Mutagenesis studies on conserved histidine residues identified as possible metal binding ligands in clavaminic acid synthase isozyme 2 were consistent with His-145 and His-280 acting as iron Ligands, in support of crystallographic and previous mutagenesis studies. Mutagenesis of the four cysteines and a glutamine residue, conserved in both clavaminic acid synthase isozymes 1 and 2, demonstrated that none of these residues is essential for activity.