Ferrous Lactoperoxidase Research Articles

The role of the sulfonium linkage in the stabilization of the ferrous form of myeloperoxidase: A comparison with lactoperoxidase

In all mammalian peroxidases, the heme is covalently attached to the protein via two ester linkages between conserved aspartate (Asp94) and glutamate residues (Glu242) and modified methyl groups on pyrrole rings A and C. Only myeloperoxidase has an additional sulfonium ion linkage between the sulfur atom of the conserved methionine 243 and the β-carbon of the vinyl group on pyrrole ring A. Upon reduction from Fe(III) to Fe(II), lactoperoxidase (LPO) but not myeloperoxidase (MPO) is shown to adopt three distinct active site conformations which depend on pH and time. Comparative spectroscopic analysis (UV-Vis absorption and resonance Raman) of the ferrous forms of LPO, wild-type MPO and the variants Asp94Val, Glu242Gln, Met243Thr and Met243Val clearly demonstrate that a single, stable ferrous form of MPO is present only in those proteins which retain an intact sulfonium linkage. By contrast, both ferrous Met243Thr and Met243Val can assume two conformations. They resemble ferrous LPO, being five-coordinated high-spin species that are distinguished by the strength of the proximal Fe-histidine bond. This bond weakens with time or decreasing pH, as indicated by the Fe-histidine stretching bands.

Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous–dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2 ± 0.3) × 10 4 M −1 s −1 at pH 7.0 and 25 °C. The H 2O 2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M −1 s −1 at pH 7.0 and 25 °C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8 ± 0.2) × 10 5 M −1 s −1. Dioxygen binding is reversible and at pH 7.0 the dissociation constant ( K D) of the oxyferrous form is 6 μM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.

Evidence for the high-spin heme iron in both stable and unstable reduced forms of lactoperoxidase: low-temperature magnetic circular dichroism data

The unstable and stable ferrous lactoperoxidase at pH 6.0, 7.0 and 10.2 have been analysed using optical absorption and variable temperature MCD spectroscopy. The evidence is given that two high-spin forms of ferrous LPO are always observed when the enzyme is reduced in a buffer-glycerol mixture at low temperature (ca. −20°C) at which no spectral changes are seen for a long time after the reduction. Form 1 (the absorption band, 450 nm) dominates significantly over form 2 (the absorption band, 435 nm), but a relative content of form 2 increases on lowering the pH value. An annealing of the unstable LPO at high temperatures is followed by complete irreversible conversion of form 1 to form 2. In addition, at least one low-spin ferrous form exists in temperature-dependent equilibrium with the high-spin form(s) in both stable and unstable ferrous LPO. The reversible increase of its content is observed at least down to 140 K, suggesting that minor structural changes are sufficient for reaching the heme iron by a distal amino acid residue (presumably a histidine).

The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H 2O 2: A Spin-trapping/ESR and absorption spectroscopy study

H 2O 2 is catalytically metabolized by ferric lactoperoxidase (LPO) → compound (cpd) I → cpd II → ferric LPO cycles. An excess of the substrate, however, is degraded by a ferric LPO → cpd I → cpd II → cpd III → ferrous LPO → ferric LPO cycle. This latter pathway leads to the partial or total irreversible inactivation of the enzyme depending on the excess of H 2O 2 ( H. Jenzer, W. Jones, and H. Kohler (1986) J. Biol. Chem. 261, 15550–15556 ). Spin-trapping/ESR data indicate that in the course of the reaction superoxide ( HO 2 O − 2 ) and hydroxyl radicals (OH.) are formed. Since many substances known to scavenge radicals, such as a spin trap (e.g., 5,5-dimethyl-l-pyrroline- N-oxide) desferrioxamine, albumin, or mannitol, do not prevent enzyme inactivation, we conclude that OH. generation is a site-specific reaction at or near the active center of LPO where bulky scavenger molecules may not be able to penetrate. We suggest the formation of OH. by a Fenton-like reaction between H 2O 2 and the intermediate ferrous state of the enzyme, which substitutes for Fe 2+ in the Fenton reaction. OH. is a powerful oxidant which in turn may attack rapidly the nearest partner available, either H 2O 2 to produce HO. 2 and H 2O, or the prosthetic group to give rise to oxidative cleavage of the porphyrin ring structure of the heme moiety of LPO and thus to the liberation of iron.

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase.

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.

On the molecular mechanism of lactoperoxidase-catalyzed H2O2 metabolism and irreversible enzyme inactivation.

