A complex between native, iron(II) soybean lipoxygenase 3 and 4-nitrocatechol, a known inhibitor of the enzyme, has been detected by isothermal titration calorimetry and characterized by X-ray crystallography. The compound moors in the central cavity of the protein close to the essential iron atom, but not in a bonding arrangement with it. The iron ligands experience a significant rearrangement upon formation of the complex relative to their positions in the native enzyme; a water molecule becomes bound to iron in the complex, and one histidine ligand moves away from the iron to become involved in a hydrogen bonding interaction with the catechol. These changes in position result in a trigonal pyramid coordination geometry for iron in the complex. Molecular modeling and force field calculations predict more than one stable complex between 4-nitrocatechol and the central cavity of lipoxygenase 3, but the interaction having the small molecule in the same orientation as the one found in the crystal structure was the most favorable. These observations reveal specific details of the interaction between lipoxygenase and a small molecule and raise the possibility that changes in the ligand environment of the iron atom could be a feature of the product activation reaction or the catalytic mechanism.