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Interplay of carbon dioxide and peroxide metabolism in mammalian cells

  • Rafael Radi
    Correspondence
    For correspondence: Rafael Radi
    Affiliations
    Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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Open AccessPublished:August 09, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102358
      The carbon dioxide/bicarbonate (CO2/HCO3-) molecular pair is ubiquitous in mammalian cells and tissues, mainly as a result of oxidative decarboxylation reactions that occur during intermediary metabolism. CO2 is in rapid equilibrium with HCO3- via the hydration reaction catalyzed by carbonic anhydrases. Far from being an inert compound in redox biology, CO2 enhances or redirects the reactivity of peroxides, modulating the velocity, extent, and type of one- and two-electron oxidation reactions mediated by hydrogen peroxide (H2O2) and peroxynitrite (ONOO/ONOOH). Herein, we review the biochemical mechanisms by which CO2 engages in peroxide-dependent reactions, free radical production, redox signaling, and oxidative damage. First, we cover the metabolic formation of CO2 and its connection to peroxide formation and decomposition. Next, the reaction mechanisms, kinetics, and processes by which the CO2/peroxide interplay modulates mammalian cell redox biology are scrutinized in-depth. Importantly, CO2 also regulates gene expression related to redox and nitric oxide metabolism and as such influences oxidative and inflammatory processes. Accumulated biochemical evidence in vitro, in cellula, and in vivo unambiguously show that the CO2 and peroxide metabolic pathways are intertwined and together participate in key redox events in mammalian cells.

      Keywords

      Abbreviations:

      CA (carbonic anhydrase), NOS (nitric oxide synthase), NOX (NADPH oxidase), PPP (pentose phosphate pathway), PTP (protein tyrosine phosphatase)
      The carbon dioxide/bicarbonate (CO2/HCO3-) molecular pair is ubiquitous in mammalian cells and tissues, and its roles in key physicochemical properties, metabolic processes, and gene expression are increasingly recognized. Herein, we will specifically analyze how CO2 levels modulate peroxide-dependent reactions and as such influences redox signaling and oxidative damage (
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      • Takors R.
      CO2 - intrinsic product, essential substrate, and regulatory trigger of microbial and mammalian production processes.
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      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). Far from being an inert compound in redox biology, CO2 has been progressively shown to enhance or redirect the reactivity of peroxides, modulating the velocity, extent, and type of one- and two-electron oxidation reactions mediated by hydrogen peroxide (H2O2) and peroxynitrite
      Peroxynitrite refers to the sum of the anionic and acid forms, namely peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH), pKa = 6.8.
      1Peroxynitrite refers to the sum of the anionic and acid forms, namely peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH), pKa = 6.8.
      (
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      Carbon dioxide-catalyzed peroxynitrite reactivity – the resilience of the radical mechanism after two decades of research.
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      Peroxynitrite-induced luminol chemiluminescence.
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      • et al.
      Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades.
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      • et al.
      The biological buffer bicarbonate/CO 2 potentiates H 2O 2-mediated inactivation of protein tyrosine phosphatases.
      ). In this sense, key oxidative posttranslational modifications in proteins such as thiol oxidation and tyrosine nitration are strongly influenced by cellular CO2 levels (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Lymar S. v
      • Hurst J.K.
      Carbon dioxide: physiological catalyst for peroxynitrite-mediated cellular damage or cellular protectant?.
      ,
      • Winterbourn C.C.
      Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.
      ,
      • Peskin A.v.
      • Pace P.E.
      • Winterbourn C.C.
      Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO2.
      ,
      • Trindade D.F.
      • Cerchiaro G.
      • Augusto O.
      A role for peroxymonocarbonate in the stimulation of biothiol peroxidation by the bicarbonate/carbon dioxide pair.
      ,
      • Truzzi D.R.
      • Coelho F.R.
      • Paviani V.
      • Alves S.v.
      • Netto L.E.S.
      • Augusto O.
      The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1.
      ,
      • Radi R.
      • Denicola A.
      • Freeman B.A.
      Peroxynitrite reactions with carbon dioxide-bicarbonate.
      ). Mitochondria represent central sites of CO2 formation in mammalian cells via the oxidative decarboxylation reactions associated to the Krebs cycle (
      • Balboni E.
      • Lehninger A.L.
      Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.
      ). In the cytosol, the oxidative phase of the pentose phosphate pathway (PPP) contributes to substantial CO2 formation, many times coupled to the cellular need of NADPH for peroxide metabolism (
      • Britt E.C.
      • Lika J.
      • Giese M.A.
      • Schoen T.J.
      • Seim G.L.
      • Huang Z.
      • et al.
      Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.
      ,
      • Lambeth J.D.
      • Neish A.S.
      Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited.
      ). Once formed, CO2 in large part converts to and is in equilibrium with bicarbonate anion (HCO3-) via the (reversible) action of carbonic anhydrases (CAs) (
      • Geers C.
      • Gros G.
      Carbon dioxide transport and carbonic anhydrase in blood and muscle.
      ). CO2 levels and gradients across cellular compartments in mammalian cells can connect energy and peroxide metabolism and participate in the regulation of various intertwined cellular processes.
      Herein, the biochemical mechanisms by which CO2 engages on peroxide-dependent reactions and impacts on redox signaling and oxidative damage will be analyzed and summarized. The interactions of CO2 with biologically relevant peroxides produce a collection of reactive and short-lived one- and two-electron oxidants. For instance, the reaction of H2O2 with CO2 yields peroxymonocarbonate (HCO4-), a strong two-electron oxidant that accelerates H2O2 reactivity with key biotargets such as protein thiols. The reaction of peroxynitrite anion (ONOO-) with CO2 yields nitrosoperoxocarboxylate (ONOOCO2-) that rapidly decays into carbonate radical (CO3•-) and nitrogen dioxide (NO2), promoting one-electron oxidations and nitrations. The review will examine reaction mechanisms, kinetics, and processes by which the CO2/peroxide interplay controls mammalian cell redox biology. Moreover, the analysis will integrate the CO2-dependent regulation of gene expression related to redox and nitric oxide (NO) metabolism, which further influences oxidative and inflammatory processes.
      Detailed biochemical analysis of the CO2/peroxide interplay at the cellular and subcellular levels assists on data interpretation and refinement of experimental designs and methodologies to dissect molecular mechanisms of redox-dependent cell signaling and injury.

      CO2 at the crossroads of the energy-redox axis

      CO2 is constantly produced in aerobic mammalian cell metabolism as part of oxidation processes in biomolecules connected to cellular respiration and energy generation. For instance, mammalian mitochondria, which are central loci of aerobic hydrocarbon catabolism, are the main cellular sources of CO2 through the oxidative decarboxylation of α- and β-ketoacids. These metabolic intermediates include pyruvate, isocitrate, and α-ketoglutarate and are substrates for the enzymatic action of specific dehydrogenases. Indeed, the action of pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase results in the formation of CO2 in parallel with the two-electron reduction of NAD+ to NADH, with the latter serving as electron donor to complex I of the respiratory electron transport chain. In this way, oxidative decarboxylation reactions in mitochondria couple energy release from the catabolism of metabolic intermediates with respiratory activity, oxygen consumption, and ATP synthesis (Fig. 1).
      Figure thumbnail gr1
      Figure 1Decarboxylation reactions in redox pathways and relation with peroxide metabolism. Glycolysis yields pyruvate in the cytosol and enters mitochondria; in mitochondria pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase catalyze oxidative decarboxylation reactions that convert NAD+ to NADH and generate CO2. The reducing equivalents of NADH and FADH2 feed the mitochondrial electron transport chain in a process that finalizes with the four-electron reduction of molecular oxygen to water. In the course of electron transport there is small percentage leakage for the monovalent reduction of oxygen to O2- that in turn dismutates enzymatically to H2O2. Mitochondria can emit H2O2 to extramitochondrial compartments. In the cytosol, glucose can also follow the pentose phosphate pathway, which in its oxidative phase yields NADPH and CO2, the latter by the action of 6-phosphogluconate dehydrogenase. The reducing equivalents of NADPH can be used for a number of redox reactions, most notably in the context of this review for the formation of O2- (and subsequently H2O2) via the membrane bound NADPH oxidases (e.g., toward the phagosome), NO synthesis by NOS, and peroxide detoxification (in GSH- or thioredoxin-based peroxidatic systems). TCA cycle, tricarboxylic acid cycle.
      Metabolic CO2 generation connected to redox processes can also occur in the cytosol by the activation of the PPP; in fact, the oxidative decarboxylation of 6-phosphogluconate in the presence of NADP+ by the reaction catalyzed by 6-phosphogluconate dehydrogenase leads to the formation of ribulose 5-phosphate, CO2, and NADPH. The NADPH in turn can be used for a variety of metabolic process, most notably in the context of this review, providing the reducing equivalents needed to catabolize peroxides via the action of redox proteins and enzymes. In fact, the PPP (and therefore CO2 production) is largely accelerated under enhanced cellular oxidant formation or oxidative stress conditions (
      • Britt E.C.
      • Lika J.
      • Giese M.A.
      • Schoen T.J.
      • Seim G.L.
      • Huang Z.
      • et al.
      Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.
      ,
      • Piacenza L.
      • Irigoín F.
      • Alvarez M.N.
      • Peluffo G.
      • Taylor M.C.
      • Kelly J.M.
      • et al.
      Mitochondrial superoxide radicals mediate programmed cell death in trypanosoma cruzi: cytoprotective action of mitochondrial iron superoxide dismutase overexpression.
      ), representing an adaptive mechanism to cope with the excess amounts of, for example, H2O2 or peroxynitrite (
      • Winterbourn C.C.
      Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.
      ,
      • Zeida A.
      • Trujillo M.
      • Ferrer-Sueta G.
      • Denicola A.
      • Estrin D.A.
      • Radi R.
      Catalysis of peroxide reduction by fast reacting protein thiols.
      ). Notably, NADPH is also required to generate (1) superoxide radical (O2•-) and H2O2 by the NADPH oxidase protein family (NOX 1–5, DUOX 1–2) (
      • Lambeth J.D.
      • Neish A.S.
      Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited.
      ) and (2) NO, a precursor of peroxynitrite, by the nitric oxide synthases (NOS 1–3) (
      • Nathan C.
      Nitric oxide as a secretory product of mammalian cells.
      ). Thus, the simultaneous formation of CO2 and NADPH parallels peroxide metabolism (Fig. 1).
      CO2 metabolism also involves non-oxidative decarboxylation and carboxylation reactions; indeed, the action of decarboxylases that lead to CO2 release and carboxylases that incorporate CO2 (or bicarbonate, HCO3-) into organic molecules play central roles interconnecting catabolism, anabolism, and energy metabolism in mammalian cells (
      • Blombach B.
      • Takors R.
      CO2 - intrinsic product, essential substrate, and regulatory trigger of microbial and mammalian production processes.
      ,
      • Walsh C.T.
      Biologically generated carbon dioxide: nature’s versatile chemical strategies for carboxy lyases.
      ). However, in mammalian cells, the levels of metabolic CO2 production normally largely exceed CO2 consumption and, as a result, there is a net and significant CO2 evolution and release. Approximately, 1 kg CO2 per day is produced by one person (
      • Walsh C.T.
      Biologically generated carbon dioxide: nature’s versatile chemical strategies for carboxy lyases.
      ).
      It is well known that CO2 can promote modifications in proteins under physiological conditions by its combination with neutral amines to form carbamates (
      • Linthwaite V.L.
      • Janus J.M.
      • Brown A.P.
      • Wong-Pascua D.
      • O’Donoghue A.M.C.
      • Porter A.
      • et al.
      The identification of carbon dioxide mediated protein post-translational modifications.
      ). This posttranslational carbamylation reaction involves the nucleophilic attack by CO2 on N-terminal amino or lysine ɛ-amino groups (
      • Blake L.I.
      • Cann M.J.
      Carbon dioxide and the carbamate post-translational modification.
      ). It is now also established that CO2 also participates in oxidative posttranslational modifications reactions mediated by H2O2 and peroxynitrite; these processes require the intermediate formation of CO2-derived species such as HCO4- and ONOOCO2-, respectively, that modulate amino acid oxidation and nitration (
      • Augusto O.
      • Truzzi D.R.
      Carbon dioxide redox metabolites in oxidative eustress and oxidative distress.
      ,
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Zhou H.
      • Singh H.
      • Parsons Z.D.
      • Lewis S.M.
      • Bhattacharya S.
      • Seiner D.R.
      • et al.
      The biological buffer bicarbonate/CO 2 potentiates H 2O 2-mediated inactivation of protein tyrosine phosphatases.
      ).

