The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme—progesterone complex. Residues His181 and His184 are important in the binding of coenzyme, steroids, nitro-aromatic ligands and the substrate 2-cyclohexen-1-one.