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

Abstract

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.

Secreted heme peroxidase from Dictyostelium discoideum: Insights into catalysis, structure, and biological role

“…As already demonstrated for chordata (family 1) peroxidases ( 18 , 42 ), peroxidasin 1 (hsPxd01, family 2) ( 29 ), and a bacterial enzyme (family 6) ( 27 , 28 ), the prosthetic group of DdPoxA is post-translationally modified by an autocatalytic radical mechanism that depends on hydrogen peroxide. However, DdPoxA is unique in having only one covalent ester bond between the hydroxymethyl group on the pyrrole ring A and Glu-236, whereas all other representatives studied so far have an additional ester bond between a conserved aspartate residue and the hydroxymethyl group of pyrrole ring C. In the case of DdPoxA, this acidic amino acid is replaced by Ile-100.…”

“…This suggests that the autocatalytic post-translational modification of the heme group ( 24 ) was already established during recombinant protein production in the methylotrophic yeast. By contrast, for other members of this heme peroxidase superfamily addition of excess H 2 O 2 was necessary to complete the post-translational modification, which was reflected by changes in spectral and redox properties ( 25 – 28 ).…”

“…Only MPO is able to oxidize chloride ( E ′ 0 (HOCl/Cl − , H 2 O) = 1.28 V at pH 7.0) at reasonable rates ( 46 , 47 ). Bromide oxidation ( E ′ 0 (HOBr/Br − , H 2 O) = 1.13 V at pH 7.0) has been demonstrated for EPO ( 48 ), LspPOX ( 27 ), and hsPxd01 ( 28 ). Oxidation of iodide ( E ′ 0 (HOI/I − , H 2 O) = 0.78 V at pH 7.0) and thiocyanate ( E ′ 0 (HOSCN/SCN − , H 2 O) = 0.56 V at pH 7.0) ( 46 ) is thermodynamically less challenging and thus typically is performed at high rates by compound I of all peroxidases of this superfamily.…”

“…DdPoxA: −0.276 V) < E ′ 0 (two ester bonds, e.g. LPO: −0.183 V; EPO: −0.176 V; LspPOX: −0.158 V; hsPxd01, −0.128 V) < E ′ 0 (two ester bonds, one sulfonium ion linkage, MPO: +0.005 V) ( 27 , 28 , 43 – 45 ).…”

Peroxidasin mediates bromination of tyrosine residues in the extracellular matrix

“…Mammalian peroxidasin is a typical heme peroxidase that catalyzes the two electron oxidation of thiocyanate and iodide as well as bromide (14). It also oxidizes typical peroxidase substrates including tyrosine, urate and nitrite (15).…”