      CO2-dependent formation of bicarbonate and connection with acid-base homeostasis

      Once formed, CO2 can be slowly hydrated to carbonic acid (H2CO3) and in turn H2CO3, a weak acid, is deprotonated to HCO3- (reviewed in (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ) and references therein). The hydration reaction is reversible and therefore CO2 in solution is in equilibrium with bicarbonate:
      CO2 + H2O ⇌ H2CO3 kf = 0.03 s−1; kr = 20 s−1
      [1]


      H2CO3 ⇌ H+ + HCO3(fast) Ka = 5.7 × 10−4
      [2]


      This route of equilibration is rather slow at neutral pH, but CA, which is widely distributed in mammalian tissues and microorganisms and extremely efficient enzymes (
      • Geers C.
      • Gros G.
      Carbon dioxide transport and carbonic anhydrase in blood and muscle.
      ,
      • Sly W.S.
      • Hu P.Y.
      Human carbonic anhydrases and carbonic anhydrase deficiencies.
      ,
      • Burghout P.
      • Cron L.E.
      • Gradstedt H.
      • Quintero B.
      • Simonetti E.
      • Bijlsma J.J.E.
      • et al.
      Carbonic anhydrase is essential for Streptococcus pneumoniae growth in environmental ambient air.
      ,
      • Campestre C.
      • de Luca V.
      • Carradori S.
      • Grande R.
      • Carginale V.
      • Scaloni A.
      • et al.
      Carbonic anhydrases: new perspectives on protein functional role and inhibition in helicobacter pylori.
      ), catalyze reaction [1] and helps the system approach equilibrium in vivo. To note, while CA is mostly cytosolic, some tissues such as liver contain mitochondrial isoforms (
      • Balboni E.
      • Lehninger A.L.
      Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.
      • Sly W.S.
      • Hu P.Y.
      Human carbonic anhydrases and carbonic anhydrase deficiencies.
      ,
      • Imtaiyaz Hassan M.
      • Shajee B.
      • Waheed A.
      • Ahmad F.
      • Sly W.S.
      Structure, function and applications of carbonic anhydrase isozymes.
      ).
      The CO2/HCO3 molecular pair influences cell and tissue pH, and conversely, pH influences the CO2/HCO3 equilibrium. Indeed, the following relationship among these parameters applies according to the Henderson–Hasselbalch equation (
      • Leader D.P.
      A method of introducing the physiological carbon dioxide-bicarbonate buffer system to medical students.
      ):
      pH=pKa+log[HCO3][CO2(aq)]
      (3)


      CO2 effectively acts as the weak acid in this system, and HCO3- is its conjugate base. Importantly, dissolved CO2 is in equilibrium with gaseous CO2:
      CO2 (aq) ⇌ CO2 (g)
      [4]


      The equilibrium constant for this reaction is defined by Henry's law (i.e., the amount of dissolved CO2 in a tissue or fluid is proportional to its partial pressure, PCO2).
      Reactions 1 and 2 can be combined for the CO2/HCO3- equilibrium in solution:
      H+ + HCO3 ⇌ CO2 (aq) + H2O
      [5]


      Kapp=[H+][HCO3][CO2(aq)]
      ([6])


      The apparent pKa for this acid-base system applicable in human physiology to be used in Equation 3 is ca. 6.1 to 6.4 (
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      • Pines D.
      • Ditkovich J.
      • Mukra T.
      • Miller Y.
      • Kiefer P.M.
      • Daschakraborty S.
      • et al.
      How acidic is carbonic acid?.
      ) and is a result of the various participating equilibria (Fig. 2, panel A).
      Figure thumbnail gr2
      Figure 2The carbon dioxide–bicarbonate equilibria in mammalian tissues. A, metabolic or environmental CO2 exposure results in dissolved CO2, which via CA-catalyzed hydration yields H2CO3, in equilibrium with HCO3. CO2 in solution equilibrates with CO2 gas. The apparent pKa of the overall CO2/HCO3- equilibria is 6.1 to 6.4. All the indicated processes are readily reversible. Modified from (
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ). B, mitochondrial metabolism produces large levels of CO2 than can either hydrate to H2CO3 (nonenzymatically or enzymatically depending on tissue) or diffuse out as a function of concentration gradient. Importantly, the pH of the matrix in active mitochondria is basic due to the pumping of H+ to the intermembrane space, which generates an electrochemical gradient across the inner membrane. This basicity facilitates more dissociation of H2CO3 to HCO3- than in other cellular and extracellular compartments under physiological conditions. CA, carbonic anhydrase.
      However, the actual pKa for H2CO3 has been recently reported as 3.5 (
      • Pines D.
      • Ditkovich J.
      • Mukra T.
      • Miller Y.
      • Kiefer P.M.
      • Daschakraborty S.
      • et al.
      How acidic is carbonic acid?.
      ).
      pKa = pKapp − log KD
      [7]


      where KD=[CO2]/[H2CO3]
      [8]


      The value of KD is not known exactly and has been a major reason for the difficulties in obtaining the exact value of Ka for Equation 2.
      Under physiologically relevant conditions, [CO2] in tissues ranges in the order of 1 to 2 mM in equilibrium with [HCO3-] (
      • Geers C.
      • Gros G.
      Carbon dioxide transport and carbonic anhydrase in blood and muscle.
      ,
      • Leader D.P.
      A method of introducing the physiological carbon dioxide-bicarbonate buffer system to medical students.
      ,
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ,
      • Kregenow D.A.
      • Swenson E.R.
      The lung and carbon dioxide: implications for permissive and therapeutic hypercapnia.
      ,
      • Arthurs G.J.
      • Sudhakar M.
      Carbon dioxide transport.
      ,
      • Tucker A.M.
      • Johnson T.N.
      Acid-base disorders: a primer for clinicians.
      ,
      • Berend K.
      • de Vries A.P.J.
      • Gans R.O.B.
      Physiological approach to assessment of acid–base disturbances.
      ); the concentration of latter will ultimately depend on the local pH. For instance, in plasma at pH 7.4 and 37 °C the [CO2] and [HCO3-] are ca. 1.3 mM and 24 mM, respectively. While the [CO2]/[HCO3-] ratio is close to 1/20 at pH 7.4; this value changes in cell/tissue compartments having different pH values (e.g., cytosol ca. 7.0, mitochondrial matrix ca. 7.8–8.0, Golgi apparatus ca. 6.6) (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Llopis J.
      • Mccaffery J.M.
      • Miyawaki A.
      • Farquhar M.G.
      • Tsien R.Y.
      Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins.
      ). The high concentration of HCO3- in equilibrium with its conjugated acid (H2CO3/CO2 (aq)) plays a central role as a physiological buffer system in human biology (
      • Leader D.P.
      A method of introducing the physiological carbon dioxide-bicarbonate buffer system to medical students.
      ,
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ). Thus, acting as a homeostatic pH control mechanism, changes in tissue CO2 or H+ levels influences the equilibrium in Equation 5. Acid-base disorders (acidosis or alkalosis) result in a primary change in the arterial PCO2 (“respiratory” origin) or HCO3- concentration (“metabolic” origin) (
      • Tucker A.M.
      • Johnson T.N.
      Acid-base disorders: a primer for clinicians.
      ,
      • Berend K.
      • de Vries A.P.J.
      • Gans R.O.B.
      Physiological approach to assessment of acid–base disturbances.
      ). For example, excess metabolic formation of organic acids (e.g., lactate via anaerobic glycolysis) drives the equation to the right, consuming HCO3- and generating CO2 (
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ,
      • Tucker A.M.
      • Johnson T.N.
      Acid-base disorders: a primer for clinicians.
      ,
      • Berend K.
      • de Vries A.P.J.
      • Gans R.O.B.
      Physiological approach to assessment of acid–base disturbances.
      ). Deviations from physiological human arterial plasma CO2 and HCO3- concentrations in clinical conditions range from 1 mM to 3 mM for CO2 and 10 to 30 mM HCO3- (
      • Tucker A.M.
      • Johnson T.N.
      Acid-base disorders: a primer for clinicians.
      ,
      • Berend K.
      • de Vries A.P.J.
      • Gans R.O.B.
      Physiological approach to assessment of acid–base disturbances.
      ,
      • Jung B.
      • Rimmele T.
      • le Goff C.
      • Chanques G.
      • Corne P.
      • Jonquet O.
      • et al.
      Severe metabolic or mixed acidemia on intensive care unit admission: incidence, prognosis and administration of buffer therapy. A prospective, multiple-center study.
      ,
      • Kraut J.A.
      • Madias N.E.
      Metabolic acidosis: pathophysiology, diagnosis and management.
      ) and require medical intervention (
      • Quade B.N.
      • Parker M.D.
      • Occhipinti R.
      The therapeutic importance of acid-base balance.
      ). The relationship among CO2/HCO3- levels, cell/tissue pH, physiological acid-base regulatory mechanisms, and their disruption in disease conditions has been reviewed elsewhere (
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ,
      • Tucker A.M.
      • Johnson T.N.
      Acid-base disorders: a primer for clinicians.
      ,
      • Berend K.
      • de Vries A.P.J.
      • Gans R.O.B.
      Physiological approach to assessment of acid–base disturbances.
      ).
      Importantly, CO2 permeates across membranes as a function of concentration gradients and associated to the dynamics of formation and consumption in different compartments (
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ). In the case of mitochondria, they are usually the main sources of CO2 in mammalian cells under most metabolic conditions, with a net outflux of CO2 through membrane permeation (
      • Balboni E.
      • Lehninger A.L.
      Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.
      ,
      • Geers C.
      • Gros G.
      Carbon dioxide transport and carbonic anhydrase in blood and muscle.
      ); thus, the mitochondrial [CO2] is usually higher than cytosolic [CO2], establishing a CO2 concentration gradient (Fig. 2B).

      CO2 dynamics in mitochondria and beyond

      In a metabolically active mitochondria with a matrix pH of 7.8 to 8.0 (
      • Llopis J.
      • Mccaffery J.M.
      • Miyawaki A.
      • Farquhar M.G.
      • Tsien R.Y.
      Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins.
      ,
      • Matsuyama S.
      • Llopis J.
      • Deveraux Q.L.
      • Tsien R.Y.
      • Reed J.C.
      Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis.
      ), [CO2] can reach values ≥2 mM (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Balboni E.
      • Lehninger A.L.
      Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.
      ); the levels of mitochondrial [HCO3-] could be well above 100 mM (calculated from Equation 3), especially on those cell types where CO2 hydration is rapidly catalyzed by mitochondrial isoforms of CA (i.e., liver) (Fig. 2B). Otherwise, CO2 permeation from mitochondria to the cytosol outcompetes the nonenzymatic hydration (
      • Balboni E.
      • Lehninger A.L.
      Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.
      ). In contrast to CO2, HCO3 is not permeable across lipid bilayers, and therefore in mammalian cells, its transport across membranes relies on the existence of bicarbonate transporters (
      • Alka K.
      • Casey J.R.
      Bicarbonate transport in health and disease.
      ).
      Overall, the mitochondrial [CO2] could have fluctuations depending on the metabolic commitment to oxidative decarboxylation reactions, local pH changes
      In state 3 mitochondria, the pH value of the intermembrane space and matrix are ca. 6.8 and 7.8, respectively, due to proton pumping associated to the electron transport chain.
      , mitochondrial CA activity, and CO2 consumption via HCO3-dependent carboxylation reactions of the urea cycle (i.e., catalyzed by carbamoyl phosphate synthetase I) and gluconeogenesis (i.e., catalyzed by pyruvate carboxylase) (Fig. 3).
      Figure thumbnail gr3
      Figure 3Carbon dioxide and bicarbonate dynamics in mitochondria. Mitochondria are key cellular sources of CO2 via oxidative decarboxylation reactions linked to aerobic energy metabolism. In the case of mammalian liver mitochondria, they can be major consumers of HCO3- during ureogenesis or gluconeogenesis. Indeed, the ATP-dependent reactions catalyzed by carbamoyl phosphate synthase (CPSI) and pyruvate carboxylase (PC), respectively, use as substrate HCO3. Under appropriate metabolic requirement liver mitochondria are equipped with considerable CA activity to convert CO2 to HCO3-, which otherwise diffuses out of mitochondria. Moreover, if needed, cytosolic CO2 can diffuse into the mitochondria (
      • Balboni E.
      • Lehninger A.L.
      Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.
      ). CA, carbonic anhydrase.
      As mitochondria also constitute main intracellular sources of H2O2 in redox-dependent processes (
      • Anderson E.J.
      • Lustig M.E.
      • Boyle K.E.
      • Woodlief T.L.
      • Kane D.A.
      • Lin C. te
      • et al.
      Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans.
      ), its formation and emission in parallel with that of CO2 lays the ground for their synergistic interactions. In addition, it has been recently shown that enhanced mitochondrial-derived H2O2 release to the cytosol leads to activation of the PPP in mammalian cells (
      • Hambardikar V.
      • Guitart-Mampel M.
      • Scoma E.R.
      • Urquiza P.
      • Nagana G.G.A.
      • Raftery D.
      • et al.
      Enzymatic depletion of mitochondrial inorganic polyphosphate (polyP) increases the generation of reactive oxygen species (ROS) and the activity of the pentose phosphate pathway (PPP) in mammalian cells.
      ) (Fig. 1).

