Biophysical tools to study the oligomerization dynamics of Prx1-class peroxiredoxins

Abstract

Peroxiredoxins (Prx) are ubiquitous, highly conserved peroxidases whose activity depends on catalytic cysteine residues. The Prx1-class of the peroxiredoxin family, also called typical 2-Cys Prx, organize as head-to-tail homodimers containing two active sites. The peroxidatic cysteine C_P of one monomer reacts with the peroxide substrate to form sulfenic acid that reacts with the resolving cysteine (C_R) of the adjacent subunit to form an intermolecular disulfide, that is reduced back by the thioredoxin/thioredoxin reductase/NADPH system. Although the minimal catalytic unit is the dimer, these Prx oligomerize into (do)decamers. In addition, these ring-shaped decamers can pile-up into high molecular weight structures. Prx not only display peroxidase activity reducing H₂O₂, peroxynitrous acid and lipid hydroperoxides (antioxidant enzymes), but also exhibit holdase activity protecting other proteins from unfolding (molecular chaperones). Highly relevant is their participation in redox cellular signaling that is currently under active investigation. The different activities attributed to Prx are strongly ligated to their quaternary structure. In this review, we will describe different biophysical approaches used to characterize the oligomerization dynamics of Prx that include the classical size-exclusion chromatography, analytical ultracentrifugation, calorimetry, and also fluorescence anisotropy and lifetime measurements, as well as mass photometry.

Keywords: AUC; Anisotropy; Lifetime fluorescence; Oligomerization; Peroxiredoxins; Phasors; Quaternary structure; SEC.

Fig. 1

Catalytic cycle of Prx1-class peroxiredoxins. Steps 1 to 4 represent the peroxidase cycle. Step 1, oxidation of C_P by the peroxide substrate. Step 2 represents the fully-folded FF to locally unfolded LU conformational transition. Step 3, intermolecular disulfide formation. Step 4, reduction by the thioredoxin Trx, thioredoxin reductase TR, NADPH system. H and H’ depict the two steps of C_P hyperoxidation, while R indicates the rescue of C_P-SO₂⁻ by sulfiredoxin (Srx), back to the peroxidase cycle

Fig. 2

Oligomerization equilibria of Prx1-class peroxiredoxins. The redox state of the active site cysteines is represented above each oligomeric form. For the hyperoxidized high-molecular-weight HMW, only the redox state of the C_P was represented. Besides the redox state of the reactive cysteines, the oligomeric state can be affected by pH, ionic strength (µ) and temperature (T), among other factors that are discussed in the text. The representations were built using PDB:7KIZ as the starting structure

Fig. 3

Intrinsic fluorescence of hPrx1. A Normalized emission spectra of 20 µM (blue, mostly decamer) and 0.5 µM (orange, mostly dimer) reduced hPrx1 (λex 295 nm). The spectral center of mass (C_M) of each spectrum is indicated. B Lorentzian distributions obtained from the fitting of the emission lifetimes of hPrx1 20 µM (blue, decamer) and 0.5 µM (orange, dimer) obtained by frequency-domain fluorometry (LED 295 nm) (Villar et al. 2022)

Fig. 4

Study of hPrx1 decamer-dimer equilibrium using the fluorescence lifetime phasor analysis. A Phasor plot for three concentration points (0.5, 1, 80 µM) of reduced hPrx1. The universal circle is delimited in red, the line segment between the dimer and decamer regions at 0.5 and 80 µM hPrx1 (confirmed by SEC) is represented in black. B Dissociation curve plotted as the fraction of hPrx1 decamer vs. total protein concentration. Both figures are adapted from (Villar et al. 2022)