Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding

https://doi.org/10.1016/j.bbrc.2017.01.034Get rights and content

Highlights

  • Human peroxiredoxin-1 (PRX1) is a heme-binding protein.

  • Cys52 in PRX1 is the heme-binding site as well as the active center.

  • Heme-bound PRX1 loses the peroxidase activity.

  • Heme that is bound to PRX1 is protected from the reaction with H2O2.

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.

Introduction

Heme (iron-containing protoporphyrin IX) is an essential molecular cofactor in electron transfer [1], oxygen metabolism [2] and oxidation reactions [3], Heme also acts as an effector molecule to modulate transcription [4], [5], translation [6], [7], and protein degradation [5], [8]. Reflecting these diverse contributions of heme, hemoproteins are localized to various organelles, including the nucleus, endoplasmic reticulum, and plasma membrane [9], [10]. Because heme biosynthesis is completed in mitochondria, heme must be trafficked to other organelles via the cytosol. Cytosolic heme-binding proteins, which bind heme loosely, are thought to contribute to heme trafficking. These include fatty acid-binding proteins (FABPs), glutathione S-transferases (GSTs), and heme-binding proteins with a molecular mass of 23 kDa (HBP23) [9], [11]. Both GSTs and HBP23 have a Cys-Pro (CP) motif, which is one of the heme regulatory motifs and is found in a wide variety of proteins whose function is regulated by heme [12], [13]. The Cys residue in the CP motif is a heme ligand. GSTs and HBP23 have relatively weak heme-binding capacities, with dissociation constants (Kd,heme) of ∼0.1–1 μM, and 55 nM, respectively [14], [15], which are a much larger than those of typical hemoproteins such as myoglobin (Kd,heme ≈ 10−7 μM) [16].

HBP23 is highly conserved to an antioxidant enzyme of the peroxiredoxin (Prx) family, in which the Cys in the CP motif constitutes the active center for reduction of hydrogen peroxide (H2O2) (Fig. S1). Members of the Prx (EC 1.11.1.15) family are ubiquitous peroxidases found in almost all kingdoms [17]. The active center of Prx proteins consists of two Cys residues, and one Cys residue is reactive with H2O2; thus, members of the Prx family are termed cysteine-dependent peroxidases to distinguish them from heme peroxidases such as horseradish peroxidase [18]. Prx1 is classified as a ‘2-Cys’ Prx, whose two conserved cysteines are a hallmark of its peroxidase activity. 2-Cys Prx proteins contain an N-terminal peroxidatic Cys (CysP-SH) and a C-terminal resolving Cys (CysR-SH), both of which are contributed by CP motifs. CysP-SH is oxidized by H2O2 to cysteine sulfenic acid (CysP-SOH), and then forms an intermolecular disulfide bond in a head to tail manner with CysR-SH from an adjacent monomer. Under physiological conditions, the disulfide linkage is reduced by NADPH-dependent thioredoxin and thioredoxin reductase to regenerate CysP-SH [19], [20]. To the best of our knowledge, there are no other proteins in which the cysteine in the active center of enzymes also forms a CP motif, leading us to hypothesize that heme binding to Prx1 affects Prx1 cysteine-dependent peroxidase activity. However, the involvement of heme binding in the cysteine-dependent peroxidase activity of Prx1 remains to be elucidated.

Here, we report the purification and characterization of human PRX1, which shares 97% amino acid identity with rat HBP23 (Fig. S1). Purified PRX1 bound to heme with a stoichiometry of 1:1 and exhibited a Kd,heme of heme binding of 0.17 μM. A mutational study showed that CysP-SH, donated by one of the CP motifs, bound heme, leading to the loss of cysteine-dependent peroxidase activity. However, hemin peroxidase activity and H2O2-mediated hemin degradation of heme-PRX1 were significantly reduced compared with free hemin, properties that are beneficial for cells. Taken together, our data suggest that PRX1 acts as a “shelter” for free hemin that prevents the undesirable peroxidation of biomolecules, but at the cost of diminished cysteine-dependent peroxidase activity.

Section snippets

Materials

All chemicals were purchased from Wako Pure Chemical Industries (Japan), Nacalai Tesque (Japan) and Sigma-Aldrich (USA), and were used without further purification.

Protein expression and purification

A full-length PRX1 gene construct, codon optimized for E. coli expression, was purchased from Eurofins Genomics (Japan) and amplified by polymerase chain reaction. The amplified fragment was cloned into the modified pET-28b vector [21] (Merck Millipore, Germany) using a Gibson Assembly kit (New England Biolabs, UK). The PRX1

Expression and purification of PRX1

Human PRX1 was expressed in E. coli strain BL21(DE3) and purified. The purified PRX1 protein had an apparent molecular mass of 22 kDa and was estimated to be ∼95% pure by SDS-PAGE (Fig. S2A). Three major peaks on the size-exclusion chromatogram, with elution times of 54.8, 76.7 and 88.0 min corresponded to a decamer, dimer and monomer, respectively, based on molecular masses estimated from the migration of bands against standard proteins (Fig. S2B). Molecular mass of the fraction eluted at

Heme coordination environment of PRX1

Although HBP23 is frequently mentioned as a candidate of cytosolic heme-binding proteins, knowledge of its heme-binding site and the functional significance of its heme binding have been limited, motivating our characterization of the human homolog, PRX1. Heme titration experiments showed that PRX1 binds 1 equivalent of heme (Fig. 1A) and the Soret band for ferric heme-PRX1 strongly suggested Cys-coordinated environment (Fig. 1B). Indeed, a mutational study demonstrated that Cys52 is

Conflict-of-interest

There is no conflict of Interest.

Acknowledgement

This study was supported in part by Grants-in-Aid for Scientific Research (16K05835 to T.U., and 15H00909 to K.I.) and the Sasakawa Scientific Research Grant (to Y.W.) from The Japan Science Society.

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