Prof. Rafael Radi (PAS), Departamento de Bioquímica, Facultad de Medicina and Director of Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

Introduction

Carbon dioxide is a metabolic by-product produced by humans. One person produces approximately 1 kg of carbon dioxide (CO₂) per day (1) much of which is exhaled to the environment through respiration. Physiological levels of CO₂ are associated to the control of pH and are tightly regulated by a series of homeostatic local and systemic mechanisms. Conditions leading to excess production or exposure to CO₂ lead to toxic events and trigger pathological responses. In the context of the “Anthropocene” is important to consider that humans are exposed to higher inner and outdoor CO₂ levels than in any previous time in history and that is becoming evident that increased atmospheric CO₂ creates direct human health risks (2). In this context, I will analyze how CO₂ influences peroxide metabolism in mammalian cells (3); specifically, CO₂ enhances the chemical reactivity of biologically-relevant peroxides such as hydrogen peroxide (H₂O₂) and peroxynitrite (ONOO-). Through its reactions with peroxides, CO₂ favors biological oxidations with consequences in redox signaling, inflammation and oxidative damage. Revealing the reactions of CO₂ with peroxides in mammalian systems help to further understand how CO₂ can serve as a biological signaling molecule, stressor, or toxicant.

CO₂: biochemical and physiological aspects of its endogenous production

Mammalian cells continuously produce CO₂ (3-5). This production is mainly connected to the aerobic oxidation of nutrients such as sugars and lipids. Most of the metabolic CO₂ formation is humans occurs in mitochondria, the key organelles responsible of bioenergy generation and cellular respiration (3). Through a series of “oxidative decarboxylation reactions” on a- and b-ketoacids including pyruvate, isocitrate and  a-ketoglutarate, specific mitochondrial dehydrogenases which enzymatically catalyze the decarboxylation process in metabolic intermediates coupled to the reduction of the electron carrier NAD⁺ which evolves to NADH (Figure 1). In turn, NADH fuels electrons to the mitochondrial electron transport chain which, in healthy mitochondria, is coupled to molecular oxygen (O₂) consumption and the generation of ATP, the latter serving as a key bioenergy intermediate in mammalian metabolism. Thus, the formation of CO₂ is a continuous process and is metabolically located at the crossroads of the redox-energy axis.

The metabolic generation of CO₂ can be also achieved by metabolic processes that occur in other cellular compartments such as the cytosol (3). For example, a specific metabolic process known as the pentose phosphate pathway (PPP) carries out an oxidative decarboxylation reaction on the glucose-derived intermediate 6-phosphogluconate and leads to the reduction of NADP⁺ to NADPH (Figure 1). This latter compound plays important role on a variety of intracellular redox processes (e.g. fatty acid biosynthesis, peroxide catabolism, synthesis of DNA precursors). The metabolic formation of CO₂ also occurs in non-oxidative reactions but their contribution to overall CO₂ formation is minor. Importantly, some metabolic processes also involve the action of carboxylases that use as a substrate CO₂ (usually in the form of bicarbonate, HCO₃^-) to incorporate it into organic molecules (4). Overall, decarboxylation (oxidative and non-oxidative) and carboxylation reactions interconnect mammalian redox and energy metabolism as well as other anabolic and catabolic processes (1, 3, 4). Overall, there is a net (and large) release of CO₂ in mammalian tissues because CO₂ production normally largely exceeds CO₂ consumption (5), with mitochondria usually representing the major source of cellular CO₂ formation (3), in turn coupled to oxygen consumption (Figure 1).

Mitochondrial CO₂ concentrations usually reach values > 2 mM. Then, a concentration gradient is typically established with extramitochondrial sites, with CO₂ normally diffusing out from mitochondria to the cytosol and then, to the extracellular milieu[1].

CO₂/HCO₃^- dynamic equilibria in human physiology

Metabolically generated CO₂ can be hydrated to carbonic acid (H₂CO₃) and, in turn, H₂CO₃, a weak acid, is rapidly deprotonated ton bicarbonate (HCO₃^-) (3). Both are reversible reactions, so that CO₂ in solution is in equilibrium with HCO₃^- (Eq. 1 and 2):

CO2 + H2O		H2CO3                     [1]
H2CO3		H+ + HCO3−            [2]

Reaction 1 is largely accelerated in mammalian tissues by the action of the enzyme carbonic anhydrase which is an extremely efficient enzyme in the catalysis of the hydration reaction and therefore permits to rapidly approach equilibrium in vivo.

