title: Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding.
abstract: HBP23, a 23-kDa heme-binding protein identified in rats, is a member of the peroxiredoxin (Prx) family, the primary peroxidases involved in hydrogen peroxide catabolism. Although HBP23 has a characteristic Cys-Pro heme-binding motif, the significance of heme binding to Prx family proteins remains to be elucidated. Here, we examined the effect of heme binding to human peroxiredoxin-1 (PRX1), which has 97% amino acid identity to HBP23. PRX1 was expressed in Escherichia coli and purified to homogeneity. Spectroscopic titration demonstrated that PRX1 binds heme with a 1:1 stoichiometry and a dissociation constant of 0.17 μM. UV-vis spectra of heme-PRX1 suggested that Cys52 is the axial ligand of ferric heme. PRX1 peroxidase activity was lost upon heme binding, reflecting the fact that Cys52 is not only the heme-binding site but also the active center of peroxidase activity. Interestingly, heme binding to PRX1 caused a decrease in the toxicity and degradation of heme, significantly suppressing H2O2-dependent heme peroxidase activity and degradation of PRX1-bound heme compared with that of free hemin. By virtue of its cytosolic abundance (∼20 μM), PRX1 thus functions as a scavenger of cytosolic hemin (<1 μM). Collectively, our results indicate that PRX1 has a dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger.
authors.affiliation: Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan; Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
authors.affiliation: Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan; Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. Electronic address: uchida@sci.hokudai.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D006418:Heme / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D054464:Peroxiredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D011994:Recombinant Proteins; D006418:Heme; D006427:Hemin; C489898:Prdx1 protein, rat; C515940:PRDX1 protein, human; D054464:Peroxiredoxins; D003545:Cysteine
keywords: Cytosolic heme-binding protein; Heme; Peroxiredoxin