      Early indications of CO2 in the modulation of redox processes

      While CO2 has been classically considered as an almost unreactive product of mammalian cell redox metabolism, evidence laboriously accumulated over several decades substantiates that both CO2 and HCO3- participate in the modulation of free radical and peroxide-mediated reactions. Thus, a recapitulation of key early discoveries connecting CO2/HCO3- with oxygen free radicals and peroxide biochemistry will be provided first.

      Radiation chemistry data: Kinetics of the reaction of OH with HCO3- and the detection of carbonate radicals

      The modulatory action of the CO2/HCO3˗ in free radical and redox processes was originally hinted in the 1960s by radiation chemistry experiments. Indeed, the first observation of the carbonate radical by pulse radiolysis of aqueous solutions was in 1962, by Hart and Boag, who observed a composite spectrum of CO3•– and the hydrated electron (e-aq) after pulse radiolysis of deaerated 0.5 M sodium carbonate, with only the carbonate radical spectrum seen in aerated solution since oxygen removes the hydrated electron (
      • Hart E.J.
      • Boag J.W.
      Absorption spectrum of the hydrated electron in water and in aqueous solutions.
      ).The extinction coefficient of the carbonate radical at 600 nm is 1860 M–1 cm–1 (
      • Neta P.
      • Huie R.E.
      • Ross A.B.
      Rate constants for reactions of inorganic radicals in aqueous solution.
      ), and the reactions can be monitored using ultrafast kinetics spectrophotometry. In 1965, using pulse radiolysis of water, the rate constants of the reaction of hydroxyl radical (OH, EóOH/H2O = +2.32 V) with HCO3 were published (
      • Keene J.P.
      • Raef K.
      • Swallow A.J.
      Pulse radiolysis studies of carboxyl and related radicals.
      ,
      • Adams G.E.
      • Boag J.W.
      • Michael B.D.
      Reactions of the hydroxyl radical Part 1.-Transient spectra of some inorganic radical-anions.
      ,
      • Thomas J.K.
      Rates of reaction of the hydroxyl radical and that of OH with bicarbonate.
      ).
      CO3•– can be conveniently produced by radiolysis of water containing HCO3/CO32– and saturated with N2O (to scavenge eaq, producing OH). The reactions are:
      OH + HCO3 → H2O + CO3•–
      [9]


      OH + CO32– (+H+) → H2O + CO3•–
      [10]


      (k9 = 8.5 × 106 M–1 s–1, k10 = 3.9 × 108 M–1 s–1, that is, k10/k9 ∼ 46 at ambient temperature). These rate constants have been obtained over a wide temperature range (
      • Chen S.N.
      • Hoffman M.Z.
      Rate constants for the reaction of the carbonate radical with compounds of biochemical interest in neutral aqueous solution.
      ). About 10% of radicals are H atoms, which react much more slowly compared to OH (k11 = 4.4 × 104 M–1 s–1) (
      • Buxton G.V.
      • Greenstock C.L.
      • Helman W.P.
      • Ross A.B.
      Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (•OH/•O–) in aqueous solution.
      ):
      H + HCO3 → H2 + CO3•–
      [11]


      Under most biological conditions, the reaction of OH with HCO3 is not a predominant one
      However, conditions that result in [HCO3-] > 100 mM favor that a fraction of OH produced in tissue radiation exposure could evolve to CO3•−.
      since the rate constant (k9) is much lower than the rate constants of OH with most biological targets that are close to the diffusion-control limit, ca. 109 M–1 s–1; on the other hand, the reaction of OH with CO32–, although with a higher rate constant, is also of little biological relevance because of the marginal amounts of CO32– existing within the pH range in mammalian cells.
      HCO3 ⇌ H+ + CO32– pK12 = 10.3
      [12]


      A compilation of rate constants for reactions of CO3•– with 181 substances has been published (
      • Neta P.
      • Huie R.E.
      • Ross A.B.
      Rate constants for reactions of inorganic radicals in aqueous solution.
      ), but this is now outdated and incomplete. Some additional rate constants can be obtained in more recent works, which include the reaction of CO3•– with lipoic acid, desferrioxamine, and 5,5-dimethyl−1−pyrroline-N-oxide, among other target molecules (
      • Alvarez M.N.
      • Peluffo G.
      • Folkes L.
      • Wardman P.
      • Radi R.
      Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide in chemical and cellular systems: pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies.
      ,
      • Bartesaghi S.
      • Trujillo M.
      • Denicola A.
      • Folkes L.
      • Wardman P.
      • Radi R.
      Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals.
      ,
      • Trujillo M.
      • Folkes L.
      • Bartesaghi S.
      • Kalyanaraman B.
      • Wardman P.
      • Radi R.
      Peroxynitrite-derived carbonate and nitrogen dioxide radicals readily react with lipoic and dihydrolipoic acid.
      ,
      • Carballal S.
      • Trujillo M.
      • Cuevasanta E.
      • Bartesaghi S.
      • Möller M.N.
      • Folkes L.K.
      • et al.
      Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest.
      ). CO3•– can promote both protein and DNA oxidation (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.X.
      • de Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ).
      The actual existence of CO3•– in the anionic form at physiological pH was for some time a subject of debate and depends on the pKa value for:
      HCO3 ⇌ H+ + CO3•–
      [13]


      While pK13 values of 9.6 or 7.9 were reported in some early studies (summarized in (
      • Neta P.
      • Huie R.E.
      • Ross A.B.
      Rate constants for reactions of inorganic radicals in aqueous solution.
      )), a later study, using a flow system to irradiate mixtures of H2CO3 and HCO3- within 50 ms of their formation, has demonstrated that the HCO3 is a strong acid, pK13 < 0, contrary to the earlier reports (the rate constant for reaction of OH with H2CO3 is 7 × 104 M–1 s–1 at about 5 °C) (
      • Czapski G.
      • Lymar S.V.
      • Schwarz H.A.
      Acidity of the carbonate radical.
      ).
      Although less oxidizing than OH, CO3•– (EóCO3⋅−/HCO3- = +1.78 V) is a strong one-electron oxidant that acts by both electron transfer and hydrogen abstraction mechanisms to produce radicals from the oxidized targets (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.X.
      • de Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ). The one-electron oxidation of HCO3- by OH is thermodynamically favored with the net value for the reaction 9 of + 0.54 V (
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.X.
      • de Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      • Armstrong D.A.
      • Huie R.E.
      • Koppenol W.H.
      • Lymar S.v.
      • Merenyi G.
      • Neta P.
      • et al.
      Standard electrode potentials involving radicals in aqueous solution: inorganic radicals (IUPAC Technical Report).
      ).

      Production of carbonate radical secondary to xanthine oxidase–dependent reactions

      In the biochemical literature, work by Hodgson and Fridovich in 1976 postulated the formation of CO3•– during the turnover of xanthine oxidase in the presence of CO2/HCO3- (
      • Hodgson E.K.
      • Fridovich I.
      The mechanism of the activity-dependent luminescence of xanthine oxidase.
      ). Utilizing acetaldehyde and molecular oxygen as substrates, xanthine oxidase catalyzes the oxidation of acetaldehyde to acetic acid and the concomitant formation of O2•- and H2O2 as follows
      The reduction of molecular oxygen at the xanthine oxidase active site can occur via one- or two-electron mechanisms to yield O2- and H2O2, respectively. The relative formation of O2- as a function of molecular oxygen consumption is commonly known as percent univalent flux; this value varies depending on substrate type and concentration, oxygen tension, and general reaction conditions. For instance, low turnover substrates increase percent univalent flux (
      • Nagano T.
      • Fridovich I.
      Superoxide radical from xanthine oxidase acting upon lumazine.
      ,
      • Rubbo H.
      • Radi R.
      • Prodanov E.
      Substrate inhibition of xanthine oxidase and its influence on superoxide radical production.
      ).
      :
      Acetaldehyde + O2xanthineoxidase acetic acid + H2O2+ O2-
      (14)


      In the presence of carbonated solutions at pH = 10, the xanthine oxidase turnover resulted in spontaneous chemiluminescence, which was dependent on carbonate in a concentration-dependent manner (
      • Hodgson E.K.
      • Fridovich I.
      The mechanism of the activity-dependent luminescence of xanthine oxidase.
      ). As in the presence of transition metal traces such as iron, O2•– and H2O2 evolve to OH via the Haber–Weiss mechanism (Equation 15) (
      • Kehrer J.P.
      The Haber-Weiss reaction and mechanisms of toxicity.
      ); light emission was attributed to the following reaction steps:
      O2-+ H2O2Fe OH-+OH+O2
      (15)


      OH + CO32– → ΟΗ + CO3•–
      [16]


      2 CO3•– → → hν + products
      [17]


      Indeed, the OH-dependent formation of CO3•– (Equation 16) is followed by its recombination reaction (Equation 17) to yield excited species that decay with light emission in the blue/green region (400–550 nm) (
      • Hodgson E.K.
      • Fridovich I.
      The mechanism of the activity-dependent luminescence of xanthine oxidase.
      ,
      • Stauff J.
      • Sanders U.
      • Jaesche W.
      Chemiluminescence.
      ,
      • Denicola A.
      • Freeman B.A.
      • Trujillo M.
      • Radi R.
      Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations.
      ). The rate constant of reaction 17 determined at 0.1 M Na2CO3 is 2.0 × 107 M−1 s−1 (
      • Czapski G.
      • Holcman J.
      • Bielski B.H.J.
      Reactivity of nitric oxide with simple short-lived radicals in aqueous solutions.
      ).
      The presence of carbonated solutions also increases luminol chemiluminescence induced during xanthine oxidase turnover (
      • Radi R.
      • Rubbo H.
      • Thomson L.
      • Prodanov E.
      Luminol chemiluminescence using xanthine and hypoxanthine as xanthine oxidase substrates.
      ), likely by enhancing luminol oxidation by CO3•– (
      • Michelson A.M.
      • Maral J.
      Carbonate anions; effects on the oxidation of luminol, oxidative hemolysis, γ-irradiation and the reaction of activated oxygen species with enzymes containing various active centres.
      ). A similar stimulatory effect of bicarbonate was later observed during peroxynitrite-dependent luminol chemiexcitation (vide infra) (
      • Radi R.
      • Cosgrove P.
      • Beckman J.S.
      • Freeman B.A.
      Peroxynitrite-induced luminol chemiluminescence.
      )

      Peroxynitrite, an unstable peroxide in carbonated solutions

      Peroxynitrite is the product of the diffusion-controlled reaction between O2•- and NO.
      O2•– + NO → ONOO-
      [18]


      ONOO- + H+ ⇌ ONOOH
      [19]


      Peroxynitrite anion is in equilibrium with peroxynitrous acid (ONOOH) with a pKa = 6.8, meaning that both species coexist under biologically relevant conditions. The biological chemistry of peroxynitrite has been reviewed recently (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). One of the key reactions of peroxynitrite is that with CO2, this reaction was first hinted in 1969 by Keith and Powell (
      • Keith W.G.
      • Powell R.E.
      Kinetics of decomposition of peroxynitrous acid.
      ),who observed the instability and rapid decay of peroxynitrite in carbonated buffers, which the authors referred to as experimentally “intolerable.” This observation was later substantiated in specific studies that revealed the change in reactivity that bicarbonate buffers imposed on peroxynitrite-mediated reactions, starting with a study in which the intermediate formation of a ONOOCO2- adduct was proposed (
      • Radi R.
      • Cosgrove P.
      • Beckman J.S.
      • Freeman B.A.
      Peroxynitrite-induced luminol chemiluminescence.
      ). Mechanistic and fast kinetic studies revealed the reaction to occur strictly between ONOO and CO2 and not with other acid-base species (
      • Lymar S.v.
      • Hurst J.K.
      Rapid reaction between peroxonitrite ion and carbon dioxide: implications for biological activity.
      ) (recently reviewed in (
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • et al.
      Carbon dioxide-catalyzed peroxynitrite reactivity – the resilience of the radical mechanism after two decades of research.
      ), vide infra).