Importantly, dissolved carbon dioxide is in equilibrium with gaseous CO₂:

CO2 (aq)

CO2 (g)                        [3]

Thus, while the actual pKa for H₂CO₃ is ca. 3.5 (6), the apparent pKa for this acid-base system applicable to human physiology is ca. 6.1-6.4 (7) due to various participating equilibria and physiological systems. Thus, although continuous formation of CO₂ tends to acidify biological milieu (Eqs. 1 and 2) the CO₂/ HCO₃⁻ pair with the concerted action of physiological processes such as CO₂ exhalation at the lungs and renal management of HCO₃^-, constitutes an important element for the regulation of cellular and extracellular acid-base homeostasis (7).

Normal CO₂ concentrations in tissues is in the order of 1-2 mM. n plasma of healthy individuals, the CO₂ and HCO₃^- concentrations are ca. 1.3 mM and 24 mM at pH 7.4 and 37^oC, respectively. Significant deviations from physiological CO₂ and HCO₃^- levels[2] (e.g. < 1 mM or > 3 mM for CO₂ and <10 or > 30 mM for HCO₃^- require medical intervention.

Oxygen metabolism and peroxide formation in mammalian cells

Most O₂ consumed by mammalian cells is utilized as the final electron acceptor of the mitochondrial electron transport chain in the process of cell respiration. In this process, O₂ interacts at the terminal oxidase called cytochrome c oxidase which reduces O₂ in a four-electron process to H₂O at the expense of electrons transferred by cytochrome c, by (Eq. 4).

                         cytochrome c oxidase

4 Cyt c²⁺ + O_{2     ––––––––>}         4 Cyt c³⁺ + 2H₂O                                         [4]

 

Under physiological conditions more than 99.5% O₂ is consumed via this reaction in human cells.

Additionally, O₂ is also utilized as substrate in other redox reactions[3] where it serves as a one- or two-electron acceptor to yield superoxide radical (O₂^•–) or hydrogen peroxide (H₂O₂), respectively (8). A prime example of the former is the reaction catalyzed by membrane-bound NADPH oxidase (NOX) that generates O₂^•– in macrophages and neutrophils for oxidative killing (Eq. 5).

                                 NAPDH oxidase

NADPH + 2O_{2        ––––––––>}         NADP⁺+ 2O₂^•–                                                       [5]

An example of the second type of O₂-consuming reaction is the oxidative deamination of L-amino acids to the corresponding a-keto acids and the concomitant formation of ammonia and H₂O₂ (Eq. 6).

 

[6]

Notably, xanthine oxidase, the terminal enzyme of purine metabolism in humans catalyzes the O₂-dependent oxidation of hypoxanthine and xanthine to uric acid, concomitantly producing both O₂^•– and H₂O₂ at variable ratios depending on reaction conditions (Eq. 7) (8).

                        xanthine oxidase

xanthine + O_{2    ––––––––>}          uric acid + H₂O₂ (or 2O₂^•–)                        [7]

Other non-enzymatic processes may promote the one-electron reduction of O₂ to O₂^•–, including the autoxidation of quinones, reduced flavins, ascorbate and glutathione, and “electron leakage” at complexes of the mitochondrial electron transport chain, among other sources.

Once formed, both O₂^•– and H₂O₂ are unstable and transient intermediates in mammalian cells. For a start, O₂^•– readily dismutates to H₂O₂ by the near diffusion-controlled reaction catalyzed by (widely distributed) superoxide dismutases:

                     superoxide dismutase

2 O₂^•– + 2H⁺      _{––––––––>}           O₂ + H₂O₂                                               [8]

Alternatively, O₂^•– can react with a limited number of biomolecular targets. In this sense, one of the most important of such reactions is the combination of O₂^•– with nitric oxide (•NO)[4], a reaction that is at the diffusional limit and the only reported one that can really outcompete O₂^•– dismutation in vivo (8). This reaction decreases the bioavailability of •NO and at the same time leads to the formation of a cytotoxic nucleophile and oxidant, peroxynitrite anion (Eq. 9) (9).

