Inhibition of Lactoperoxidase by Its Own Catalytic Product: Crystal Structure of the Hypothiocyanate-Inhibited Bovine Lactoperoxidase at 2.3-Å Resolution

To the best of our knowledge, this is the first report on the structure of product-inhibited mammalian peroxidase. Lactoperoxidase is a heme containing an enzyme that catalyzes the inactivation of a wide range of microorganisms. In the presence of hydrogen peroxide, it preferentially converts thiocyanate ion into a toxic hypothiocyanate ion. Samples of bovine lactoperoxidase containing thiocyanate (SCN⁻) and hypothiocyanate (OSCN⁻) ions were purified and crystallized. The structure was determined at 2.3-Å resolution and refined to R_cryst and R_free factors of 0.184 and 0.221, respectively. The determination of structure revealed the presence of an OSCN⁻ ion at the distal heme cavity. The presence of OSCN⁻ ions in crystal samples was also confirmed by chemical and spectroscopic analysis. The OSCN⁻ ion interacts with the heme iron, Gln-105 N^ɛ1, His-109 N^ɛ2, and a water molecule W96. The sulfur atom of the OSCN⁻ ion forms a hypervalent bond with a nitrogen atom of the pyrrole ring D of the heme moiety at an S–N distance of 2.8 Å. The heme group is covalently bound to the protein through two ester linkages involving carboxylic groups of Glu-258 and Asp-108 and the modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is significantly distorted from planarity, whereas pyrrole rings A, B, C, and D are essentially planar. The iron atom is displaced by ≈0.2 Å from the plane of the heme group toward the proximal site. The substrate channel resembles a long tunnel whose inner walls contain predominantly aromatic residues such as Phe-113, Phe-239, Phe-254, Phe-380, Phe-381, Phe-422, and Pro-424. A phosphorylated Ser-198 was evident at the surface, in the proximity of the calcium-binding channel.