RESUMO
Typical 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous Cys-based peroxidases, which are stable as decamers in the reduced state, and may dissociate into dimers upon disulfide bond formation. A peroxidatic Cys (CP) takes part of a catalytic triad, together with a Thr/Ser and an Arg. Previously, we described that the presence of Ser (instead of Thr) in the active site stabilizes yeast 2-Cys Prx as decamers. Here, we compared the hyperoxidation susceptibilities of yeast 2-Cys Prx. Notably, 2-Cys Prx containing Ser (named here Ser-Prx) were more resistant to hyperoxidation than enzymes containing Thr (Thr-Prx). In silico analysis revealed that Thr-Prx are more frequent in all domains of life, while Ser-Prx are more abundant in bacteria. As yeast 2-Cys Prx, bacterial Ser-Prx are more stable as decamers than Thr-Prx. However, bacterial Ser-Prx were only slightly more resistant to hyperoxidation than Thr-Prx. Furthermore, in all cases, organic hydroperoxide inhibited more the peroxidase activities of 2-Cys Prx than hydrogen peroxide. Moreover, bacterial Ser-Prx displayed increased thermal resistance and chaperone activity, which may be related with its enhanced stability as decamers compared to Thr-Prx. Therefore, the single substitution of Thr by Ser in the catalytic triad results in profound biochemical and structural differences in 2-Cys Prx.
RESUMO
2-Cys peroxiredoxins (Prxs) rapidly reduce H2O2, thereby acting as antioxidants and also as sensors and transmitters of H2O2 signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H2O2 levels. Under these conditions, H2O2 oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (CPSOH) to the sulfinate (CPSO2-) and sulfonated (CPSO3-) forms, redirecting the CPSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the CPSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H2O2 and was selected here to investigate the effect of a physiological concentration of HCO3-/CO2 (25 mm) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO3-/CO2 increases Prx1 hyperoxidation and inactivation both in the presence of excess H2O2 and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO3-/CO2 was due to HCO4-, a peroxide present in equilibrated solutions of H2O2 and HCO3-/CO2 Indeed, additional experiments and calculations uncovered that HCO4- oxidizes CPSOH to CPSO2- with a second-order rate constant 2 orders of magnitude higher than that of H2O2 ((1.5 ± 0.1) × 105 and (2.9 ± 0.2) × 103 m-1·s-1, respectively) and that HCO4- is 250 times more efficient than H2O2 at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO3-/CO2 pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity.
Assuntos
Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Sequência de Aminoácidos , Antioxidantes/química , Antioxidantes/metabolismo , Bicarbonatos/química , Bicarbonatos/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Catálise , Cisteína/química , Cisteína/metabolismo , Humanos , Cinética , NADP/química , NADP/metabolismo , Oxirredução , Peróxidos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108â¯M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.
Assuntos
Oxirredução , Peroxirredoxinas/genética , Processamento de Proteína Pós-Traducional/genética , Tirosina/genética , Domínio Catalítico/genética , Cisteína/genética , Dissulfetos/química , Humanos , Mutação/genética , Nitratos/metabolismo , Peroxidase/genética , Peróxidos/metabolismo , Peroxirredoxinas/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Peroxiredoxins are thiol-dependent peroxidases that function in peroxide detoxification and H2 O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H2 O2 and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H2 O2 than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H2 O2 with rate constants of ca 2 × 103 M-1 s-1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s-1 for PRDX1, 0.2 s-1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.