      The formation of peroxymonocarbonate

      In 1986, Flanagan et al. established for the first time that HCO3-containing solutions in the pH range 7.0 to 9.5 under excess H2O2 lead to the formation of peroxymonocarbonate (HCO4-) as inferred by Raman and 13C-NMR spectroscopy (
      • Flanagan J.
      • Jones D.P.
      • Griffith W.P.
      • Skapski A.C.
      • West A.P.
      On the existence of peroxocarbonates in aqueous solution.
      ). The possibility that HCO4- could participate in biochemical reactions was postulated as early as in 1978 (
      • Peter Esnouf B.M.
      • Green M.R.
      • Allen H.
      • Brent Irvine Hill.G.
      • Walter S.J.
      Evidence for the involvement of superoxide in vitamin K-dependent carboxylation of glutamic acid residues of prothrombin.
      ). Later on, the actual reactions, kinetics, and equilibria involving the CO2/HCO3- molecular pair and H2O2 that lead to HCO4- formation was disclosed (
      • Richardson D.E.
      • Yao H.
      • Frank K.M.
      • Bennett D.A.
      Equilibria, kinetics, and mechanism in the bicarbonate activation of hydrogen peroxide: oxidation of sulfides by peroxymonocarbonate.
      ,
      • Bakhmutova-Albert E.v.
      • Yao H.
      • Denevan D.E.
      • Richardson D.E.
      Kinetics and mechanism of peroxymonocarbonate formation.
      ) and will be analyzed in detail later in the text.

      Interactions of bicarbonate with transition metal centers: Oxidation and disproportionation reactions

      Bicarbonate may also modulate transition metal–dependent oxidation processes. On one hand, HCO3- may promote transition metal–dependent site-specific oxidation of biotargets; in particular, HCO3- facilitates Fenton-type reactions during amino acid oxidation by H2O2 (
      • Berlett B.S.
      • Chock P.B.
      • Yim M.B.
      • Stadtman E.R.
      Manganese(II) catalyzes the bicarbonate-dependent oxidation of amino acids by hydrogen peroxide and the amino acid-facilitated dismutation of hydrogen peroxide.
      ,
      • Stadtman E.R.
      • Berlett B.S.
      Fenton chemistry: amino acid oxidation.
      ,
      • Illés E.
      • Mizrahi A.
      • Marks V.
      • Meyerstein D.
      Carbonate-radical-anions, and not hydroxyl radicals, are the products of the Fenton reaction in neutral solutions containing bicarbonate.
      ). On the other hand, HCO3-–Mn complexes catalyze the disproportionation of H2O2 in a catalase-like manner (
      • Stadtman E.R.
      • Berlett B.S.
      • Chock P.B.
      Manganese-dependent disproportionation of hydrogen peroxide in bicarbonate buffer (superoxide anion/oxygen radicals/hydroxyl radicals/catalase mimic).
      ). Likewise, it is possible that HCO3-–Mn complexes may favor O2•– dismutation (
      • Archibald F.S.
      • Fridovich I.
      The scavenging of superoxide radical by manganous complexes: in vitro.
      ).
      The participation of HCO3- in transition metal–dependent redox reactions triggered by H2O2 and O2•– in biology remains largely unexplored. Thus, while the formation of redox active complexes of HCO3- with transition metals is possible and opens possibilities for their participation in the modulation of oxidative reactions in vitro, their role in vivo remains only speculative.

      Chemical aspects of the reaction of CO2 with peroxides

      Herein, we will analyze how the reactions of CO2 with peroxynitrite and/or H2O2 lead to CO2-derived reactive intermediates that promote one- and two-electron oxidations.

      Peroxynitrite and carbonate radical

      The nucleophilic character of peroxynitrite anion (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Radi R.
      Peroxynitrite, a stealthy biological oxidant.
      ) enables its fast reaction with CO2. Indeed, the pH-independent rate constant for the reaction between ONOO and CO2 has been determined as k20 = 3 × 104 M−1 s−1 (25 °C) (
      • Lymar S.v.
      • Hurst J.K.
      Rapid reaction between peroxonitrite ion and carbon dioxide: implications for biological activity.
      ) or 5.8 × 104 M−1 s−1 (37 °C) (
      • Denicola A.
      • Freeman B.A.
      • Trujillo M.
      • Radi R.
      Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations.
      ); the product of the reaction is a transient adduct (eq.[20]), nitrosoperoxocarboxylate (ONOOCO2-) that readily undergoes homolysis to yield NO2 and CO3 in 35% yields (Equation 21), with the rest isomerizing to nitrate (NO3-) (recently reviewed in (
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • et al.
      Carbon dioxide-catalyzed peroxynitrite reactivity – the resilience of the radical mechanism after two decades of research.
      ,
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      )).
      ONOO- + CO2 → ONOOCO2-
      [20]


      ONOOCO2-NO2 + CO3
      [21]


      Because of the velocity of the reaction 20 which is a function of the product of k20 times [CO2], the biological chemistry of peroxynitrite is highly influenced by the existing levels of CO2 in cells and tissues; CO2 represents a key biological target of peroxynitrite (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). For a comparative analysis of the relative weight of the CO2 reaction on the fate of peroxynitrite versus that of other biotargets, see (
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • Carballal S.
      • Bartesaghi S.
      • Radi R.
      Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite.
      ). For example, the reaction of peroxynitrite with cytosolic concentrations of CO2 (ca. 1.3 mM) yields a pseudo–first order rate constant in the order of 60 s−1, a reference value that is utilized to assess the relevance of alternative routes of peroxynitrite consumption (
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ). For example, this value is much larger than that of the reaction of peroxynitrite with cytosolic GSH (ca. 10 s−1) and usually smaller than that of the reactions with peroxiredoxins
      Concentration values of different peroxiredoxins largely vary depending on cell type, with over a 100-fold change from values in the range of 2 to 240 μM (
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • de Armas M.I.
      • Esteves R.
      • Viera N.
      • Reyes A.M.
      • Mastrogiovanni M.
      • Alegria T.G.P.
      • et al.
      Rapid peroxynitrite reduction by human peroxiredoxin 3: implications for the fate of oxidants in mitochondria.
      ,
      • Moore R.B.
      • Mankad M.v.
      • Shriver S.K.
      • Mankad V.N.
      • Plishker G.A.
      Reconstitution of Ca2+-dependent K+ transport in erythrocyte membrane vesicles requires a cytoplasmic protein.
      ); therefore, its competition with CO2 for peroxynitrite goes from modest to substantial.
      (ca. > 100 s−1) (
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • de Armas M.I.
      • Esteves R.
      • Viera N.
      • Reyes A.M.
      • Mastrogiovanni M.
      • Alegria T.G.P.
      • et al.
      Rapid peroxynitrite reduction by human peroxiredoxin 3: implications for the fate of oxidants in mitochondria.
      ).
      CO3•− and NO2 are good one-electron oxidants (EóNO2/NO2- = 0.9 V); in addition, NO2 can participate in nitration reactions (
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.X.
      • de Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ,
      • Folkes L.K.
      • Bartesaghi S.
      • Trujillo M.
      • Wardman P.
      • Radi R.
      Radiolysis studies of oxidation and nitration of tyrosine and some other biological targets by peroxynitrite-derived radicals.
      ). In this respect, the presence of CO2 usually promotes peroxynitrite-dependent protein tyrosine nitration, as CO3•− readily oxidizes tyrosine to tyrosyl radical, which then undergoes a fast recombination reaction with NO2 to yield protein 3-nitrotyrosine (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Radi R.
      Nitric oxide, oxidants, and protein tyrosine nitration.
      ). Also, thiol oxidation by peroxynitrite in the presence of CO2 shifts from the direct two-electron process (i.e., to sulfenic acid) towards CO3- and NO2-mediated one-electron oxidations to thiyl radical (
      • Trujillo M.
      • Folkes L.
      • Bartesaghi S.
      • Kalyanaraman B.
      • Wardman P.
      • Radi R.
      Peroxynitrite-derived carbonate and nitrogen dioxide radicals readily react with lipoic and dihydrolipoic acid.
      ,
      • Trujillo M.
      • Alvarez B.
      • Radi R.
      One- and two-electron oxidation of thiols: mechanisms, kinetics and biological fates.
      ,
      • Trujillo M.
      • Carballal S.
      • Zeida A.
      • Radi R.
      Comparative analysis of hydrogen peroxide and peroxynitrite reactivity with thiols.
      ) (Fig. 4)
      Figure thumbnail gr4
      Figure 4Carbon dioxide in peroxide-dependent oxidation reactions. Superoxide radical can yield peroxynitrite anion (ONOO-) or H2O2 by its reactions with NO or another O2•- molecule (catalyzed by SOD), respectively. Peroxynitrite anion protonates to peroxynitrous acid (ONOOH) which reacts in a two-electron oxidation process with thiols to yield the corresponding sulfenic acid (RSOH). Alternatively, ONOO- reacts with CO2 to yield a transient species, ONOOCO2-, that undergoes homolysis to the free radicals CO3•- and NO2. Peroxynitrite-derived radicals promote one-electron oxidations in biomolecules to yield, for example, tyrosyl and thiyl radicals which subsequently evolve to stable products such as 3-nitrotyrosine (a specific biomarker of NO-derived oxidants) or sulfinic acid (RSO2H). H2O2 and HCO4- (formed in the presence of CO2, see (Equations [22], [23], [24])) can also directly oxidize thiols to sulfenic acid. Moreover, they can also promote thiol hyperoxidation to sulfinic acid, a process that is typically faster for HCO4- than H2O2. Finally, in the presence of transition metal centers HCO4- can evolve to CO3•- and promote one-electron oxidations. SOD, superoxide dismutase.

      Hydrogen peroxide and peroxymonocarbonate

      CO2 reacts slowly with H2O2 to yield HCO4- (pKa = 3.4, corresponding to the dissociation of the carboxylate group) in two different reactions (
      • Bakhmutova-Albert E.v.
      • Yao H.
      • Denevan D.E.
      • Richardson D.E.
      Kinetics and mechanism of peroxymonocarbonate formation.
      ). On one hand, CO2 can undergo perhydration with H2O2 (Equation 22), in a reaction analogous to that of hydration (addition of water) described previously (Equation 1). The process can be decomposed in two separate reactions, namely the perhydration reaction per se 22 to yield H2CO4, followed by deprotonation to reach an acid-base equilibrium 23.
      CO2 (aq) + H2O2 ⇌ H2CO4 kf = 0.02 M−1 s−1; kr = 22 s−1
      [22]


      H2CO4 ⇌ H+ + HCO4(fast) Ka = 2.9 ×10−4 M
      [23]


      H2O2 reacts more rapidly than water with CO2 (0.02 M−1s−1 versus ∼8 × 10−5 M−1s−1).
      Alternatively, HO2-, the conjugated base of H2O2 (pKa = 11.7), adds as a nucleophile to CO2 (the carbon atom is an electrophilic Lewis acid center in CO2) (Equation 24), in a reaction analogous to that of ONOCO2- formation (Equation 20).
      CO2 (aq) + HO2- ⇌ HCO4- kf = 280 M−1 s−1; kr = 1.8 × 10−3 s−1
      [24]


      The estimated rate constant for the reaction of HO2- with CO2 (280 M−1s−1) at 25 °C is substantially smaller than that for OH- (8500 M−1s−1) (
      • Bakhmutova-Albert E.v.
      • Yao H.
      • Denevan D.E.
      • Richardson D.E.
      Kinetics and mechanism of peroxymonocarbonate formation.
      ) and ONOO- (3 × 104 M−1s−1) (
      • Lymar S.v.
      • Hurst J.K.
      Rapid reaction between peroxonitrite ion and carbon dioxide: implications for biological activity.
      ).
      Both reactions 22 and 24 are rather slow and contribute to HCO4- formation under physiologically relevant conditions; indeed, while the rate constant of reaction 24 is higher than that of reaction 22, the opposite occurs is terms of concentration of peroxide (i.e., H2O2 versus HO2-) at physiologically relevant pH. Thus, at pH 7.4, it is estimated that reactions 22 and 24 contribute in 59% and 41% to HCO4- formation, respectively (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      ). Recognizably, pH changes will affect both the CO2/HCO3- and H2O2/HO2- ratios and will be reflected on the relative contribution and velocity of reactions 22 and 24. In this regard, the contribution of HO2- to initial HCO4- formation increases with increasing pH, dominating above pH 8. The elementary reactions and their equilibrium and rate constants for peroxymonocarbonate formation have been comprehensively reported in (
      • Bakhmutova-Albert E.v.
      • Yao H.
      • Denevan D.E.
      • Richardson D.E.
      Kinetics and mechanism of peroxymonocarbonate formation.
      ).
      For practical and experimental purposes, the following overall equilibrium applies:
      H2O2 + HCO3- ⇌ HCO4- + H2O
      [25]


