The crystal structure of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 was determined at 1.3 angstrom resolution. The two cysteine residues (alpha Cys(112) and alpha Cys(114)) equatorially coordinated to the ferric ion were post-translation ally modified to cysteine sulfinic acids. A glutamine residue (alpha Gln(90)) in the active center gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion. (C) 2007 Elsevier Inc. All rights reserved.