The status of half-cystine residues and locations of N-glycosylated asparagine residues in human eosinophil peroxidase

The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.     

A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase,built on the scaffold of the myeloperoxidase X-ray structure

 Lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) belong to the class of haloperoxidases, a group of mammalian enzymes able to catalyze the peroxidative oxidation of halides and pseudohalides, such as thiocyanate. They all play a key role in the development of antibacterial activity. The homology in their functional role is emphasized by the striking similarity of their primary structures. A theoretical model for the three-dimensional structure of LPO and EPO has been developed on the basis of the X-ray structure of MPO, a high degree of similarity having been found in their sequences. Evidence supporting the hypothesis of an ester linkage between heme and apoprotein in LPO and EPO, originally proposed by Hultquist and Morrison is discussed. Received: 2 May 1996 / Accepted: 25 July 1996     

Design of anti‐thyroid drugs: Binding studies and structure determination of the complex of lactoperoxidase with 2‐mercaptoimidazole at 2.30 Å resolution

Lactoperoxidase (LPO) belongs to mammalian heme peroxidase superfamily, which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO catalyzes the oxidation of a number of substrates including thiocyanate while TPO catalyzes the biosynthesis of thyroid hormones. LPO is also been shown to catalyze the biosynthesis of thyroid hormones indicating similar functional and structural properties. The binding studies showed that 2‐mercaptoimidazole (MZY) bound to LPO with a dissociation constant of 0.63 µM. The inhibition studies showed that the value of IC₅₀ was 17 µM. The crystal structure of the complex of LPO with MZY showed that MZY bound to LPO in the substrate‐binding site on the distal heme side. MZY was oriented in the substrate‐binding site in such a way that the sulfur atom is at a distance of 2.58 Å from the heme iron. Previously, a similar compound, 3‐amino‐1,2,4‐triazole (amitrole) was also shown to bind to LPO in the substrate‐binding site on the distal heme side. The amino nitrogen atom of amitrole occupied the same position as that of sulfur atom in the present structure indicating a similar mode of binding. Recently, the structure of the complex of LPO with a potent antithyroid drug, 1‐methylimidazole‐2‐thiol (methimazole, MMZ) was also determined. It showed that MMZ bound to LPO in the substrate‐binding site on the distal heme side with 2 orientations. The position of methyl group was same in the 2 orientations while the positions of sulfur atom differed indicating a higher preference for a methyl group.     

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.     

Resonance Raman characterization of the heme prosthetic group in eosinophil peroxidase

The resonance-enhanced Raman spectrum of eosinophil peroxidase (EPO) from horse and human eosinophils is reported. Based upon the spectral energies, distribution and depolarization ratios of the high-frequency skeletal modes and upon the presence of weak bands assignable to vinyl substituent groups, we conclude that the heme prosthetic group is high-spin, 6 coordinate protoporphyrin. The Raman spectrum reveals clear differences from lactoperoxidase (LPO), an enzyme which appears nearly structurally isomorphous by other physical techniques; the data indicate a stronger axial 6th ligand in EPO. Mechanistic implications are discussed in relation to LPO and myeloperoxidase, an enzyme present in neutrophils and monocytes which contains a unique functional active-site chlorin.     

Effect of covalent links on the structure, spectra, and redox properties of myeloperoxidase--a density functional study

The enzyme myeloperoxidase shows several unusual properties compared to other peroxidases, e.g. a red-shifted absorption spectrum and a peroxidase activity towards chloride. It has been suggested that this is caused by the unusual covalent links between the heme group and the surrounding protein, but whether it is caused by the two ester links to Glu-242 and Asp-94 or the sulfonium ion linkage to Met-243 is unclear. To investigate these suggestions, we have used density functional theory to study the structure, spectra, and reduction potential of 25 models of myeloperoxidase in the reduced (Fe^II) and oxidized (Fe^III) states, as well as in the compound I (formally Fe^VO) and II (Fe^IVO or Fe^IVOH) states, using appropriate models of the linkages to the Asp, Glu, and Met residues (including the back-bone connection between Glu-242 and Met-243) in varying combinations. The calculated spectral shifts indicate that both the ester and sulfonium linkages play a role in the spectral shift. On the other hand, the sulfonium linkage seems to be mainly responsible for the high positive reduction potential for the both ferric/ferrous and compound I/II couples of myeloperoxidase.     

Interaction between the heavy and the regulatory light chains in smooth muscle myosin subfragment 1.