      [HCO4][H2O2] x [HCO3]= Keq= 0.31 M-1at 25°C.
      (26)


      Thus, at equilibrium, HCO4- concentration can be calculated as follows:
      [HCO4-] = 0.31 × [H2O2] × [HCO3-]
      [27]


      which for 25 mM HCO3- (0.025 M) typically represents ca. 1% of initial H2O2 (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      ).
      HCO4- (Eó= +1.8 V) is a strong two-electron oxidant, with a redox potential similar to that of H2O2 (Eó= +1.77 V). However, HCO4- typically reacts with target molecules at rates 100 to 1000 times faster than those of H2O2 (
      • Trujillo M.
      • Carballal S.
      • Zeida A.
      • Radi R.
      Comparative analysis of hydrogen peroxide and peroxynitrite reactivity with thiols.
      ,
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      ,
      • Richardson D.E.
      • Regino C.A.S.
      • Yao H.
      • Johnson J.v.
      Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide.
      ). For instance, the second order rate constants of H2O2 and HCO4- with GSH [Equations 28a and 28b] are 1.9 and 1.6 × 102 M−1s−1, respectively (reviewed in (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      )). This reactivity inversely correlates with the pKa of the conjugated acid of the peroxide leaving group
      For the reactions of H2O2 and HCO4-, the pKa of the corresponding leaving groups (i.e., OH-, CO32-) are 15.7 and 10.3, respectively.
      .
      H2O2 + GS- → HO- + GSOH
      [28a]


      HCO4- + GS- → CO32- + GSOH
      [28b]


      HCO4- also reacts faster with both free and protein-bound methionine (e.g., in α1-proteinase inhibitor) than H2O2, to yield the corresponding two-electron oxidation product methionine sulfoxide (
      • Richardson D.E.
      • Regino C.A.S.
      • Yao H.
      • Johnson J.v.
      Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide.
      ). As methionine oxidation represents a reaction that can regulate protein function in vitro and in vivo (e.g., pyruvate kinase M2) (
      • Walker E.J.
      • Bettinger J.Q.
      • Welle K.A.
      • Hryhorenko J.R.
      • Molina Vargas A.M.
      • O’Connell M.R.
      • et al.
      Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues.
      ,
      • He D.
      • Feng H.
      • Sundberg B.
      • Yang J.
      • Powers J.
      • Christian A.H.
      • et al.
      Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis.
      ), direct assessment of the role of CO2 on peroxide-dependent methionine oxidation (
      • Richardson D.E.
      • Regino C.A.S.
      • Yao H.
      • Johnson J.v.
      Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide.
      ,
      • Pryor W.A.
      • Jin X.
      • Squadrito G.L.
      One- and two-electron oxidations of methionine by peroxynitrite.
      ,
      • Perrin D.
      • Koppenol W.H.
      The quantitative oxidation of methionine to methionine sulfoxide by peroxynitrite.
      ,
      • Tien M.
      • Berlett B.S.
      • Levine R.L.
      • Boon Chock P.
      • Stadtman E.R.
      Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation.
      ,
      • Alvarez B.
      • Ferrer-Sueta G.
      • Freeman B.A.
      • Radi R.
      Kinetics of peroxynitrite reaction with amino acids and human serum albumin.
      ) becomes necessary
      In the case of the reaction of methionine with peroxynitrite, the presence of CO2 would divert the two-electron oxidation of the amino acid toward one-electron oxidations that yield methional and ethylene (
      • Pryor W.A.
      • Jin X.
      • Squadrito G.L.
      One- and two-electron oxidations of methionine by peroxynitrite.
      ,
      • Perrin D.
      • Koppenol W.H.
      The quantitative oxidation of methionine to methionine sulfoxide by peroxynitrite.
      ). CO3•- reacts very fast with methionine 1.2 × 108 M−1s−1 (
      • Neta P.
      • Huie R.E.
      • Ross A.B.
      Rate constants for reactions of inorganic radicals in aqueous solution.
      ,
      • Zhang M.M.
      • Rempel D.L.
      • Gross M.L.
      A fast photochemical oxidation of proteins (FPOP) platform for free-radical reactions: the carbonate radical anion with peptides and proteins.
      ).
      .
      On the other hand, HCO4- can be reduced by one electron via transition metal centers to yield CO3•-, likely
      HCO4- + Men+ → CO3•- + Me (n−1)+ + OH-
      [29]


      Thus, the CO2-dependent formation of HCO4- leads to the activation of H2O2 for both two electron [28b], [28a] and radical 29 chemistry, the latter in combination with transition metals (
      • Bakhmutova-Albert E.v.
      • Yao H.
      • Denevan D.E.
      • Richardson D.E.
      Kinetics and mechanism of peroxymonocarbonate formation.
      ,
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      )
      Peroxymonocarbonate can be also generated at the active site of redox enzymes and promote CO2-dependent oxidations and peroxidations (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      ,
      • Liochev S.I.
      • Fridovich I.
      The role of CO2 in metal-catalyzed peroxidations.
      ,
      • Liochev S.I.
      • Fridovich I.
      CO 2, not HCO 3, facilitates oxidations by Cu,Zn superoxide dismutase plus H 2 O 2.
      ,
      • Medinas D.B.
      • Augusto O.
      Mechanism of the peroxidase activity of superoxide dismutase 1.
      ,
      • Zhang H.
      • Joseph J.
      • Felix C.
      • Kalyanaraman B.
      Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical.
      ,
      • Medinas D.B.
      • Toledo J.C.
      • Cerchiaro G.
      • Do-Amaral A.T.
      • De-Rezende L.
      • Malvezzi A.
      • et al.
      Peroxymonocarbonate and carbonate radical displace the hydroxyl-like oxidant in the Sod1 peroxidase activity under physiological conditions.
      ,
      • Bonini M.G.
      • Miyamoto S.
      • Di Mascio P.
      • Augusto O.
      Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate.
      ). Two well-known examples of these processes have been described for Cu/Zn superoxide dismutase (
      • Liochev S.I.
      • Fridovich I.
      CO 2, not HCO 3, facilitates oxidations by Cu,Zn superoxide dismutase plus H 2 O 2.
      ,
      • Zhang H.
      • Joseph J.
      • Felix C.
      • Kalyanaraman B.
      Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical.
      ,
      • Medinas D.B.
      • Toledo J.C.
      • Cerchiaro G.
      • Do-Amaral A.T.
      • De-Rezende L.
      • Malvezzi A.
      • et al.
      Peroxymonocarbonate and carbonate radical displace the hydroxyl-like oxidant in the Sod1 peroxidase activity under physiological conditions.
      ) and xanthine oxidase (
      • Bonini M.G.
      • Miyamoto S.
      • Di Mascio P.
      • Augusto O.
      Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate.
      ). Once formed, the reduction of HCO4- at the enzyme active sites yields CO3•-, which is the proximal oxidant responsible for the CO2-dependent one-electron oxidations. Formation of ternary complexes at the enzyme active site with HO2- and CO2 to yield metal-bound HCO4- has been invoked to explain how bicarbonate buffers accelerate H2O2-dependent oxidations (reviewed in (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      )).

      CO2 in the modulation of peroxynitrite-dependent and hydrogen peroxide–dependent oxidations in biology

      Kinetic considerations

      CO2 is a relevant biological target of peroxynitrite (Equation 20) and determines part of its fate and half-life in different biological compartments (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • Carballal S.
      • Bartesaghi S.
      • Radi R.
      Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite.
      ). The “CO2 pathway” of peroxynitrite decomposition is in competition with other relevant reactions including its catabolism by peroxiredoxins (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Zeida A.
      • Trujillo M.
      • Ferrer-Sueta G.
      • Denicola A.
      • Estrin D.A.
      • Radi R.
      Catalysis of peroxide reduction by fast reacting protein thiols.
      ). Indeed, kinetic analysis taking into consideration existing concentrations of CO2 in intracellular compartments, pH, and the second order rate constant of reaction 20 indicates a pseudo–first order rate constant of peroxynitrite decay by CO2 in the order of 60 to 100 s−1. This k′ value translates into a half-life (t1/2 = ln2/k′) in the order of 10 ms, a time scale that in extracellular compartments allows the diffusion of peroxynitrite a mean distance >5 μm (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Denicola A.
      • Souza J.M.
      • Radi R.
      Diffusion of peroxynitrite across erythrocyte membranes.
      ,
      • Romero N.
      • Denicola A.
      • Souza J.M.
      • Radi R.
      Diffusion of peroxynitrite in the presence of carbon dioxide.
      ). Obviously, the half-life of peroxynitrite significantly shortens intracellularly where other fast reacting targets such as peroxiredoxins (k′ > 100 s−1) react (for a detailed kinetic analysis see (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • Carballal S.
      • Bartesaghi S.
      • Radi R.
      Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite.
      )). Obviously, changing levels of CO2 will affect the route and the extent by which peroxynitrite decomposes into CO3•- and NO2 radicals (and nitrate, NO3-). It is important to note that in extracellular environments, the CO2 pathway exerts significant control on the reactivity and diffusion of peroxynitrite due to the scarcity of other biotargets that can compete at significant rates (
      • Lymar S. v
      • Hurst J.K.
      Carbon dioxide: physiological catalyst for peroxynitrite-mediated cellular damage or cellular protectant?.
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ). Indeed, as the half-lives of NO2 and CO3•– are very short (< ms to μs time scale), in extracellular compartments, the CO2 reaction with peroxynitrite usually represents a major “decay” pathway that partially limits peroxynitrite diffusion to cells and focusses its oxidative chemistry in extracellular or plasma membrane targets (
      • Romero N.
      • Denicola A.
      • Souza J.M.
      • Radi R.
      Diffusion of peroxynitrite in the presence of carbon dioxide.
      ,
      • Romero N.
      • Peluffo G.
      • Bartesaghi S.
      • Zhang H.
      • Joseph J.
      • Kalyanaraman B.
      • et al.
      Incorporation of the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester to red blood cell membranes to study peroxynitrite-dependent reactions.
      ,
      • Winterbourn C.C.
      Reconciling the chemistry and biology of reactive oxygen species.
      ,
      • Kennett E.C.
      • Davies M.J.
      Glycosaminoglycans are fragmented by hydroxyl, carbonate, and nitrogen dioxide radicals in a site-selective manner: implications for peroxynitrite-mediated damage at sites of inflammation.
      ,
      • Kennett E.C.
      • Rees M.D.
      • Malle E.
      • Hammer A.
      • Whitelock J.M.
      • Davies M.J.
      Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains.
      ,
      • Degendorfer G.
      • Chuang C.Y.
      • Mariotti M.
      • Hammer A.
      • Hoefler G.
      • Hägglund P.
      • et al.
      Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function.
      ) (Fig. 5). In the case of mitochondria, the high levels of CO2 promote the organelle-specific oxidation and nitration of mitochondrial proteins by peroxynitrite, even under basal conditions (
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • de Armas M.I.
      • Esteves R.
      • Viera N.
      • Reyes A.M.
      • Mastrogiovanni M.
      • Alegria T.G.P.
      • et al.
      Rapid peroxynitrite reduction by human peroxiredoxin 3: implications for the fate of oxidants in mitochondria.
      ,
      • Sacksteder C.A.
      • Qian W.J.
      • Knyushko T.v.
      • Wang H.
      • Chin M.H.
      • Lacan G.
      • et al.
      Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease.
      ,
      • Kameritsch P.
      • Singer M.
      • Nuernbergk C.
      • Rios N.
      • Reyes A.M.
      • Schmidt K.
      • et al.
      The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity.
      ).
      Figure thumbnail gr5
      Figure 5Peroxide reactions with CO2 in competition with diffusion and transport across the plasma membrane. Carbon dioxide is a strong contender for the diffusion of peroxynitrite and its transport across membranes via anion channels or passive diffusion (
      • Romero N.
      • Denicola A.
      • Souza J.M.
      • Radi R.
      Diffusion of peroxynitrite in the presence of carbon dioxide.
      ). The fast reaction of peroxynitrite with CO2 limits peroxynitrite diffusion and focusses its reactivity and decay. The rapid extracellular homolysis of ONOOCO2- results in the formation of radicals that may recombine to NO3- before reaching target molecules or a membrane. Hydrogen peroxide can cross membranes by passive diffusion or facilitated by aquaporins (
      • Erudaitius D.
      • Huang A.
      • Kazmi S.
      • Buettner G.R.
      • Rodgers V.G.J.
      Peroxiporin expression is an important factor for cancer cell susceptibility to therapeutic H2O2: implications for pharmacological ascorbate therapy.
      ,
      • Bestetti S.
      • Galli M.
      • Sorrentino I.
      • Pinton P.
      • Rimessi A.
      • Sitia R.
      • et al.
      Human aquaporin-11 guarantees efficient transport of H2O2 across the endoplasmic reticulum membrane.
      ). Events related to the H2O2 plus CO2-dependent formation and consumption of HCO4- are shown. Peroxynitrite and H2O2 kinetics, equilibria, and transport will be influenced by the levels of CO2/HCO3-, which in turn is dictated by metabolic CO2 formation (or CO2 exposure) and dynamic aspects that involve CA-catalyzed reactions, diffusion, and transport. The figure exemplifies extracellular peroxides diffusing toward an intracellular compartment. CA, carbonic anhydrase.
      In the case of H2O2, due to the kinetic and equilibria properties of its reactions with CO2 (either with H2O2 22 or HO2- 24), at any given time, only a small fraction would be present as HCO4. The H2O2/CO2 pathway will be in competition with other H2O2-consuming processes, in particular, those with peroxiredoxins, catalase and GSH peroxidase that occur at very fast rates (
      • Winterbourn C.C.
      Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.
      ,
      • Zeida A.
      • Trujillo M.
      • Ferrer-Sueta G.
      • Denicola A.
      • Estrin D.A.
      • Radi R.
      Catalysis of peroxide reduction by fast reacting protein thiols.
      ,
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      ,
      • Sies H.
      • Belousov V.v.
      • Chandel N.S.
      • Davies M.J.
      • Jones D.P.
      • Mann G.E.
      • et al.
      Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology.
      ) and with H2O2 diffusion and transport across membranes (Fig. 5). The slow velocity of formation of HCO4- and the fractional amount found at equilibrium questions, at first glance, the possible role of HCO4- in biological oxidations. However, additional factors must be taken into consideration. First, consumption of HCO4- by reactions with target molecules will continuously shift the reactions 22 and 24 to the right. Second, the formation of HCO4- can be accelerated by CA (
      • Bakhmutova-Albert E.v.
      • Yao H.
      • Denevan D.E.
      • Richardson D.E.
      Kinetics and mechanism of peroxymonocarbonate formation.
      ). Also, HCO4- formation is favored in the presence of lipids and proteins, shifting the equilibrium of reaction 25 to the right and increasing Keq values (
      • Trindade D.F.
      • Cerchiaro G.
      • Augusto O.
      A role for peroxymonocarbonate in the stimulation of biothiol peroxidation by the bicarbonate/carbon dioxide pair.
      ). Thus, it is kinetically possible for the reaction of H2O2 with CO2 to generate a biologically relevant flux of HCO4.
      In cellular compartments such as the macrophage phagosome, the drop of pH values and increase of CO2 levels observed during the activation of cytotoxic processes (
      • Martínez A.
      • Prolo C.
      • Estrada D.
      • Rios N.
      • Alvarez M.N.
      • Piñeyro M.D.
      • et al.
      Cytosolic Fe-superoxide dismutase safeguards Trypanosoma cruzi from macrophage-derived superoxide radical.
      ,
      • Borregaard N.
      • Schwartz J.H.
      • Tauber A.I.
      Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.
      ,
      • Westman J.
      • Grinstein S.
      Determinants of phagosomal pH during host-pathogen interactions.
      ) are expected to be influential on the biological chemistry of both peroxynitrite and H2O2 (Fig. 6).
      Figure thumbnail gr6
      Figure 6Carbon dioxide–derived oxidants in the macrophage phagosome. Engulfment of pathogens in the phagosome leads to a series of metabolic events directed to cause oxidative killing of the invader microbial cell. In this regard, activation of NOX leads to the formation of O2•- and H2O2 and in the case of immunostimulated cells, the concomitant generation of NO and peroxynitrite. These processes are coupled with the activation of the PPP that generates NADPH for the catalytic action of NOX and NOS and CO2 that can diffuse inside the phagosomal lumen and contribute to the formation of HCO4- and ONOOCO2. At the same time, membrane-bound ATP-dependent pumps release H+ toward the phagosome causing a drop in pH. NOS, nitric oxide synthase; NOX, NADPH oxidase; PPP, pentose phosphate pathway.

