The facial 2-His-1-carboxylate (Asp/Glu) motif has emerged as the structural paradigm formetal binding in the
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-ketoglutarate (
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-KG)-dependent nonheme iron oxygenases. Clavaminate synthase(CS2) is an unusual member of this enzyme family that mediates three different, nonsequential reactionsduring the biosynthesis of the
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-lactamase inhibitor clavulanic acid. In this study, covalent modificationof CS2 by the affinity label
N-bromoacetyl-
L-arginine near His297,
which is
within the HRV signature ofa His-2 motif, suggested this histidine could play a role in metal coordination. Ho
wever, site-specificmutagenesis of eight His residues to Gln identified His145 and His280, but not His297, as involved iniron binding. Weak homology of His145 and its flanking sequence and the presence of Glu147 fitting thecanonical acidic residue of the His-Xaa-Asp/Glu signature are consistent
with His145 being a coordinatingligand (His-1). His280 and its flanking sequence,
which give poor alignments to most other members ofthis enzyme family, are similar among a subset of these enzymes and notably to CarC, an apparentoxygenase involved in carbapenem biosynthesis. The separation of His145 and His280 is more than t
wicethat 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 oxidativecyclization/desaturation and hydroxylation assays, establish that no histidine ligand s
witching occurs duringthe catalytic cycle. These results are confirmed in a recent X-ray crystal structure of CS1, a highly similarisozyme of CS2 (81% identical). Tyr299, Tyr300 in CS2 modified by
N-bromoacetyl-
L-arginine, is hydrogenbonded to Glu146 (Glu147 in CS2) in this structure and
well-positioned for reaction
with the affinitylabel.