Lactoperoxidase-catalyzed H2O2 metabolism proceeds through one of three different pathways, depending on the nature and the concentration of the second substrate as an e- donor and/or on pH conditions. In the lactoperoxidase (LPO)-H2O2 system, at low H2O2 concentrations and/or alkaline conditions the peroxidatic cycle involves ferric LPO----compound I----compound II----ferric LPO conversion, whereas high H2O2 concentrations and/or acidic conditions favor the ferric LPO----compound I----compound II----compound III----ferrous LPO----ferric LPO pathway. The compound III/ferroperoxidase states are associated with irreversible enzyme inactivation by cleavage of the heme moiety and liberation of iron. It is likely that either singlet oxygen or superoxide and hydroxyl radicals are involved in the attack on heme iron, because inactivation correlates with oxygen production and can be decreased to a certain degree by scavengers such as ethanol, 1-propanol, 2-propanol, or mannitol. In the LPO-H2O2-I- system, the enzyme may also be inactivated by I2 generated in the course of enzymatic I- oxidation (i.e. during ferric LPO----compound I----ferric LPO cycles).

The nitrosyl compounds of ferrous animal haloperoxidases

Human myeloperoxidase, human eosinophil peroxidase and bovine lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) reduced with ascorbic acid form nitrosyl compounds which show rhombic EPR signals centered at g = 2. Using 14NO ( I N = 1), the central resonance signal exhibited a hyperfine structure of nine lines originating from a triplet with a small hyperfine splitting ( A II( z) = 0.69 mT for myeloperoxidase and 0.73 mT for eosinophil peroxidase and lactoperoxidase) superimposed upon a triplet with a larger hyperfine splitting ( A I ( z) = 2.34, 2.32 and 2.09 mT for myeloperoxidase, eosinophil peroxidase and lactoperoxidase, respectively). Using 15NO ( I N = 1 2 ), the nitrosyl compound of ferrous myeloperoxidase and ferrous lactoperoxidase showed a doublet of triplets superimposed upon the central resonance signal. These results demonstrate that a nitrogen nucleus is present at the fifth ligand position of the haem iron in these peroxidases.

The nitrosyl compound of ferrous lactoperoxidase

The nitrosyl compound of reduced bovine lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) has been prepared using two methods of initial reduction prior to exposure to NO; namely, dithionite and electrochemical reduction. In both cases, a rhombic EPR spectrum with a three-line hyperfine splitting of the central band was obtained at 80 K. This indicates an interaction between the unpaired electron and single 14N nucleus of the NO radical. It is concluded that the iron-histidine bond, shown by magnetic circular dichroism (Sievers, G., Gadsby, P.M.A., Peterson, J. and Thomson, A.J. (1983) Biochim. Biophys. Acta 742, 659–668) to be present in the oxidised state of the enzyme, is broken in the ferrous nitrosyl derivative.

Structure of milk lactoperoxidase. A study using circular dichroism and difference absorption spectroscopy

Circular dichroism spectra of milk lactoperoxidase, its fluoride and cyanide derivatives, and those of ferrous lactoperoxidase and its carbonyl and cyanide compounds, were recorded in the wavelength region 200–670 nm. All derivatives have split ellipticity bands, suggesting that lactoperoxidase has a narrow heme pocket that prevents ligands forming linear iron-ligand bonds. Difference absorption spectroscopy of the enzyme in the far-ultraviolet region supports the previously held view that the fifth ligand of the heme iron is histidine. The secondary structure of lactoperoxidase, calculated from the far-ultraviolet circular dichroism spectrum, contains 65% β-structure, 23% α-helix and 12% unorderded structure. Reduction of lactoperoxidase with dithionite gives two forms, indicating that after reduction some compound arising from dithionite binds in the vicinity of the heme iron.

The kinetics of fluoride binding by lactoperoxidase

The kinetics of the reversible binding of fluoride by ferric lactoperoxidase has been studied over the pH range 3.8–5.4 by means of a stopped-flow apparatus. Because of small absorbance changes upon the binding of fluoride by lactoperoxidase, the reaction was driven to completion by addition of a large excess of fluoride. Under these conditions the binding reaction obeys pseudo-first-order kinetics and an accurate analysis of the dissociation rate data is not possible. The results are consistent with a mechanism in which HF binds to the active site which contains no groups capable of ionization in the pH range of the present study. The specific rate constant for the binding reaction of HF to lactoperoxidase is (9.7 ± 0.4) × 102 M−1s−1.