In addition to its role in global warming and its harmful health effects at high levels, carbon dioxide (CO₂) may have an important physiological function in mammalian organisms, hitherto little investigated. Continuously produced through respiration – we produce about 1.0 kg of CO₂ per day – it forms the bicarbonate/CO₂ pair, our main physiological buffer, responsible for maintaining blood and cellular pH between 7.0 and 7.4. But CO₂ is not an inert gas whose only physiological role is to control pH. In fact, the main physiological buffer is active in redox processes, influencing cellular signaling.

This is proposed by RIDC Redoxoma scientists, led by Professor Ohara Augusto from Instituto de Química at Universidade de São Paulo (USP), based on a study showing that the bicarbonate/carbon dioxide pair increases human peroxiredoxin 1 (Prx1) hyperoxidation, by a mechanism involving peroximonocarbonate, an oxidant formed by the reaction between hydrogen peroxide (H₂O₂) and carbon dioxide (CO₂). Hyperoxidized peroxiredoxins lose their antioxidant and redox transmitter function but may operate in other redox signaling pathways. The results of the work were published in the Journal of Biological Chemistry (JBC).

“Our results raise some very interesting questions involving the role of CO₂ in hydrogen peroxide mediated cell signaling and the versatility of peroxiredoxins. These enzymes have evolved to be highly efficient as antioxidants. However, especially in mammals, they are susceptible to hyperoxidation and thus able to assume other functions. The importance of our study is to clearly demonstrate the molecular mechanism by which bicarbonate/CO₂ increases this hyperoxidation and thus, show that CO₂ can influence cellular signaling. Redox mechanisms are the focus of RIDC Redoxoma”, Ohara Augusto said.

There are six human Prxs (Prx1 to Prx6), classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys), which vary in their intracellular localization and catalytic mechanisms. The 2-Cys Prx1 investigated in this study is found in the mammalian cells cytosol. The antioxidant function of peroxiredoxins in plants, animals, and bacteria is to break down peroxides, mainly hydrogen peroxide. As a redox transmitter, these enzymes, when oxidized by hydrogen peroxide, oxidize other proteins, thereby transmitting the signal and allowing other molecules to “feel” the peroxide.

Hydrogen peroxide (H₂O₂) is a powerful two-electron oxidant continuously produced in our body from electrons escaping the mitochondrial electron transport chain during respiration and by reactions catalyzed by several oxidases such as NADPH oxidases (NOX), xanthine oxidase (XO), and monoamine oxidase (MAO). Cells of the immune system, such as neutrophils, use H₂O₂ to produce hypochlorous acid and attack invading microorganisms, fighting infections. As a stable and diffusible molecule, H₂O₂ is considered a signaling molecule in cellular communication. Fine regulation of hydrogen peroxide levels by 2-Cys Prxs, considered sensors of this peroxide, may be related to processes such as tumor suppression, neuronal differentiation, and cardiovascular disease.

Interestingly, according to the researchers, the eukaryotic 2-Cys Prxs can be hyperoxidized at high levels of H₂O₂. Hyperoxidation is the process in which H₂O₂ oxidizes the sulfenic acid derivative of the Prx peroxidatic cysteine to the sulfinate and sulfonated forms. Prxs enzymes in these forms are not recycled by reducers, which inactivates their peroxidase and redox transmitter activity. Among other possible functions, hyperoxidized 2-Cys Prxs may potentiate signaling pathways that depend on H₂O₂-mediated oxidation of proteins that contain less reactive cysteines than peroxidase cysteine.

“Our data show that, in the presence of bicarbonate/CO₂, the deviation for peroxiredoxin hyperoxidation is greater than just in the presence of hydrogen peroxide. This led us to investigate what would be the action of CO₂ in this increase”, explained Professor Daniela Ramos Truzzi, also from the Instituto de Química at USP and a member of RIDC Redoxoma, first author of the article.

The bicarbonate/ CO₂ pair and oxidants production

Animals under high CO₂ stress have lung, cardiovascular and nervous system disorders. Increased levels of CO₂ also occur in clinical situations such as emphysema, respiratory muscle paralysis, and pulmonary fibrosis. These clinical conditions are generally associated with oxidative damage, but the relation between such damage and CO₂ levels was overlooked.

The picture began to change with the discovery of peroxynitrite, a potent nitric oxide-derived oxidant, which has CO₂ as one of its main biological targets. “This was the first clear demonstration that CO₂ could participate in oxidation reactions. My group has strongly contributed to show that the reaction between peroxynitrite and CO₂ generates the carbonate radical, which is the second most potent biological oxidant after the hydroxyl radical. With this, we found that CO₂ is responsible for the deleterious effects of peroxynitrite and therefore of nitric oxide”, Ohara Augusto explained. According to the researcher, this renewed the interest in the pathogenic roles of oxidants derived from the main physiological buffer, the bicarbonate/carbon dioxide pair.

“Concerning CO₂, we can draw an analogy with oxygen, which is a fundamental molecule for vital processes, yet it is toxic,” she says.

In 2007, Augusto's group published a review on oxidants derived from the bicarbonate buffer, which emphasized radical carbonate and peroximonocarbonate, an oxidant whose existence in equilibrium with H₂O₂ and the bicarbonate/CO₂ pair is known by the chemists since the 1980s, but which didn't arouse much interest in biology. The review helped to increase this interest, but the group focused on the deleterious effects of the bicarbonate/CO₂ pair. In 2016-2017, Augusto and Truzzi wrote a book chapter in which they emphasized the possible participation of peroximonocarbonate in biological oxidations, especially in the oxidation of protein cysteine ​​residues.

According to Truzzi, “This critical analysis of the literature, published in 2018, and the fact that H₂O₂ and CO₂, peroximonocarbonate precursors, are continuously produced in cells, led us to the now published study. We performed in vitro experiments using techniques such as immunoblotting, kinetics and mass spectrometry. And our data indicate that peroximonocarbonate is the intermediate that stimulates human Prx1 hyperoxidation.”

Ohara points out that in a recent article also published in JBC, New Zealand researcher Christine Winterbourn and colleagues showed in cells that the bicarbonate/CO₂ pair increases the inactivation of the phosphatase involved in EGF (epidermal growth factor) mediated signaling, and also proposed that would be by action of peroximonocarbonate. “I believe that the impact of these two papers published at almost the same time is to demonstrate that CO₂ influences redox signaling and that peroximonocarbonate is an important biological oxidant that needs further investigation,” she said.

The article The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1, by Daniela R. Truzzi, Fernando R. Coelho, Veronica Paviani, Simone V. Alves, Luis ES Netto, and Ohara Augusto, can be accessed here