      Effect of CO2 on peroxynitrite-dependent processes

      Initial work assessing the effect of CO2 in the microbicidal effect of extracellularly added peroxynitrite on bacteria and parasites showed a protective effect in cell killing (
      • Lymar S. v
      • Hurst J.K.
      Carbon dioxide: physiological catalyst for peroxynitrite-mediated cellular damage or cellular protectant?.
      ,
      • Zhu L.
      • Gunn C.
      • Beckman J.S.
      Bactericidal activity of peroxynitrite.
      ,
      • Denicola A.
      • Rubbo H.
      • Rodriguez D.
      • Radi R.
      Peroxynitrite-mediated cytotoxicity to Trypanosoma cruzi.
      ). These observations are due to the fact that at low microbial suspension densities (intercellular distances of several micrometer among them), the reaction of extracellularly added peroxynitrite with CO2 (i.e., bicarbonate-containing solutions) and its decay by isomerization and radical (self-coupling and crosscoupling) recombination reactions outcompetes peroxynitrite diffusion to cells (
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • et al.
      Carbon dioxide-catalyzed peroxynitrite reactivity – the resilience of the radical mechanism after two decades of research.
      ,
      • King D.A.
      • Sheafor M.W.
      • Hurst J.K.
      Comparative toxicities of putative phagocyte-generated oxidizing radicals toward a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae).
      ) (Fig. 5). However, when considering the interactions of macrophage-derived peroxynitrite with pathogens located inside phagosomes (diffusion distances << 1 μm), extracellular CO2 only partially competes with peroxynitrite permeation to cells and leads to enhanced nitration of the microbial membrane by the localized action of peroxynitrite-derived CO3•- and NO2 (
      • Martínez A.
      • Prolo C.
      • Estrada D.
      • Rios N.
      • Alvarez M.N.
      • Piñeyro M.D.
      • et al.
      Cytosolic Fe-superoxide dismutase safeguards Trypanosoma cruzi from macrophage-derived superoxide radical.
      ,
      • Alvarez M.N.
      • Peluffo G.
      • Piacenza L.
      • Radi R.
      Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity.
      ,
      • Piacenza L.
      • Trujillo M.
      • Radi R.
      Reactive species and pathogen antioxidant networks during phagocytosis.
      ). Moreover, once peroxynitrite has reached inside the pathogen, intracellular CO2 is in competition with enzymatic systems that catabolize peroxynitrite (e.g., microbial peroxiredoxins), and therefore, CO3•- and NO2 promote microbicidal effects via oxidation and nitration reactions bypassing the peroxiredoxin detoxification pathway (
      • Martínez A.
      • Prolo C.
      • Estrada D.
      • Rios N.
      • Alvarez M.N.
      • Piñeyro M.D.
      • et al.
      Cytosolic Fe-superoxide dismutase safeguards Trypanosoma cruzi from macrophage-derived superoxide radical.
      ,
      • Alvarez M.N.
      • Peluffo G.
      • Piacenza L.
      • Radi R.
      Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity.
      ,
      • Piacenza L.
      • Trujillo M.
      • Radi R.
      Reactive species and pathogen antioxidant networks during phagocytosis.
      ,
      • Wolcott R.G.
      • Franks B.S.
      • Hannum D.M.
      • Hurst J.K.
      Bactericidal potency of hydroxyl radical in physiological environments.
      ). Thus, the “peroxynitrite-CO2” toxicity experiment in diluted cell suspensions, if not analyzed within the actual biological context, may lead to erroneous interpretations: the fact that CO2 may be a “protectant” from cytotoxic or microbicidal effects of peroxynitrite in vitro does not usually extrapolates to biologically relevant situations in vivo involving close cell-to-cell interactions and in microcompartments such as phagosomes and mitochondria (
      • King D.A.
      • Sheafor M.W.
      • Hurst J.K.
      Comparative toxicities of putative phagocyte-generated oxidizing radicals toward a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae).
      ,
      • Lymar S. v
      • Hurst J.K.
      Role of compartmentation in promoting toxicity of leukocyte-generated strong oxidants.
      ,
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      Macrophage-derived peroxynitrite diffusion and toxicity to Trypanosoma cruzi.
      )
      The extracellular half-life of peroxynitrite with or without CO2 is very different. It is about one 1 s without versus 10 ms with physiological [CO2]. The much shorter half-life is the reason for why the presence of CO2 may decrease toxicity when cells are in suspension. These considerations help explain some discrepancies in the literature.
      . In essence, CO2 in vivo focusses the reactivity of peroxynitrite to a very narrow region within the micrometer distance scale.
      On the other hand, there has been a recent debate on whether urease-dependent CO2 formation in Helicobacter pylori serves to neutralize the cytotoxic effects of peroxynitrite released by inflammatory cells in the stomach (
      • Kuwahara H.
      • Miyamoto Y.
      • Akaike T.
      • Kubota T.
      • Sawa T.
      • Okamoto S.
      • et al.
      Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production.
      ,
      • Gobert A.P.
      • Wilson K.T.
      The immune battle against Helicobacter pylori infection: NO offense.
      ,
      • Tsikas D.
      • Hanff E.
      • Brunner G.
      Helicobacter pylori, its urease and carbonic anhydrases, and macrophage nitric oxide synthase.
      ). H. pylori is usually considered an extracellular pathogen but it can be also found intracellularly, which has been associated to the persistence of the bacteria in the stomach. The discussion is quite interesting because, in effect, urease activity blunts peroxynitrite-dependent cytotoxicity in H. pylori in vitro. Close inspection to the data also shows that (1) addition of HCO3- (but not NH4+) and (2) urease-dependent CO2 formation decrease bacterial protein tyrosine nitration and peroxynitrite-dependent toxicity. This latter result indicates that the protective effects are occurring extracellularly; indeed, the effects of CO2 were intracellular, protein tyrosine nitration should have increased and peroxynitrite partially spared from its detoxification by bacterial peroxiredoxins. H. pylori contains CA that acts synergistically with urease for pH acclimation and colonization in the gastric mucosa. Thus, H. pylori CO2 emission toward the extracellular milieu and in the context of intercellular distances in the micrometer range promotes extracellular peroxynitrite decay and spares the bacterium from oxidative toxicity. However, the overall relevance of these elegant findings to the pathophysiology of H. pylori infection to the stomach remains, in this author’s opinion, undefined. It is important to note, however, that H. pylori CA and urease participate in the control of bacterial CO2 levels and counteract macrophage-derived oxidative killing constituting key factors for the establishment, progression, and/or control of infection (
      • Campestre C.
      • de Luca V.
      • Carradori S.
      • Grande R.
      • Carginale V.
      • Scaloni A.
      • et al.
      Carbonic anhydrases: new perspectives on protein functional role and inhibition in helicobacter pylori.
      ,
      • Gobert A.P.
      • Wilson K.T.
      The immune battle against Helicobacter pylori infection: NO offense.
      ,
      • Tsikas D.
      • Hanff E.
      • Brunner G.
      Helicobacter pylori, its urease and carbonic anhydrases, and macrophage nitric oxide synthase.
      ).
      In turn, CO2 is a substantial target of peroxynitrite inside mitochondria and influenced by changes in the rates of the Krebs cycle and oxidative stress conditions where the concentrations of reduced mitochondrial peroxiredoxins (Prx3 and Prx5) (i.e., peroxide catabolism) fall (
      • de Armas M.I.
      • Esteves R.
      • Viera N.
      • Reyes A.M.
      • Mastrogiovanni M.
      • Alegria T.G.P.
      • et al.
      Rapid peroxynitrite reduction by human peroxiredoxin 3: implications for the fate of oxidants in mitochondria.
      ,
      • Kameritsch P.
      • Singer M.
      • Nuernbergk C.
      • Rios N.
      • Reyes A.M.
      • Schmidt K.
      • et al.
      The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity.
      ,
      • Radi R.
      • Cassina A.
      • Hodara R.
      • Quijano C.
      • Castro L.
      Peroxynitrite reactions and formation in mitochondria.
      ). The reaction of mitochondrial peroxynitrite with CO2 leads to protein oxidation and nitration, even under basal physiological conditions (
      • Sacksteder C.A.
      • Qian W.J.
      • Knyushko T.v.
      • Wang H.
      • Chin M.H.
      • Lacan G.
      • et al.
      Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease.
      ). Accordingly, several nitrated proteins were detected in control mitochondria isolated from rat liver as well as in mitochondria from the heart of mice suffering diabetes, a disease known to be associated with increase nitro-oxidative stress (reviewed in (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Castro L.
      • Demicheli V.
      • Tórtora V.
      • Radi R.
      Mitochondrial protein tyrosine nitration.
      ). In the latter case, Prx3
      Peroxiredoxin 3 is exclusively located in mitochondria. Its nitration underscores that idea that part of mitochondrial peroxynitrite escapes Prx3 and Prx5-dependent catabolism by reaction with CO2, which, in turn, results in the formation of nitrating and oxidizing species.
      was among the nitrated proteins detected (
      • Turko I.v.
      • Li L.
      • Aulak K.S.
      • Stuehr D.J.
      • Chang J.Y.
      • Murad F.
      Protein tyrosine nitration in the mitochondria from diabetic mouse heart: implications to dysfunctional mitochondria in diabetes.
      ). Also, recent work in vivo in a model of vascular dysfunction shows that mitochondrial peroxiredoxins become overoxidized under conditions of excess peroxynitrite formation (
      • Kameritsch P.
      • Singer M.
      • Nuernbergk C.
      • Rios N.
      • Reyes A.M.
      • Schmidt K.
      • et al.
      The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity.
      ). In an in vitro experiment using isolated Prx3, enzyme nitration and hyperoxidation is only observed when exposed to excess peroxynitrite; this observation is consistent with the fast and dominant reaction of peroxynitrite with the peroxidatic thiol of Prx3 and that hyperoxidation rates are usually ∼103 slower than oxidation rates (
      • de Armas M.I.
      • Esteves R.
      • Viera N.
      • Reyes A.M.
      • Mastrogiovanni M.
      • Alegria T.G.P.
      • et al.
      Rapid peroxynitrite reduction by human peroxiredoxin 3: implications for the fate of oxidants in mitochondria.
      ). However, in the presence of CO2, a fraction of peroxynitrite bypasses the Prx3 and Prx5 detoxification routes and promote oxidative posttranslational modifications in vivo (
      • de Armas M.I.
      • Esteves R.
      • Viera N.
      • Reyes A.M.
      • Mastrogiovanni M.
      • Alegria T.G.P.
      • et al.
      Rapid peroxynitrite reduction by human peroxiredoxin 3: implications for the fate of oxidants in mitochondria.
      ,
      • Kameritsch P.
      • Singer M.
      • Nuernbergk C.
      • Rios N.
      • Reyes A.M.
      • Schmidt K.
      • et al.
      The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity.
      ).

