Biochemical and Biophysical Research Communications
Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding
Graphical abstract
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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