The crystal structure of cobalt-containing nitrile hydratase from Pseudonocardia thermophila JCM 3095 at 1.8 Angstrom resolution revealed the structure of the non-corrin cobalt at the catalytic center. Two cysteine residues (alpha Cys(111) and alpha Cys(113)) coordinated to the cobalt were posttranslationally modified to cysteine-sulfinic acid and to cysteine-sulfenic acid, respectively, like in iron-containing nitrile hydratase. A tryptophan residue (beta Trp(72)), which may be involved in substrate binding, replaced the tyrosine residue of iron-containing nitrile hydratase. The difference seems to be responsible for the preference for aromatic nitriles rather than aliphatic ones of cobalt-containing nitrile hydratase.