Inhibition of lactoperoxidase by its own catalytic product: crystal structure of the hypothiocyanate-inhibited bovine lactoperoxidase at 2.3-A resolution

Abstract

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-A 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(epsilon1), His-109 N(epsilon2), 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 A. 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 approximately 0.2 A 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.

Figure 1

(a) OSCN⁻ fitted into 2.5 σ (|F_o − F_c|) electron density. The heme group and distal Gln-105 and His-109 are also shown. (b) SCN⁻ fitted into 2.5 σ (|F_o − F_c|) electron density. Residues in the vicinity are also indicated.

Figure 2

Schematic diagram of bovine lactoperoxidase complex with OSCN⁻ ion, indicating positions of α-helices (cylinders 1–19), heme moiety (green online and medium gray in print), hypothiocyanate ion, and glycan chains (yellow online and light gray in print). Side chains of Asn-95, Asn-205, Asn-241, and Asn-332, covalently linked to glycan chains, are also shown. Figure was drawn using Pymol (39).

Figure 3

Electron density (|F_o − F_c|) for hypothiocyanate ion bound in distal heme cavity, contoured at 2.5 σ level. Residues Gln-105 and His-109 on distal site and His-351 on proximal site are also shown. Positions of α-helices H2 and H8 with respect to heme group are also indicated. Two opposite orientations (a and b) are shown for clarity of interactions with OSCN⁻ ion.

Figure 4

Electron density (|2F_o − F_c|) for heme moiety contoured at 1.2 σ level. Electron densities for residues Asp-108 and Glu-258, covalently linked to the heme group, are also shown. Iron, oxygen, and nitrogen atoms of the heme group are labeled. The four pyrrole rings are also labeled.

Figure 5

View of substrate channel connecting distal heme cavity to surface of protein. Positions of calcium and OSCN⁻ ions, and of His-109, W2–W6, and His-266, are shown. Position of SCN⁻ ion is also indicated. Channel walls contain predominantly hydrophobic residues, which are also indicated.

Figure 6

Relative positions of (a) OSCN⁻ ion in LPO and (b) SCN⁻ ion in MPO. The bound water molecule (W209) is present in MPO structure. Corresponding water molecule is displaced by OSCN⁻ ion in LPO. The OSCN⁻ ion seems to bind more preferentially than does the SCN⁻ ion.

Figure 7

Interactions involving OSCN⁻ ion and heme group in distal heme cavity of LPO, as indicated by dashed line.

Figure 8

Some interactions are shown between protein residues in proximity of heme group and with heme group, which are unique in LPO.