The interaction between the heavy and the regulatory light chains within chicken gizzard myosin heads was investigated by using a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC). The chicken gizzard subfragment 1 (S-1) used was treated with papain so that the heavy chain was partly cleaved into the NH2-terminal 72K and the COOH-terminal 24K fragments and the regulatory light chain into the 16K fragment. S-1 was reacted with EDC either alone or in the presence of ATP or F-actin. In all cases, the 16K fragment of the regulatory light chain formed a covalent cross-link with the 24K heavy chain fragment but not with the 72K fragment. The 38K cross-linked peptide, which was the product of cross-linking between the 16K light chain and the 24K heavy chain fragments, was isolated and further cleaved with cyanogen bromide and arginylendopeptidase. Smaller cross-linked peptides were purified by reverse-phase HPLC and then characterized by amino acid analysis and sequencing. The results indicated that cross-linking occurred between Lys-845 in the heavy chain and Asp-168, Asp-170, or Asp-171 in the regulatory light chain. The position of the cross-linked lysine was only three amino acid residues away from the invariant proline residue mapped as the S-1-rod hinge by McLachlan and Karn [McLachlan, A. D., & Karn, J. (1982) Nature (London) 299, 226-231]. We propose that the COOH-terminal region of the regulatory light chain is located in the neck region of myosin and that this region and the phosphorylation site of the regulatory light chain together may play a role in the phosphorylation-induced conformational change of gizzard myosin.     

Asp-225 and glu-375 in autocatalytic attachment of the prosthetic heme group of lactoperoxidase.

The heme in lactoperoxidase is attached to the protein by ester bonds between the heme 1- and 5-methyl groups and Glu-375 and Asp-275, respectively. To investigate the cross-linking process, we have examined the D225E, E375D, and D225E/E375D mutants of bovine lactoperoxidase. The heme in the E375D mutant is only partially covalently bound, but exposure to H(2)O(2) results in complete covalent binding and a fully active protein. Digestion of this mutant shows that the heme is primarily bound through its 5-methyl group. Excess H(2)O(2) increases the proportion of the doubly linked species without augmenting enzyme activity. The D225E mutant has little covalently bound heme and a much lower activity, neither of which are significantly increased by the addition of heme and H(2)O(2). The heme is linked to this protein through a single bond to the 1-methyl group. The D225E/E375D mutant has no covalently bound heme and no activity. A small amount of iron 1-hydroxymethylprotoporphyrin IX is obtained from the wild-type enzyme along with the predominant dihydroxylated derivative. The results establish that a single covalent link suffices to achieve maximum catalytic activity and suggest that the 5-hydroxymethyl bond may form before the 1-hydroxymethyl bond.     

Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.     

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474–7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.     

The sulfonium ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.

The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met(243). Mutation of Met(243) into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met(243) mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the (13)CD(3)-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu(242), it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.     

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes.

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.     

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.     

Conjugation and modeled structure/function analysis of lysozyme on glycine esterified cotton cellulose-fibers

The antimicrobial activity of lysozyme covalently bound to glycine-derivatized cotton cellulose was assessed in a 96-well format. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through both a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against Bacillus subtilis was assessed as a suspension of pulverized cotton fibers in microtiter wells. Inhibition of B. subtilis growth was observed to be optimal within a range of 0.14-0.3 mM (equivalent to 4-20 mg of lysozyme-bound cotton/mL) of lysozyme. Enhancement of activity over soluble lysozyme may result from the solid-phase protection afforded by the cellulose linkage of the glycoprotein against proteolytic lysis. Computational models of lysozyme based on its crystal structure attached through aspartate, glutamate, and COOH-terminal residues to cellopentaose-(3) Gly-O-6-glycyl-glycine ester were constructed. The models demonstrate no steric constraints to the active-site cleft from the glycine-conjugated cellulose chain when lysozyme is bound at the carboxylates of Asp-87, Glu-7, Asp-119, Asp-18, and COOH-terminal Leu-129. The more robust antibacterial activity of the enzyme when bonded to cotton fibers suggests good potential for biologically active enzymes on cotton-based fabrics.     

Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.     

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.     

Crystal structure of lactoperoxidase at 2.4 A resolution

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to R_cryst and R_free factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by anti-phosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined α-helices of varying lengths including a helix, H_2a, unique to LPO, and two short antiparallel β-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 Å from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 Å. Two histidine residues and six buried water molecules are connected through a hydrogen-bonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry.     

Lactoperoxidase: structural insights into the function,ligand binding and inhibition

Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.     

Primary structure of bovine lactoperoxidase, a fourth member of a mammalian heme peroxidase family

Much is known about bovine lactoperoxidase but no data are available on its primary structure. In this work its main active fraction was isolated from cow's milk and sequenced using a conventional strategy. A clear similarity was found with human myeloperoxidase, eosinophil peroxidase and thyroperoxidase, the sequences of which were recently elucidated from those of their cDNAs and/or genes. The single peptide chain of bovine lactoperoxidase contains 612 amino acid residues, including 15 half-cystines and 4 or 5 potential N-glycosylation sites. The corresponding peptide segments of human myeloperoxidase, eosinophil peroxidase and thyroperoxidase display 55%, 54% and 45% identity with bovine lactoperoxidase, respectively, with 14 out of the 15 half-cystines present in each of the four enzymes being located in identical positions. The occurrence of an odd number of half-cystines in bovine lactoperoxidase supports the recent finding of a heme thiol released from this enzyme by a reducing agent, suggesting that the heme is bound to the peptide chain via a disulfide linkage, since the absence of free thiol in the enzyme was reported long ago.