A self-subunit swapping chaperone is crucial for cobalt incorporation into nitrile hydratase. However, further information about its structural features is not available. The flexibility and positive charge of the C-terminal domain of the self-subunit swapping chaperone (P14K) of nitrile hydratase from Pseudomonas putida NRRL-18668 play an important role in cobalt incorporation. C-terminal domain truncation, alternation of C-terminal domain flexibility through mutant P14K(G86I), and elimination of the positive charge in the C-terminal domain sharply affected nitrile hydratase cobalt content and activity. The flexible, positively charged C-terminal domain most likely carries out an external action that allows a cobalt-free nitrile hydratase to overcome an energetic barrier, resulting in a cobalt-containing nitrile hydratase.