Hydrogen peroxide (H 2 O 2 ) is an important biological oxidant that can be formed both enzymatically and non-enzymatically, and several enzymatic systems are capable of rapidly decomposing it. During its short lifetime, H 2 O 2 can participate in cell signaling at low concentrations or induce irreparable molecular damage at high concentrations. The diffusion of H 2 O 2 in cells and tissues remains under debate, particularly regarding the role of cellular membranes as barriers to its diffusion and the mechanisms involved in its transport. Diffusion across lipid-only membranes occurs approximately five orders of magnitude more slowly than through an equivalent layer of water and can be influenced by lipid composition, temperature, and membrane fluidizers. This low permeability is attributed to the limited solubility of H 2 O 2 in the hydrophobic core of the lipid bilayer. The permeability of biological membranes to H 2 O 2 is within a similar range, with reported permeability coefficients between 2 × 10 -4 and 3 × 10 -3 cm s -1 . Given the similarity between H 2 O 2 and water, several aquaporins have been found to facilitate H 2 O 2 diffusion across cellular membranes. In humans, aquaporins 3 and 8 are considered the main facilitators of H 2 O 2 transport, while the role of aquaporin 1 remains controversial. Although membrane permeability to H 2 O 2 is relatively low and limits its transmembrane flux, diffusion remains rapid, and only a few cellular systems-most notably peroxiredoxins-can effectively compete with H 2 O 2 efflux. This review provides quantitative information on H 2 O 2 diffusion within cells and across cellular membranes and contrasts these processes with its predominant biochemical reactions.

Entamoeba histolytica, a unicellular parasite, has negligible glutathione levels and instead relies on cysteine as its primary intracellular thiol. It has a functional thioredoxin (TRX) system, which consists of four canonical TRXs and thioredoxin reductase (TRXR). We identified a coding sequence for a putative non-canonical TRX with a WCKDC redox-active motif (EhTRX212, Uniprot M2QBU7) within the E. histolytica HM-1:IMSS genome. We produced the recombinant protein and conducted its biochemical characterization. Steady-state kinetic assays revealed that EhTRX212 is not reduced by EhTRXR nor directly reduces protein disulfides. Instead, EhTRX212 catalyzes the reduction of cystine, S-nitrosocysteine, and cysteine-derived heterodisulfides through a coupled reaction with EhTRX8 (a canonical TRX) and EhTRXR. Complementary pre-steady-state kinetics, using stopped-flow methodology, showed that the reduction of cystine by EhTRX212 follows a biphasic temporal progression, consistent with a thiol-disulfide exchange mechanism. The first phase (nucleophilic cysteine attack on the cystine disulfide) followed second-order kinetics (k = 2.4 × 106 M-1 s-1), while the second phase (mixed disulfide resolution) followed first-order kinetics (k = 11 s-1). Chemical modifications, on the amino group of the cysteine in disulfide substrates, impaired reduction by EhTRX212. Finally, confocal microscopy and digitonin subcellular fractionation experiments localized EhTRX212 to the trophozoite cytoplasm. This study strongly supports the existence of a novel class of TRXs with high specificity for cystine reduction, expanding our understanding of TRX function in E. histolytica and suggesting important roles for these proteins in the redox metabolism of this parasitic pathogen.

Human peroxiredoxin 1 and peroxiredoxin 2 (HsPrx1 and HsPrx2), both cytosolic antioxidant enzymes share more than 90% sequence similitude, peroxide substrate specificity, reactivity, and an oligomeric ensemble of five homodimers forming a decamer. However, it is suggested that they serve different purposes in the cell. The question, whether the decamer-dimer equilibrium is relevant to the peroxidase activity and signaling functions has a long-standing history within the field, yet assessing its significance is still a challenge. We have studied the oligomerization dynamics of HsPrx1 and HsPrx2 in their dithiol and disulfide forms to find differences that could provide an explanation for their distinct functions. In this study, we performed analytic size exclusion chromatography (SEC) and fluorescence emission lifetime phasor analysis (FELPA) at different protein concentrations and quantified the relative fraction of the decamer species. We observed that reduced HsPrx2 forms stable decamers that do not fully dissociate, while HsPrx1 exhibits a highly cooperative transition from dimers to decamers with increasing concentration. Disulfide formation at the active site has a larger disruptive effect on the oligomerization equilibrium of HsPrx2 than that of HsPrx1. By performing kinetic measurements using FELPA, we observed that HsPrx2 goes from oxidized dimers to reduced decamers almost 20 times faster than HsPrx1 upon addition of DTT. Lastly, both SEC and FELPA results revealed that the mixture of reduced HsPrx1 and HsPrx2 yields hybrid decamers, that have not been looked for in vivo yet.