O₂^.- + •NO -> ONOO^-                                                                                                                                                               [9]

As O₂^.- and •NO formation are ubiquitous in mammalian cells, their metabolism leads to a continuous biological flow of H₂O₂ and peroxynitrite,[5] even under relatively low physiological oxygen tensions (e.g. 2% O₂) (10). Homeostatic levels of H₂O₂ and peroxynitrite participate in redox signaling pathways (e.g. reversible oxidation/inactivation of protein tyrosine phosphatases) while excessive levels promote oxidative stress conditions that may ultimately cause biological oxidative damage (e.g. protein overoxidation, lipid peroxidation) (3). Peroxides are largely decomposed by strong peroxidatic systems (e.g. peroxiredoxins) (11) and therefore their steady-state concentrations are kept typically low and their toxic actions minimized; however, we now understand that their catabolism is in competition and/or interfered with their reactions with CO₂.

Reactions of CO₂ with H₂O₂ and ONOO^- and biological oxidations

Despite the mM concentration of CO₂ present in mammalian cells and tissues, it has been normally considered a relatively poor reactant in biological milieu, except for enzyme-catalyzed carboxylation reactions and the relatively slow reaction with neutral amines in amino acids and proteins. In the latter case, the reaction of CO₂ with N-terminal-amino or lysine ɛ-amino groups results in the formation of carbamates (3). This carbamoylation reaction it is well known in the case of hemoglobin (i.e. carbamino-Hb); other proteins carrying this reversible posttranslational modification are increasingly revealed (12).

With respect to the role of CO₂ on the chemical biology of redox reactions, there had been several hints in the literature (reviewed in Ref. 3). However, only recently CO₂ has become increasingly appreciated as redox modulator in human biology via H₂O₂ and ONOO^--dependent reactions. The influence of CO₂ ranges from redox signaling to oxidative damage and encompasses both one- and two-electron oxidation processes.

The interplay of CO₂ with H₂O₂ and ONOO^- (3) leads to the formation of CO₂-derived adducts (i.e. peroxymonocarbonate, HCO₄^-; nitrosoperoxocarboxylate, ONOOCO₂^-, Figure 2) which efficiently mediate oxidation reactions in vitro, in cellula and in vivo. Due to the transient nature of these reactive species (biological half-life typically in the ms to µs time scale, respectively), their detection is quite challenging, still in evolution, and requires either the use of probes or the detection of molecular footprints.

Current chemical and biological evidence support the idea that CO₂ enhances the oxidative potency of both H₂O₂ and ONOO^-, albeit via different molecular mechanisms (3).

For instance, HCO₄^- is a stronger two-electron oxidant than H₂O₂, typically oxidizing thiol groups at 100-1000 faster reaction rates (3). It is important to consider that the reaction of CO₂ with H₂O₂ is reversible and that under physiological conditions, only ca. 1% of total H₂O₂ would be present as HCO₄^-. However, as HCO₄^- is consumed by reacting with target molecules, a re-equilibration process continuously occurs and, under excess CO₂ levels, there will be a continuous flow of HCO₄^- from H₂O₂. In addition to participate in two-electron oxidations, HCO₄^- can also evolve to carbonate radicals (CO₃^•-) in the presence of oxidized transition metal centers (Figure 2), although the relevance of this reaction biology has not been yet demonstrated.

In the case of ONOO^-, the reaction with CO₂ is quite fast and leads to the formation of the rather unstable ONOOCO₂^- which immediately undergoes homolysis to nitrogen dioxide (•NO₂) and CO₃^•-. Both •NO₂ and CO₃•- are strong one-electron oxidants and initiate free radical processes in mammalian systems (13); in addition, •NO₂ mediates nitration reactions in biomolecules, most notably in protein tyrosine residues to yield 3-nitrotyrosine. Protein tyrosine nitration is a quantifiable oxidative post-translational modification evidenced in vivo associated to human disease and aging; CO₂ plays a key role on modulating the extent and site-specificity of peroxynitrite-dependent tyrosine nitration in proteins (13).

In summary, the chemical reactivity of CO₂ with H₂O₂ and ONOO^- can have profound influence on the biology of redox signaling and oxidative stress, much of which still needs to be revealed and characterized.

