Abstract
Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions.
Highlights
Peroxynitrite (ONOOH/ONOO−, pKa = 6.9) is an oxidant formed by a diffusion-controlled reaction of nitric oxide (NO ) with superoxide anion (O2 − ) radicals (Equation (1), k1 = 1.9 × 1010
Thiol peroxidases (TP) members reduce peroxynitrite with high rate constants via a conserved peroxidatic peroxynitrite with high rate constants via a conserved cysteine (CP ), which is oxidized to sulfenic acid in the process (Scheme 1, step 1)
We showed that coumarin boronic acid (CBA) oxidation in ∆8 strain exposed to fluxes of peroxynitrite decreases with increasing cell densities (Figure 2) and attenuates in glycerol adapted ∆8 cells when compared to ∆8 control cells (Figure 6B)
Summary
Peroxynitrite (ONOOH/ONOO− , pKa = 6.9) is an oxidant formed by a diffusion-controlled reaction of nitric oxide (NO• ) with superoxide anion (O2 •− ) radicals (Equation (1), k1 = 1.9 × 1010M−1 s−1 ) [1]. Peroxynitrite formation under normal conditions is minimized by keeping the NO•and O2 •− concentrations low, but probably increases in episodes of infection and inflammation [2].Upon protonation or reaction with CO2 , peroxynitrite may generate other aggressive species, such as Antioxidants 2020, 9, 434; doi:10.3390/antiox9050434 www.mdpi.com/journal/antioxidantsAntioxidants 2020, 9, 434 hidroxyl (OH• ), nitrogen dioxide (NO2 • ), and carbonate anion (CO3 •− ) radicals (Equations (2) and (3)) [3,4]. Peroxynitrite and its downstream radical products potentially oxidize and damage many biological targets and may be involved in the onset of multiple pathophysiological conditions [5].NO• + O2 •− → ONOO− (1)ONOO− + H+ → 0.3(NO2 • + OH• ) + 0.7(NO3 − + H+ ) (2)ONOO− + CO2 → ONOOCO2 − → 0.35(NO2 • + CO3 •− ) + 0.65(NO3 − + CO2 ) (3)
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