Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K m and K i values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of bTyr68 formed hydrogen bonds with both n-butyric acid and aSer112, which is located in the active center. The bY68F mutant showed an elevated K m value and a significantly decreased k cat value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between aCys108 and aCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (aT109S and aY114T) were constructed, each residue being replaced with an iron-containing one. The aT109S mutant showed similar characteristics to the wild-type enzyme. However, the aY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of aCys111 and aCys113 residues were not observed. The aTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.