Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase

The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N-3(-), CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (K-D increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N-3(-), and CN-(that bind at the iron site). K-D values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as K-D; K-M for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket. (C) 1997 Elsevier Science Inc.