All cells contain antioxidant systems to prevent chemical damage to their components. Among these systems, peroxiredoxins are considered the main detoxifying enzymes of peroxides such as hydrogen peroxide, an oxidant continuously produced in our body. To maintain the antioxidant activity, peroxiredoxins are recycled by reducing agents, usually thiols. Thiols (or molecules containing –SH groups) are sulfur-derived compounds central to redox physiology. However, ascorbate, or vitamin C (a compound that does not contain sulfur = S), can also recycle these enzymes, reducing sulfenic acid (R-SOH), which is the initial product of thiol oxidation by peroxides. Now, researchers have determined the rate constants of the reaction between ascorbate and sulfenic acid in several proteins, which is an important step to investigate the biological relevance of a thiol-independent redox pathway.

“Many central processes in biology, such as the synthesis of DNA precursors, involve redox reactions between sulfur-containing molecules, such as thiols (SH-R), sulfenic acids (SOH-R) and disulfides (R-SS-R). We are proposing a new mechanism by which ascorbate can interact with molecules that contain sulfur (S). This is another interface, by which sulfur-containing molecules can talk to other systems via ascorbate. Ascorbate in turn is involved in a number of other processes such as collagen synthesis. This reaction of ascorbate with sulfenic acid serves as a new route for redox metabolism”, said Professor Luis ES Netto, from the Instituto de Biociências at Universidade de São Paulo (USP) and member of the RIDC Redoxoma, coordinator of the research published in the journal Free Radical Biology and Medicine (FRBM).

Sulfenic acids are generated in the cysteine ​​residues of the peroxiredoxins active site. Peroxiredoxins are a family of antioxidant enzymes present in all living organisms. Mammalian cells express six forms of the enzyme, classified as 2-Cys and 1-Cys, based on the number of cysteine ​​residues that participate in catalysis. In both cases, the reactions of peroxiredoxins with peroxides generate sulfenic acid in the reactive cysteine (Figure 1). In 2-Cys peroxiredoxins, sulfenic acid forms a disulfide with another catalytic cysteine, which is then reduced by a biological thiol, thioredoxin, closing the catalytic cycle; the enzyme returns to its initial form and the cycle begins again. In the case of 1-Cys peroxiredoxins, as there is only one cysteine, sulfenic acid cannot form a disulfide. The 1-Cys peroxiredoxins are less studied and the identities of their biological reducers are not clear.

In 2007, Netto and his group showed that ascorbate reduced the peroxiredoxin 1-Cys sulfenic acid, challenging the existing paradigm that the antioxidant activity of these enzymes was strictly dependent on thiols. This work was published in PNAS and had great repercussions, as it revealed a new function of vitamin C. The researcher always said, however, that it would be required to determine the rate of this process to assess its biological relevance. “The current work is an unfolding of the 2007 study. We now have seen that this reaction proceeds at moderate rates. This indicates that the reduction of protein sulfenic acids is relevant in biological compartments where ascorbate concentration is high.”

According to Netto, evidence of the importance of this reaction in vivo was provided by an investigation employing a mutant mice that developed a phenotype similar to scurvy, which is a disease caused by vitamin C deprivation, leading to bleeding, altered gums and decreased resistance to infections. According to the authors, these animals had scurvy by a different mechanism. Their cells had a high concentration of sulfenic acid that consumed ascorbate, causing the disease. This is also an example that the reduction of sulfenic acids by ascorbate can also occur in proteins other than 1-Cys peroxiredoxins.

To determine rate constants between ascorbate and protein sulfenic acids, the researchers conducted a competition assay using a compound called DCPIP (dichlorophenolindophenol) as competing oxidant. The method was developed by the researcher Gerardo Ferrer-Sueta, from the Universidad de La República, Montevideo, Uruguay, co-author of the article. The results obtained by Valesca Anschau, a former PhD student, indicated that the rate constants lie in the 0.4-2.2 × 10³ M^-1 s^-1 range.

Plants, fungi, and bacteria

This mechanism can also be of great importance in plants, fungi and bacteria. In humans, peroxiredoxins can be reduced by different systems that compete with each other, being difficult to attribute the relevance of ascorbate in the recycling of these enzymes. But in organisms such as plants, fungi and bacteria, reduction by ascorbate is the only mechanism demonstrated for recycling of these enzymes so far.

In plants, 1-Cys peroxiredoxins are located in the nucleus of the seed cells. In general, plants have very high levels of ascorbate, which has been implicated with seed development and germination. “We are studying plant organisms. We believe that 1-Cys peroxiredoxin and ascorbate have an important role in the germination of seeds”, Netto said.

In bacteria, 1-Cys peroxiredoxin is involved in virulence. “We published a work in collaboration with the group of professor Regina Baldini, in which we showed that if you the gene that encodes the peroxiredoxin from Pseudomonas aeruginosa is deleted, this bacteria is no longer virulent in mice,” said the researcher. And the only reductant demonstrate for this enzyme so far is ascorbate.

Likewise, pathogenic fungi of the Aspergillus genus have three 1-Cys peroxiredoxins and so far the only reductant proposed for them is ascorbate.

The article Reduction of sulfenic acids by ascorbate in proteins, connecting thiol-dependent to alternative redox pathways, by Valesca Anschau, Gerardo Ferrer-Sueta, Rogerio Luis Aleixo-Silva, Renata Bannitz Fernandes, Carlos A. Tairum, Celisa Caldana Costa Tonoli, Mario Tyago Murakami, Marcos Antonio de Oliveira and Luis Eduardo Soares Netto, can be accessed at: https://authors.elsevier.com/a/1bRIZ3AkHATbLM