Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1.
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.
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Martina
M
Paumann-Page
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Romy-Sophie
RS
Katz
the Departments of Life Sciences and.
Marzia
M
Bellei
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Irene
I
Schwartz
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Eva
E
Edenhofer
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Benjamin
B
Sevcnikar
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Monika
M
Soudi
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and.
Stefan
0000-0003-3375-7715
S
Hofbauer
Chemistry and Geology, University of Modena and Reggio Emilia, 41125 Modena, Italy.
Gianantonio
0000-0003-4716-5745
G
Battistuzzi
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and paul.furtmueller@boku.ac.at.
Paul G
0000-0002-1199-2469
PG
Furtmüller
From the Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria and christian.obinger@boku.ac.at.
Christian
0000-0002-7133-3430
C
Obinger
D001965:Bromides / Q000378:metabolism; D020134:Catalytic Domain; D002712:Chlorides / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism; D054879:Halogenation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D013379:Substrate Specificity; D013861:Thiocyanates / Q000378:metabolism; D000097423:Peroxidasin
D001965:Bromides; D002712:Chlorides; D024141:Collagen Type IV; D016326:Extracellular Matrix Proteins; D005290:Ferric Compounds; D006861:Hydrogen Peroxide; D007454:Iodides; D009195:Peroxidase; D013861:Thiocyanates; D000097423:Peroxidasin; C031760:thiocyanate
enzyme catalysis; extracellular matrix; heme; kinetics; peroxidase