CO₂ reactions with peroxides in cellula and in vivo

Recent evidence supports that at least some of the redox signaling actions of H₂O₂ are in fact due to HCO₄^-. These new discoveries tend to reconcile the relatively poor direct chemical reactivity of H₂O₂ with H₂O_2-dependent redox signaling events that depend on protein thiol oxidation are difficult to envision if considering H₂O₂ as the proximal redox mediator. Indeed, recent elegant work on epidermal growth factor (EGF) stimulation of an adenocarcinoma cell line, a process known to use H₂O₂ as intracellular messenger, showed that increasing intracellular HCO₃^- concentrations enhanced total protein phosphotyrosine levels (14). The actions of EGF are initially mediated through receptor tyrosine kinase (RTK) activation which in turn triggers H₂O₂ production by membrane-bound NADPH oxidase and promotes PTP1B oxidation/inactivation. While it was previously established that the formation of H₂O₂ is a central requisite for protein tyrosine phosphate 1B (PTP1B) inactivation, the new work reveals that this type of H₂O₂-dependent redox signaling requires the presence of HCO₃^- (and likely through HCO₄^- formation) to mediate EGF-induced cellular oxidation of PTP1B.

Related observations were made in the case of a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), that contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation (15). We have shown H₂O₂-induced inactivation in vitro and in cellula was strongly enhanced in the presence of CO₂/HCO₃^- and inferred that HCO₄^- was playing a central role on GAPDH oxidation and inactivation. The reversible oxidative inactivation of GAPDH is being recognized as a “redox switch” that can facilitate redirection of glucose to the pentose phosphate pathways.

While the reaction of CO₂ with H₂O₂ to yield HCO₄^- and the oxidative properties of HCO₄^- have been well documented in the chemical and biochemical literature, its detection in biological systems has been mostly inferential. We have just provided the first documented evidence that a type of mammalian cell, activated macrophages, produce HCO4^- under biologically relevant concentrations of H₂O₂ and CO₂/HCO₃^- (16). The detection was possible due to the use of boronate-based probes that react with HCO₄^- and yield a stable (fluorescent) product that can be visualized by a variety of techniques. The comparative cytotoxic capacity of macrophage-derived HCO₄^- vs H₂O₂ to invading pathogens such as bacteria or intracellular parasites remains to be established. Preliminary data support that the presence of CO₂ potentiates H₂O₂-dependent oxidative killing, consistent with the higher reactivity of HCO₄^- (reviewed in Ref. 3).

The reaction of ONOO^- with CO₂ has been analyzed recently in the context of the mitochondrial metabolism peroxynitrite and the competition with mitochondrial peroxiredoxins in vitro, in cellula e in vivo (9, 17, 18). Mitochondrial peroxiredoxins are key enzymes in the catabolism of ONOO^- to NO₂^- (11). However, mitochondrial CO₂ kinetically competes well with peroxiredoxins (17), in which case •NO₂ and CO₃•- will be formed. Thus, among other factors, the fate, and actions of mitochondrial ONOO^- depends on the levels of CO₂, peroxiredoxins and associated reducing systems (e.g. thioredoxin, thioredoxin reductase; Ref. 18). The presence of CO₂ promotes mitochondrial protein nitration, peroxiredoxin inactivation and overoxidation and, depending on metabolic conditions, may lead to mitochondrial oxidative stress and mitochondrial dysfunction (19) in human disease and aging (for a recent analysis, see also Refs. 3 and 9). New evidence on the key role of peroxynitrite in mitochondrial dysfunction and disruption of cell physiology in vivo has been just reported (20).

Immunostimulated macrophages can yield ONOO- upon activation by invading pathogens. The presence of CO2 can promote pathogen oxidation and nitration inside the phagosome (reviewed in Ref. 21). The fast reaction of CO₂ with ONOO^- and the short half-life of ONOOCO₂^- determines that CO₂ focusses the reactivity of peroxynitrite in cellula and in vivo to a very narrow region within the mm distance scale. The putative role of CO₂ on HCO₄^- and ONOOCO₂^- formation and oxidative pathogen killing inside the macrophage phagosome is shown in Figure 3.