      Effect of CO2 on hydrogen peroxide–mediated processes

      The reaction of CO2 with H2O2 results on HCO4- that oxidizes biomolecules such as thiols, typically more readily than H2O2 alone. In fact, while the second order rate constants for the reaction of H2O2 with the single protein thiol (Cys34) of human serum albumin and GSH are in the order of 1 to 2 M−1s−1, respectively, these values increase ca. 100-fold for their reaction with HCO4- (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      ). The accelerating effect of CO2 on H2O2-dependent thiol oxidation was proposed to explain the oxidative inactivation of protein tyrosine phosphatases (PTPs) in cells (
      • Zhou H.
      • Singh H.
      • Parsons Z.D.
      • Lewis S.M.
      • Bhattacharya S.
      • Seiner D.R.
      • et al.
      The biological buffer bicarbonate/CO 2 potentiates H 2O 2-mediated inactivation of protein tyrosine phosphatases.
      ). PTPs are known to be important molecular targets in redox signal transduction processes with their inactivation leading to increased intracellular phosphorylation (
      • Sies H.
      • Belousov V.v.
      • Chandel N.S.
      • Davies M.J.
      • Jones D.P.
      • Mann G.E.
      • et al.
      Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology.
      ). Indeed, one of the most enigmatic problems in redox biology has been how H2O2 inactivates PTPs, as the direct reaction is quite slow (ca. 10–40 M−1s−1 for PTP1B). Elegant biochemical and crystallographic work showed that HCO4- was capable of promoting the two-electron oxidation of the catalytic cysteine in PTP1B with an apparent rate constant one order of magnitude higher than that of H2O2 (i.e., 396 M−1s−1 at 25 mM HCO3-, pH 7.0 and 37 °C (
      • Zhou H.
      • Singh H.
      • Parsons Z.D.
      • Lewis S.M.
      • Bhattacharya S.
      • Seiner D.R.
      • et al.
      The biological buffer bicarbonate/CO 2 potentiates H 2O 2-mediated inactivation of protein tyrosine phosphatases.
      )). Considering that at 25 mM HCO3-, only ca. 1% of H2O2 would be present as HCO4-, the actual rate constant of the reaction would be in the range of 4 × 104 M−1s−1.
      While the disparate reactivity of HCO4- versus H2O2 over thiols is seen for low molecular weight thiols and some protein thiols
      The peroxidatic thiol in AhpE from M. tuberculosis reacts with H2O2 and HCO4- with k of 8.2 × 104 M−1s−1 and 1.1 × 107 M−1 s−1 (
      • Hugo M.
      • Turell L.
      • Manta B.
      • Botti H.
      • Monteiro G.
      • Netto L.E.S.
      • et al.
      Thiol and sulfenic acid oxidation of AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: kinetics, acidity constants, and conformational dynamics.
      ), respectively. In the case of the AhpE sulfenic acid, the k values for the hyperoxidation reaction with H2O2 and HCO4- are 40 M−1 s−1 and 2.1 × 103 M−1 s−1 (
      • Reyes A.M.
      • Hugo M.
      • Trostchansky A.
      • Capece L.
      • Radi R.
      • Trujillo M.
      Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation.
      ) at pH 7.4 and 25 °C, respectively
      , this characteristic is lost for peroxidatic protein thiols (e.g., peroxiredoxins), in which rate constants for both H2O2 and HCO4- (and also for ONOOH) converge in upper values in the order of 107 to 108 M−1s−1 (
      • Zeida A.
      • Trujillo M.
      • Ferrer-Sueta G.
      • Denicola A.
      • Estrin D.A.
      • Radi R.
      Catalysis of peroxide reduction by fast reacting protein thiols.
      ). Interestingly, peroxide-mediated thiol hyperoxidation
      The terms thiol “hyperoxidation” or “overoxidation” are used interchangeably in the literature to denote the oxidation of thiols over the sulfenic acid redox state toward sulfinic or sulfonic acid derivatives.
      (i.e., by the reaction with sulfenic acid intermediates, Equations 30a and 30b) is faster for HCO4- than H2O2, representing a mechanism of peroxiredoxin oxidative inactivation.
      RSOH + H2O2 → RSO2H + H2O
      [30a]


      RSOH + HCO4- → RSO2H + HCO3-
      [30b]


      For instance, HCO4-mediated human cytosolic (Prx1 and 2) and mitochondrial (Prx3) peroxiredoxin hyperoxidation occurs significantly faster than the corresponding reaction with H2O2 (
      • Peskin A.v.
      • Pace P.E.
      • Winterbourn C.C.
      Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO2.
      ,
      • Truzzi D.R.
      • Coelho F.R.
      • Paviani V.
      • Alves S.v.
      • Netto L.E.S.
      • Augusto O.
      The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1.
      ). For instance, the reaction of Prx1 with HCO4- and H2O2 occur with estimated k of 1.5 × 105 and 2.9 × 103 M−1s−1, respectively (
      • Truzzi D.R.
      • Coelho F.R.
      • Paviani V.
      • Alves S.v.
      • Netto L.E.S.
      • Augusto O.
      The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1.
      ).
      Studies on the relative toxicity of H2O2 versus HCO4- to microorganisms are almost lacking. One report indicates enhanced H2O2-dependent cytoxicity in the presence of CO2 to Pseudomona aeruginosa (
      • Yang Y.
      • Kitajima M.
      • Pham T.P.T.
      • Yu L.
      • Ling R.
      • Gin K.Y.H.
      • et al.
      Using Pseudomonas aeruginosa PAO1 to evaluate hydrogen peroxide as a biofouling control agent in membrane treatment systems.
      ). This result points to a potential increased microbicidal action of HCO4- over H2O2 in preventing bacterial growth and removing biofilms for disinfection purposes. The contribution of these reaction chemistries to the control of invading pathogens at the immune cell phagosomes was somehow hinted in early work on radiolytic inactivation of bacteria (
      • King D.A.
      • Sheafor M.W.
      • Hurst J.K.
      Comparative toxicities of putative phagocyte-generated oxidizing radicals toward a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae).
      ,
      • Wolcott R.G.
      • Franks B.S.
      • Hannum D.M.
      • Hurst J.K.
      Bactericidal potency of hydroxyl radical in physiological environments.
      ) and remains to be elucidated (Fig. 6). It is relevant to appreciate that the respiratory burst accompanying foreign body recognition by phagocytic cells promotes CO2 generation by the PPP (
      • Koo S. jie
      • Szczesny B.
      • Wan X.
      • Putluri N.
      • Garg N.J.
      Pentose phosphate shunt modulates reactive oxygen species and nitric oxide production controlling Trypanosoma cruzi in Macrophages.
      ), with a quotient respect to O2 consumption close to one (
      • Borregaard N.
      • Schwartz J.H.
      • Tauber A.I.
      Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.
      ) (Fig. 6).

      In addition to peroxide reactivity: CO2 and gene expression during inflammatory oxidative stress

      While this review underscores reaction mechanisms by which CO2 directly influences rates, fate, and yields of peroxide-mediated oxidations, it is important to note that CO2 can also modulate gene expression of proteins and enzymes linked to redox metabolism. Thus, the in cellula and in vivo effects of CO2 in oxidative modifications that can take place in the period of hours or days must take into consideration the fact that CO2 can provoke a specific repertoire of transcriptional events in a dose-dependent manner. In particular, genes associated with inflammation, immunity, and metabolism are CO2 sensitive and the process evolutionarily conserved (
      • Cummins E.P.
      • Strowitzki M.J.
      • Taylor C.T.
      Mechanisms and consequences of oxygen and carbon dioxide sensing in mammals.
      • Cummins E.P.
      • Oliver K.M.
      • Lenihan C.R.
      • Fitzpatrick S.F.
      • Bruning U.
      • Scholz C.C.
      • et al.
      NF-κB links CO 2 sensing to innate immunity and inflammation in mammalian cells.
      ,
      • Taylor C.T.
      • Cummins E.P.
      Regulation of gene expression by carbon dioxide.
      ,
      • Wang N.
      • Gates K.L.
      • Trejo H.
      • Favoreto S.
      • Schleimer R.P.
      • Sznajder J.I.
      • et al.
      Elevated CO 2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage.
      ,
      • Vohwinkel C.U.
      • Lecuona E.
      • Sun H.
      • Sommer N.
      • Vadász I.
      • Chandel N.S.
      • et al.
      Elevated CO2 levels cause mitochondrial dysfunction and impair cell proliferation.
      ,
      • Shigemura M.
      • Welch L.C.
      • Sznajder J.I.
      Hypercapnia regulates gene expression and tissue function.
      ).
      Early indications of intertwined events between the effects of CO2 on redox reaction chemistry and gene expression originated from studies in immunostimulated alveolar macrophages that generate significant levels of NO and peroxynitrite; this cellular system caused surfactant protein A tyrosine nitration in a process enhanced by the presence of CO2 (
      • Zhu S.
      • Basiouny K.F.
      • Crow J.P.
      • Matalon S.
      Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages.
      ). In addition to the CO2-catalyzed formation of nitrating species from peroxynitrite (i.e., CO3•- and NO2), CO2 may induce or upregulate NOS activity, which can additionally contribute to the enhanced protein tyrosine nitration. In fact, a 30 to 60 min exposure to 1.2 mM CO2 (i.e., cells incubated in a buffered media supplemented with 25 mM NaHCO3, under 5% CO2, 95% air; PCO2 ∼40 Torr, pH = 7.4) led to a significantly higher NOS activity in lipopolysaccharide-stimulated alveolar macrophages (
      • Zhu S.
      • Basiouny K.F.
      • Crow J.P.
      • Matalon S.
      Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages.
      ). In line with this observation, cytokine plus lipopolysaccharide stimulated alveolar epithelial cells exposed to high levels of CO2 (e.g., 5%–15%, hypercapnia) for 3 to 48 h revealed an increased production of NO and NOS expression and activity; this process was associated to cell injury and protein tyrosine nitration, underscoring that the interplay of NO-derived species with CO2 participate in inflammatory processes (
      • Lang J.D.
      • Chumley P.
      • Eiserich J.P.
      • Estevez A.
      • Bamberg T.
      • Adhami A.
      • et al.
      Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway.
      ).
      The mechanisms by which CO2 may regulate gene expression and transcriptional responses involving NOS are yet to be fully disclosed. A complex interaction exists between CO2 signaling, NF-κB, IKKα, and NOS expression (
      • Cummins E.P.
      • Strowitzki M.J.
      • Taylor C.T.
      Mechanisms and consequences of oxygen and carbon dioxide sensing in mammals.
      ,
      • Shigemura M.
      • Welch L.C.
      • Sznajder J.I.
      Hypercapnia regulates gene expression and tissue function.
      ,
      • Keogh C.E.
      • Scholz C.C.
      • Rodriguez J.
      • Selfridge A.C.
      • von Kriegsheim A.
      • Cummins E.P.
      Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.
      ,
      • Connelly L.
      • Palacios-Callender M.
      • Ameixa C.
      • Moncada S.
      • Hobbs A.J.
      Biphasic regulation of NF-κB activity underlies the pro- and anti-inflammatory actions of nitric oxide.
      ,
      • Grumbach I.M.
      • Chen W.
      • Mertens S.A.
      • Harrison D.G.
      A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription.
      ,
      • Taniguchi K.
      • Karin M.
      NF-B, inflammation, immunity and cancer: coming of age.
      ). CO2-dependent regulation of inflammatory signaling is in part dependent of the CO2 sensitivity of the NF-κB pathway and usually associated to the suppression of proinflammatory cytokines (
      • Keogh C.E.
      • Scholz C.C.
      • Rodriguez J.
      • Selfridge A.C.
      • von Kriegsheim A.
      • Cummins E.P.
      Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.
      ); however, CO2-induced inflammation has been also reported (
      • Abolhassani M.
      • Guais A.
      • Chaumet-Riffaud P.
      • Sasco A.J.
      • Schwartz L.
      Carbon dioxide inhalation causes pulmonary inflammation.
      ). In turn, the relationship between the NF-κB pathway and NOS is intricate: while the regulation of NOS expression is governed predominantly by the transcription factor NF-κB, NO exerts a biphasic regulation of the NF-κB pathway (
      • Connelly L.
      • Palacios-Callender M.
      • Ameixa C.
      • Moncada S.
      • Hobbs A.J.
      Biphasic regulation of NF-κB activity underlies the pro- and anti-inflammatory actions of nitric oxide.
      ). Thus, in the context of inflammatory oxidative stress, CO2 can directly modulate peroxide-mediated oxidations and also influence both redox and NO metabolism via the regulation of gene expression.

