Kinetic analysis of structural influences on the susceptibility of peroxiredoxins 2 and 3 to hyperoxidation

Abstract

Mammalian 2-cysteine peroxiredoxins (Prxs) are susceptible to hyperoxidation by excess H2O2. The cytoplasmic family member Prx2 hyperoxidizes more readily than mitochondrial Prx3 due to slower dimerization of the sulfenic acid (SpOH) intermediate. Four variant amino acids near the C-terminus have been shown to contribute to this difference. We have performed kinetic analysis of the relationship between hyperoxidation and disulfide formation, using whole-protein MS and comparing wild-type (WT) Prx2 and Prx3 with tail-swap mutants in which the four amino acids were reversed. These changes make Prx3 more sensitive and Prx2 less sensitive to hyperoxidation and accounted for ~70% of the difference between the two proteins. The tail swap mutant of Prx3 was also more susceptible when expressed in the mitochondria of HeLa cells. The hyperoxidized product at lower excesses of H2O2 was a semi-hyperoxidized dimer with one active site disulfide and the other a sulfinic acid. For Prx2, increasing the H2O2 concentration resulted in complete hyperoxidation. In contrast, only approximately half the Prx3 active sites underwent hyperoxidation and, even with high H2O2, the predominant product was the hyperoxidized dimer. Size exclusion chromatography (SEC) showed that the oligomeric forms of all redox states of Prx3 dissociated more readily into dimeric units than their Prx2 counterparts. Notably the species with one disulfide and one hyperoxidized active site was decameric for Prx2 and dimeric for Prx3. Reduction and re-oxidation of the hyperoxidized dimer of Prx3 produced hyperoxidized monomers, implying dissociation and rearrangement of the subunits of the functional homodimer.

Experimentally Dissecting the Origins of Peroxiredoxin Catalysis

“…Our work on StAhpC revealed that disulfide bond formation weakened the decamers and favored dissociation to dimers (Fig. 2C) (66), and this tendency is also seen for other Prx1-class enzymes (2,4,29,55). Furthermore, we showed through making decamerdestabilizing (T77I) or -stabilizing (T77V) mutations of StAhpC that decamerization strongly promotes peroxidase activity by enhancing the stability of the FF substrate-ready form (41), agreeing with computational analyses describing activating influences of adjacent subunits in tryparedoxin peroxidase, another Prx1-class enzyme (69).…”

Cellular Timekeeping: It’s Redox o’Clock

“…Interestingly, in vitro studies have revealed that PRDX2, which is located in the cytosol, is more sensitive to hyperoxidation than mitochondrial-located PRDX3 owing to the 10fold slower formation of a disulfide bond with its resolving cysteine (Haynes et al 2013;Poynton et al 2016). In addition to the intrinsic biochemical properties of PRDXs, the hyperoxidation is, in fact, dependent on a functional reducing system, which allows the enzymes to spend more time in the sulfenic acid form.…”

Mapping the phenotypic repertoire of the cytoplasmic 2-Cys peroxiredoxin – Thioredoxin system. 1. Understanding commonalities and differences among cell types

“…For simplicity, we consider each Prx's proximate C P -C R pair as an independent functional unit. The Tpx1 peroxiredoxin of Schizosaccharomyces pombe [19] and the human mitochondrial Prx3 [35] may violate this assumption. However, there is no evidence that the same happens extensively for other eukaryotic Prx in the cytoplasm [35] .…”

“…The Tpx1 peroxiredoxin of Schizosaccharomyces pombe [19] and the human mitochondrial Prx3 [35] may violate this assumption. However, there is no evidence that the same happens extensively for other eukaryotic Prx in the cytoplasm [35] . Further, even in the noted exceptions, the PTTRS shows qualitatively the same overall dynamics.…”