Three decades of research on the biochemistry of peroxynitrite (ONOOH/ONOO−) have established that this stealthy oxidant is formed in biological systems, and that its main targets are carbon dioxide (CO2), metalloproteins and thiols (RSH). Peroxynitrous acid reacts directly with thiols (precisely, with thiolates, RS−), forming sulfenic acids (RSOH). In addition, the free radicals derived from peroxynitrite, mainly carbonate radical (CO3•−) and nitrogen dioxide (NO2•) formed from the reaction of peroxynitrite anion with CO2, oxidize thiols to thiyl radicals (RS•). These two pathways are under kinetic competition. The primary products of thiol oxidation can follow different decay routes; sulfenic acids usually react with other thiols forming disulfides, while thiyl radicals can react with oxygen, with other thiols and with other reductants such as ascorbic acid. Peroxynitrite is also able to oxidize hydrogen sulfide (H2S/HS−) and persulfides (RSSH/RSS−). Among the different biological thiols, peroxiredoxins stand out as main peroxynitrite reductases due to their very high rate constants of reaction with peroxynitrite together with their abundance. Rooted in kinetic concepts, evidence is emerging for the role of peroxiredoxins in peroxynitrite detoxification, with potential implications in diseases in which peroxynitrite is involved.

In this work, the structural, solution, electrochemical, and catalytic properties of the complexes with ligands derived from imidazole and pyridines were studied. A comparative study of five bioinspired copper catalysts with or without coordinated imidazole and with different chelate ring sizes is presented. Catalytic efficiency on the oxidation of 3,5-di-tert-butylcatechol (DTBC) and ortho-aminophenol (OAP) in a MeOH/H2O medium was assessed by means of the Michaelis-Menten model. Catalysts comprising imidazole-containing ligands and/or a six-membered chelate ring proved to be more efficient in both oxidation reactions. Determination of stability constants and electrochemical parameters of the copper complexes supported the explanation of the catalytic behavior. A catalytic cycle similar for both reactions has been proposed. The results of density functional theory (DFT) free energy calculations for all five complexes and both catalytic reactions agree with the experimental results.

We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped‐flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105–106 m−1 s−1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN) in the Trx is greatly affected by the formation of the Prx–Trx complex. The protein–protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol‐disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN. This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

Peroxiredoxins (Prx), thiol-dependent peroxidases, were first identified as H2O2 detoxifiers, and more recently as H2O2 sensors, intermediates in redox-signaling pathways, meta-bolism modulators, and chaperones. The multifaceted nature of Prx is not only dependent on their peroxidase activity but also strongly associated with specific protein-protein interactions that are being identified, and where the Prx oligomerization dynamics plays a role. Their oxidation by a peroxide substrate forms a sulfenic acid that opens a route to channel the redox signal to diverse protein targets. Recent research underscores the importance of different Prx isoforms in the cellular processes behind disease development with potential therapeutic applications.

The acidity of small alkylthiols depends mainly on the inductive effect of substituents, and the ionization state of neighbor functional groups. Protein thiols have a wide range of pKas, which depend, qualitatively, on the electrostatic environment, due to charged neighboring groups and α-helix macrodipoles and hydrogen bonds. The desolvation of the thiol/thiolate group also causes major alterations of the pKa. Nucleophilicity is the most important kinetic property of the thiolate catalytic cysteines in proteins, it is not related to pKa and depends on the regulation of the solvation as well as hydrogen bonding of protein thiolates. In general, aqueous environments greatly diminish the nucleophilicity of thiolates but the protein environment can delicately modulate it by tweaking its nonbonding and solvation interactions, and importantly enhancing its reactivity when the right substrate is bound and ready to react. This explains why protein thiolate reactivity is highly selective.

Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitations, meaning that at least two or more different approaches are needed to characterize a case study. Herein, we present a new method to study protein associations using intrinsic fluorescence lifetime with phasors. In this case, the method is applied to determine the equilibrium dissociation constant (KD) of human peroxiredoxin 1 (hPrx1), an efficient cysteine-dependent peroxidase, that has a quaternary structure comprised of five head-to-tail homodimers non-covalently arranged in a decamer. The hPrx1 oligomeric state not only affects its activity but also its association with other proteins. The excited state lifetime of hPrx1 has distinct values at high and low concentrations, suggesting the presence of two different species. Phasor analysis of hPrx1 emission lifetime allowed for the identification and quantification of hPrx1 decamers, dimers, and their mixture at diverse protein concentrations. Using phasor algebra, we calculated the fraction of hPrx1 decamers at different concentrations and obtained KD (1.1 × 10−24 M4) and C0.5 (1.36 μM) values for the decamer–dimer equilibrium. The results were validated and compared with size exclusion chromatography. In addition, spectral phasors provided similar results despite the small differences in emission spectra as a function of hPrx1 concentration. The phasor approach was shown to be a highly sensitive and quantitative method to assess protein oligomerization and an attractive addition to the biophysicist’s toolkit.

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.

We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped‐flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105–106 m−1 s−1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN) in the Trx is greatly affected by the formation of the Prx–Trx complex. The protein–protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol‐disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN. This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 +/- 0.08 x 10(3) M-1 s(-1) through a competition assay developed here, employing 2,6-dichlorophenolindophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4-2.2 x 10(3) M-1 s(-1) range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4-2.2 x 10(3) M-1 s(-1) range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The K-cat/K-M(Asc) was 7.4 +/- 0.07 x 10(3) M-1 s(-1), which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high.