Other mechanisms by which CO₂ influences redox biology and inflammation

In addition to the direct actions of CO2 on peroxide reactivity, oxidative processes may be modulated by CO₂ due to its actions on the regulation of genes related to inflammation (22-24). A complex interaction exists between CO₂ signaling, NF-kB and NOS expression (3). Depending on the CO2 exposure protocol and the organism, both anti- and pro-inflammatory actions of CO2 have been observed (including changes on protein levels of target genes for NF-kB such as IL-6 and TNFα), although a definitive and mechanistic-based picture is yet to be provided. CO₂ exposure conditions reportedly leading to inflammation involve activation of the nucleotide-binding domain-like receptor 3 (NLRP3) inflammasome and elevated interleukin (IL)-1β production. Recent reports of the role of CO₂ signaling, transcriptional responses and inflammation have been communicated elsewhere (2, 22-24).

Exogenous CO2 exposure to human tissues: Perspectives for the Anthropocene

A recent report has analyzed the direct health effects of increasing atmospheric CO₂ levels (2). Environmentally relevant elevations in CO₂ (<5,000 ppm) may pose direct risks for human health. While most of the evolution of humans has been carried out under atmospheric CO₂ levels in the order of 250 ppm, recent increases in the 400-500 ppm range indicates that this could be of toxicological concern. Conditions leading to acute or chronic exposure to high CO₂ levels (extended indoor life and poor ventilation, populated cities, and excess combustion of fossil fuels, all of which can lead to exposures > 1000 ppm) are increasingly observed and its impact in various organs and physiological systems revealed. Shockingly, reports also indicate that HCO₃^- levels in human serum among the general US population appears be increasing (2, 25, 26) (still within the normal range) which may reflect increased CO₂ exposure to humans. Among other health issues, CO₂ may promote inflammation and oxidative stress pathways, some of which may depend on the reactions of CO₂ with peroxides.

Cities contribute substantially to anthropogenic CO₂ emissions. An aspect that is not particularly appreciated is that even the respiration of humans and livestock can represent a significant CO₂ source in some cities (27). In a context of increased of the world population to ca. 11 billion people by 2100, the metabolic-derived formation of CO₂ may not be considered negligible for city carbon budget analysis. This challenging idea must be taken into consideration in the context of the One Health concept.

Thus, from a medical and toxicological perspective, the environmentally relevant elevations in CO₂ may affect human health in common everyday activities: the molecular mechanisms may imply redox processes arising from the reactions of CO₂ with H₂O₂ and peroxynitrite. Importantly, both H₂O₂ and nitrogen oxides (NO_x) are present in urban air and their local concentrations in aerosols can be quite high (28-32). Thus, there is a possibility for corporal surfaces such as skin, mucous membranes, cornea, and respiratory epithelium to be exposed to environmental CO₂/ H₂O₂ and CO₂/peroxynitrite mixtures.

Future in cellula and in vivo experiments under well-controlled CO₂ and O₂ levels in conjunction with additional studies in humans (2) will provide new clues on how the CO₂/peroxide interplay participates in human physiology and disease.

 

Acknowledgements

I thank Drs. Lucía Piacenza and Natalia Ríos for their assistance with the artwork.

This work was supported by grants from Universidad de la República (EI_2020), The Richard Lounsbery Foundation and Programa de Alimentos y Salud Humana (PAyS) IDB-R.O.U. (4950/OC-UR). Additional funding was obtained from Programa de Desarrollo de Ciencias Básicas (PEDECIBA).

 

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[1] In liver and kidney mitochondria sometimes there is an uptake of CO₂ for carboxylation reactions carried out in mitochondria related to gluconeogenesis and ureogenesis (Ref. 3).

[2] Deviations of CO₂ levels in blood below or above the physiological range are defined as hypocapnia or hypercapnia, respectively.

[3] Molecular oxygen also participates in oxygenation reactions, namely the incorporation of oxygen to a biomolecule, but these reactions are out of the scope of this manuscript.

[4] Nitric oxide is a free radical and signal transducing agent that participates in a variety of physiological processes including vasodilation, neurotransmission, and immune responses. It is mostly generated by the enzymatic action of nitric oxide synthases (NOS) which use as substrates L-arginine, O₂ and NADPH.

[5] Peroxynitrite refers to the sum of peroxynitrite anion and peroxynitrous acid (ONOOH, pKa = 6.8).