<p>Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.</p>