Enhancing hypothiocyanite production by lactoperoxidase – mechanism and chemical properties of promotors

Background: The heme enzyme lactoperoxidase is found in body secretions where it significantly contributes to the humoral immune response against pathogens. After activation the peroxidase oxidizes thiocyanate to hypothiocyanite which is known for its microbicidal properties. Yet several pathologies are accompanied by a disturbed hypothiocyanite production which results in a reduced immune defense. Methods: The results were obtained by measuring enzyme-kinetic parameters using UV–vis spectroscopy and a standardized enzyme-kinetic test system as well as by the determination of second order rate constants using stopped-flow spectroscopy. Results: In this study we systematically tested thirty aromatic substrates for their efficiency to promote the lactoperoxidase-mediated hypothiocyanite production by restoring the native ferric enzyme state. Thereby hydrophobic compounds with a 3,4-dihydroxyphenyl partial structure such ashydroxytyrosol and selected flavonoids emerged as highly efficient promotors of the (pseudo-)halogenating lactoperoxidase activity. Conclusions: This study discusses important structure-function relationships of efficient aromatic LPO substrates and may contribute to the development of new agents to promote lactoperoxidase activity in secretory fluids of patients. Significance: This study may contribute to a better understanding of the (patho-)physiological importance of the (pseudo-)halogenating lactoperoxidase activity. The presented results may in future lead to the development of new therapeutic strategies which, by reactivating lactoperoxidase-derived hypothiocyanite production, promote the immunological activity of this enzyme

A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase

AbstractSpectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi-mixing stopped-flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9×107 M−1 s−1, pH 7 and 15°C) and reduction of compound I to compound II (3.0×104 M−1 s−1, pH 7 and 15°C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6×104 M−1 s−1, 1.4×106 M−1 s−1, 1.4×107 M−1 s−1 and 1.4×107 M−1 s−1, respectively), whereas the endogenous one-electron donor ascorbate mediated transformation of compound I to compound II (2.3×105 M−1 s−1) and of compound II back to the resting enzyme (5.0×103 M−1 s−1). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently

Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) may 34 contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite “dismutase”, Cld). Beside the water-splitting manganese complex of photosystem II this metalloenzyme is the second known enzyme that catalyzes the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in E. coli and shown to efficiently degrade chlorite with an activity optimum at pH 5 (kcat 1144 ± 23.8 s-1, KM 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 106 M-1 s-1). The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7. Cyanide mediates the formation of a low-spin complex with kon = (1.6 ± 0.1) × 105 M-1 s-1 and koff = 1.4 ± 2.9 s-1 (KD ~ 8.6 μM). Both, thermal and chemical unfolding follows a non-two state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2-producing proteins in (nitrogen-fixing) cyanobacteria

From chlorite dismutase towards HemQ -the role of the proximal H-bonding network in haeme binding

Synopsis Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from 'Candidatus Nitrospira defluvii' (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV-vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure-function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria

Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution

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