoglutarate (alpha-KG)-dependent nonheme iron oxygenases. Clavaminate synthase (CS2) is an unusual member of this enzyme family that mediates three different, nonsequential reactions during the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In this study, covalent modification of CS2 by the affinity label N-bromoacetyl-L-arginine near His297, which is within the HRV signature of a His-2 motif, suggested this histidine could play a role in metal coordination. However, site-specific mutagenesis of eight His residues to Gln identified His145 and His280, but not His297, as involved in iron binding. Weak homology of His145 and its flanking sequence and the presence of Glu147 fitting the canonical acidic residue of the His-Xaa-Asp/Glu signature are consistent with His145 being a coordinating ligand (His-1). His280 and its flanking sequence, which give poor alignments to most other members of this enzyme family, are similar among a subset of these enzymes and notably to CarC, an apparent oxygenase involved in carbapenem biosynthesis. The separation of His145 and His280 is more than twice that seen in the current 2-His-1-carboxylate model and may define an alternative iron binding motif, which we propose as His-3. These ligand assignments, based on kinetic measurements of both oxidative cyclization/desaturation and hydroxylation assays, establish that no histidine ligand switching occurs during the catalytic cycle. These results are confirmed in a recent X-ray crystal structure of CS1, a highly similar isozyme of CS2 (81% identical). Tyr299, Tyr300 in CS2 modified by N-bromoacetyl-L-arginine, is hydrogen bonded to Glu146 (Glu147 in CS2) in this structure and well-positioned for reaction with the affinity label.