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 ε1 , His-109 N ε2 , 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. © 2009 by the Biophysical Society.
Singh, A. K., Singh, N., Sharma, S., Shin, K., Takase, M., Kaur, P., … Singh, T. P. (2009). Inhibition of lactoperoxidase by its own catalytic product: Crystal structure of the hypothiocyanate-inhibited bovine lactoperoxidase at 2.3-Å resolution. Biophysical Journal, 96(2), 646–654. https://doi.org/10.1016/j.bpj.2008.09.019