Structure of bovine lactoperoxidase with a partially linked heme moiety at 1.98 Å resolution

Highlights

• In mature LPO, the heme moiety is linked to protein through two covalent bonds.

• In the present structure, the linkage involving Glu258 is observed in 50% molecules.

• In other 50% molecules, the side chain of Glu258 does not form covalent linkage.

• The side chain of unlinked Glu258 is located in the substrate binding site.

• The unlinked Glu258 blocks the substrate binding site.

Abstract

Lactoperoxidase (LPO) is a member of mammalian heme peroxidase superfamily whose other members are myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). In these enzymes, the heme moiety is linked to protein through two or three covalent bonds. In the mature LPO, the heme moiety is linked to protein through two ester bonds with highly conserved glutamate and aspartate residues. The previously reported structures of LPO have confirmed the formation of two covalent linkages involving Glu258 and Asp108 with 1-methyl and 5-methyl groups of pyrrole rings A and C respectively. We report here a new form of structure of LPO where the covalent bond between Glu258 and 1-methyl group of pyrrole ring A is present only in a fraction of protein molecules. In this case, the side chain of Glu258 occupies two distinct positions, each of which has a 0.5 occupancy. In one position, it forms a normal ester covalent linkage while in the second position, the side chain of Glu258 is located in the middle of the substrate binding site on the distal heme side. In this position, the atom of the side chain of Glu258 forms several contacts with atoms of other residues and heme moiety. Out of the two observed positions of the side chain of Glu258, the former contributes to the stabilization of heme position and improved catalytic action of LPO while the latter is responsible for the reduced stability of the heme position as well as it blocks the substrate binding site.

Introduction

Heme peroxidases catalyze the H₂O₂-dependent oxidation of a number of inorganic [1], [2], [3] and small organic substrates [4], [5], [6], [7], [8]. These enzymes can be broadly divided into two super families consisting of (i) bacterial, fungal and plant (non-animal) heme peroxidases and (ii) mammalian heme peroxidases which are represented by myeloperoxoidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). Evolutionarily, the two families emerged independently [9] and differ greatly in their amino acid sequences with sequence identities between the members of two superfamilies ranging from 10% to 20% only. They also differ in their three-dimensional structures although carry out similar functional roles. However, the most striking difference between the members of the two superfamilies is attributed to the way their prosthetic heme groups are associated with the protein. There are also further differences in the fine architectures of their substrate binding sites as well as between the hydrogen bonding patterns involving the important His351 (LPO numbering scheme) on the proximal side. The functionally critical ferroprotophorphyrin IX moiety is held non-covalently in the enzymes of non-animal heme peroxidases while in the mammalian heme peroxidases, as a result of post-translational modification, the heme moiety is bound firmly with the help of two or three covalent bonds. In LPO, EPO and TPO, the heme moiety is covalently linked to the protein via two ester bonds through conserved aspartate and glutamate residues [10] while in MPO, it has an additional sulfonium ion linkage between the 2-vinyl group of the heme moiety and a methionine residue of the protein [11]. These linkages contribute to the rigidification of the prostheic heme group and the formation of a well defined substrate binding site on the distal heme side which may, in turn, be responsible for the peculiar actions of mammalian heme peroxidases. The studies have also shown that the heme moiety of lactoperoxidase can be cleaved using various procedures [12].

The fine differences and similarities in the functional properties of mammalian heme peroxidases have been summarized in a recent publication [13]. The amino acid sequence identities among the four members of the mammalian heme peroxidase superfamily range from 46% to 57%. Several crystal structures of LPO [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] and MPO [25], [26], [27], [28], [29], [30], [31] in both bound and unbound forms have been determined. In spite of ongoing significant efforts, the crystal structures of EPO and TPO have not yet been determined. As revealed by structures of LPO and MPO, the overall-folds of protein chains in these two members of the mammalian heme peroxidase superfamily are identical. The stereochemical features of their substrate binding sites on the distal heme side are also very similar. Because of the highly conserved amino acid sequences of the substrate binding sites and similar functional properties, the substrate binding sites of all the four proteins of the mammalian heme peroxidase superfamily are expected to have similar heme environments.

The biological activities of LPO, MPO and EPO are concerned primarily with their roles in the host defence [32], [33], [34] while TPO is involved in the biosynthesis of thyroid hormones, thyroxine and tri-iodothyronine [35]. The substrates for these four enzymes include small anions such as chloride, bromide, iodide, thiocynate and nitrite [3]. As shown by crystal structure of LPO with four inorganic substrates [21], there is a subtle difference in the preference for the binding of these substrates. The role of two covalent linkages between protein and heme moiety involving conserved glutamate and aspartate residues is considered to be essential for fulfilling the structural requirements of the substrate binding site. It was presumed that these two ester linkages are firmly formed for stabilizing the structure. The roles of glutamate and aspartate residues in linking to heme moiety through two ester linkages via 1-methyl and 5-methyl groups respectively were investigated biochemically and spectroscopically [36], [37], [38], [39]. Here, we report a new structure of LPO in which the covalent bond between Glu258 and 1-methyl group of heme moiety is present only in 50% of protein molecules. In the rest of the 50% molecules, the side chain of Glu258 occupies a position in the middle of the substrate binding site on the distal heme side. At this position, the side chain of Glu258 forms hydrogen bonded and van der Waals contacts with several other residues thus making it a stable arrangement for Glu258. At this position, the side chain of Glu258 occupies a significant space of the distal heme cavity thus blocking the substrate binding site. This may prevent the binding of ligands in the substrate binding site. This is the first structural observation that demonstrates the absence of an ester covalent bond involving Glu258 and 1-methyl group of pyrrole ring C of the heme moiety in LPO. Because of structural and functional similarities, the similar conditions may be present in other members of the mammalian heme peroxidase superfamily.