Disruption of the Aspartate to Heme Ester Linkage in Human Myeloperoxidase

Zederbauer, Martina; Furtmüller, Paul G.; Bellei, Marzia; Stampler, Johanna; Jakopitsch, Christa; Battistuzzi, Gianantonio; Moguilevsky, Nicole; Obinger, Christian

doi:10.1074/jbc.m610685200

Cited by 26 publications

(7 citation statements)

References 48 publications

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“…Full establishment of the covalent bonds is never achieved even when native proteins are purified from natural sources (20, 22–24). Similarly, in the case of recombinantly produced members from this superfamily, there was always some heterogeneity that could be diminished to some extent by adding low micromolar amounts of H 2 O 2 (10, 19, 25–27). In human peroxidasin 1 Asp 826 and Glu 890 have been proposed to be involved in heme-protein ester bonds (3–5).…”

Section: Discussionmentioning

confidence: 99%

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

Paumann-Page

Katz

Bellei

et al. 2017

Journal of Biological Chemistry

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Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV.

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Section: Discussionmentioning

confidence: 99%

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

Paumann-Page

Katz

Bellei

et al. 2017

Journal of Biological Chemistry

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“…This is important because the recombinant proteins are partially unprocessed monomers (28). Nevertheless, they exhibit identical spectral, redox, and enzymatic features (28,(35)(36)(37)(38) thus being valuable model proteins for studying structure-function relationships.…”

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confidence: 99%

“…The purity number (A Soret /A 280 ) of the respective proteins were 0.5-0.62 (wild-type) and 0.18 -0.29 (N421D), indicating incomplete heme occupancy in the mutant protein. Thus, to guarantee comparability of enzymatic properties the concentration of active enzyme had to be calculated via Soret absorbance as has been demonstrated recently with other mutants (36,38,39) and described under "Experimental Procedures".…”

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confidence: 99%

Essential Role of Proximal Histidine-Asparagine Interaction in Mammalian Peroxidases

Carpena

Vidossich²,

Schroettner

et al. 2009

Journal of Biological Chemistry

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In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.

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“…Histidine-bound haloperoxidases do tend to show covalent modifications at the heme edge, and it is known that at least some of these modifications dramatically increase the haloperoxidase activity. However, as pointed out by experimentalists, [18][19][20][21] these covalent modifications do not abolish peroxidase activity in general and are rather associated with increased heme rigidity and better halide binding. In view of our results reported above, we also support the notion that there is no intrinsic electronic-structure rationale for heme edge modification in haloperoxidases and that these modifications are part of a mechanism to create a halide binding site near the heme -binding that apparently also requires some degree of pocket rigidity (possibly connected to the slight endergonicity of halide binding to Compound I, Figure 2).…”

Section: Role Of Heme Modifications In Halide Activationmentioning

confidence: 92%

Halide Activation by Heme Peroxidases: Theoretical Predictions on Putative Adducts of Halides with Compound I

Silaghi-Dumitrescu

2008

Eur J Inorg Chem

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The first density functional theory (DFT) results on models of the putative Fe III -OX and Fe III -HOX (X = halogen) adducts of chloroperoxidase (CPO) and myeloperoxidase (MPO) are reported, and their stability and electronic structure assessed. The oxygen-halogen bonds are computed to be particularly weak, to the extent where ferric hypohalous acid complexes with intact oxygen-halide bonds should be difficult to observe in heme peroxidases; a 'caged' adduct, featuring a ferric species in close (but not covalent) contact with

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Disruption of the Aspartate to Heme Ester Linkage in Human Myeloperoxidase

Abstract: In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here

Cited by 26 publications

References 48 publications

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

Essential Role of Proximal Histidine-Asparagine Interaction in Mammalian Peroxidases

Halide Activation by Heme Peroxidases: Theoretical Predictions on Putative Adducts of Halides with Compound I

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