      Implications of the CO2 and peroxide interplay in redox signaling and metabolism

      Mechanistic analysis on H2O2 reactivity and specificity in thiol-based cell signaling has been presented recently (
      • Winterbourn C.C.
      Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.
      ). Redox signaling typically involves the reversible H2O2-dependent oxidation of proteins (e.g., thiol-disulfide transitions) that are not themselves particularly H2O2 reactive in isolated systems. Thus, efforts are underway to reveal how the “H2O2 signal” in cells can specifically result in target protein oxidation and subsequent downstream effects. Recent evidence points to H2O2 plus CO2-derived HCO4- as a feasible contributory redox signaling intermediate. In addition, CO2 modulates peroxynitrite-mediated thiol oxidation and tyrosine nitration processes in a way that may also impact cell signaling. Thus, we will analyze proposed mechanisms of peroxide-dependent signaling in which CO2 can play an important role.

      CO2 increases the reactivity of H2O2 toward PTP1B

      Phosphorylation cascades represent central processes in cellular redox signaling, with the phosphorylation state usually reflecting the relative activity of kinases and phosphatases. In this regard, the reversible regulation of PTP1B activity via transient oxidative inactivation at the active site cysteine represents one of the hallmarks of redox events in cell signaling (
      • Winterbourn C.C.
      Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.
      • Tonks N.K.
      Protein tyrosine phosphatases: from genes, to function, to disease.
      ,
      • Frijhoff J.
      • Dagnell M.
      • Godfrey R.
      • Östman A.
      Regulation of protein tyrosine phosphatase oxidation in cell adhesion and migration.
      ). In cells, the formation of H2O2 is a central requisite for PTP1B inactivation, which in turn results in increase of the phosphorylation state induced by receptor tyrosine kinase (RTK) activation. Notably, RTK activation triggers transient H2O2 production from plasma membrane–bound NADPH oxidases (NOXs) (
      • Lambeth J.D.
      • Neish A.S.
      Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited.
      ,
      • Lambeth J.D.
      NOX enzymes and the biology of reactive oxygen.
      ). Indeed, RTK activation facilitates NOX assembly to generate extracellular O2•-, which in turn is readily converted enzymatically to H2O2 (i.e., by EC-SOD) and enters the cells to promote redox signaling. This way, the tyrosine kinase–dependent phosphorylation events are synergistically coupled to H2O2-dependent inactivation of PTP1B. However, one of the most mysterious issues in the redox signaling field has been how the rather sluggish reaction of H2O2 with PTP1B (k = 24 M−1s−1, reviewed in (
      • Truzzi R.D.
      • Augusto O.
      Influence of CO2 on hydroperoxide metabolism.
      )) would lead to cellular responses in the time range of minutes and outcompeting the much faster reactions of H2O2 with abundant peroxidatic systems such as those of peroxiredoxins and GSH peroxidases (k ca. 107–108 M−1s−1) (
      • Zeida A.
      • Trujillo M.
      • Ferrer-Sueta G.
      • Denicola A.
      • Estrin D.A.
      • Radi R.
      Catalysis of peroxide reduction by fast reacting protein thiols.
      ,
      • Sies H.
      • Belousov V.v.
      • Chandel N.S.
      • Davies M.J.
      • Jones D.P.
      • Mann G.E.
      • et al.
      Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology.
      ). Indeed, the thioredoxin reductase/thioredoxin/peroxiredoxin system can completely inhibit H2O2-dependent PTP1B oxidation in vitro in phosphate buffer systems (
      • Dagnell M.
      • Frijhoff J.
      • Pader I.
      • Augsten M.
      • Boivin B.
      • Xu J.
      • et al.
      Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-ß receptor tyrosine kinase signaling.
      ,
      • Dagnell M.
      • Pace P.E.
      • Cheng Q.
      • Frijhoff J.
      • Östman A.
      • Arnér E.S.J.
      • et al.
      Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H2O2 exposure.
      ). However, upon addition of HCO3/CO2, PTP1B is more readily oxidized by H2O2 and peroxiredoxins are not capable of completely preventing PTP1B inactivation (
      • Dagnell M.
      • Cheng Q.
      • Rizvi S.H.M.
      • Pace P.E.
      • Boivin B.
      • Winterbourn C.C.
      • et al.
      Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades.
      ). These observations are compatible with reaction of HCO4- with PTP1 (13), where peroxiredoxins cannot fully neutralize the reaction of H2O2 with CO2. In a recent elegant work, studies in an adenocarcinoma cell line stimulated with epidermal growth factor (EGF), known to act through RTK, have shown that increasing intracellular HCO3- concentrations enhanced total protein phosphotyrosine levels in parallel with the occurrence of PTP1B oxidation/inactivation. In fact, the presence of HCO3- was an absolute requirement for EGF-induced cellular oxidation of PTP1B
      The thioredoxin reductase-thioredoxin system can reduce back the thiol-oxidized PTP1 to the active state (
      • Dagnell M.
      • Pace P.E.
      • Cheng Q.
      • Frijhoff J.
      • Östman A.
      • Arnér E.S.J.
      • et al.
      Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H2O2 exposure.
      ).
      , allowing physiological steady-state levels of H2O2 to inactivate PTPs within a time scale of minutes (
      • Dagnell M.
      • Cheng Q.
      • Rizvi S.H.M.
      • Pace P.E.
      • Boivin B.
      • Winterbourn C.C.
      • et al.
      Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades.
      ,
      • Zhou H.
      • Singh H.
      • Parsons Z.D.
      • Lewis S.M.
      • Bhattacharya S.
      • Seiner D.R.
      • et al.
      The biological buffer bicarbonate/CO 2 potentiates H 2O 2-mediated inactivation of protein tyrosine phosphatases.
      ). Notably, NOX activation is typically coupled with enhanced CO2 formation by the PPP (
      • Britt E.C.
      • Lika J.
      • Giese M.A.
      • Schoen T.J.
      • Seim G.L.
      • Huang Z.
      • et al.
      Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.
      ,
      • Borregaard N.
      • Schwartz J.H.
      • Tauber A.I.
      Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.
      ) These data reconcile mechanisms of PTP1B-mediated H2O2 oxidation/inactivation in vitro and in vivo and points to CO2 and HCO4- as relevant intermediates in redox signaling.

      CO2 favors H2O2-dependent peroxiredoxin hyperoxidation and affects redox relays

      H2O2-dependent thiol hyperoxidation of peroxiredoxins is significantly accelerated in HCO3-containing buffers (
      • Peskin A.v.
      • Pace P.E.
      • Winterbourn C.C.
      Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO2.
      ,
      • Truzzi D.R.
      • Coelho F.R.
      • Paviani V.
      • Alves S.v.
      • Netto L.E.S.
      • Augusto O.
      The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1.
      ), with CO2 typically increasing the k value by two orders of magnitude. Thus, by this mechanism, a fraction of the oxidized peroxidatic protein thiol in the sulfenic acid state can react with HCO4- and evolve to hyperoxidized (and inactive) forms (i.e., sulfinic acid) in kinetic competition with its reaction with the resolving protein cysteine residue leading to the formation of an intermolecular disulfide in the “typical” peroxiredoxins (such as peroxiredoxins 2 and 3, (
      • Zeida A.
      • Trujillo M.
      • Ferrer-Sueta G.
      • Denicola A.
      • Estrin D.A.
      • Radi R.
      Catalysis of peroxide reduction by fast reacting protein thiols.
      )). Peroxiredoxin hyperoxidation jeopardizes the reversibility of redox signaling in the context of “redox relays” mediated by the peroxiredoxin sulfenic acid intermediate interacting with other thiol-containing proteins (
      • Sobotta M.C.
      • Liou W.
      • Stöcker S.
      • Talwar D.
      • Oehler M.
      • Ruppert T.
      • et al.
      Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling.
      ,
      • Stöcker S.
      • van Laer K.
      • Mijuskovic A.
      • Dick T.P.
      The conundrum of hydrogen peroxide signaling and the emerging role of peroxiredoxins as redox relay hubs.
      ). Also, peroxiredoxin hyperoxidation may further increase H2O2 levels due to the inability of these inactivated peroxiredoxins to decompose H2O2, which may secondarily favor the oxidation of less H2O2-reactive proteins. Thus, CO2 provides a feasible mechanism for the “floodgate” hypothesis of redox signaling (
      • Wood Z.A.
      • Poole L.B.
      • Karplus P.A.
      Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.
      ,
      • Cho C.S.
      • Yoon H.J.
      • Kim J.Y.
      • Woo H.A.
      • Rhee S.G.
      Circadian rhythm of hyperoxidized peroxiredoxin II is determined by hemoglobin autoxidation and the 20S proteasome in red blood cells.
      ), under which excess H2O2 levels disrupts signaling by sequestering a fraction of peroxiredoxin in an inactive state
      This hyperoxidized proteoform may, in the case of peroxiredoxins, return to the resting state by the action of ATP-dependent reducing enzymatic mechanisms such as sulfiredoxins (
      • Chang T.S.
      • Jeong W.
      • Woo H.A.
      • Sun M.L.
      • Park S.
      • Sue G.R.
      Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine.
      ).
      .

      CO2, tyrosine nitration, and redox signaling

      Protein tyrosine nitration may affect signaling pathways in a variety of ways (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). Both tyrosine nitration yields and regioselectivity are affected by the presence of CO2 (
      • Demicheli V.
      • Moreno D.M.
      • Radi R.
      Human Mn-superoxide dismutase inactivation by peroxynitrite: a paradigm of metal-catalyzed tyrosine nitration: in vitro and in vivo.
      ,
      • Demicheli V.
      • Tomasina F.
      • Sastre S.
      • Zeida A.
      • Tórtora V.
      • Lima A.
      • et al.
      Cardiolipin interactions with cytochrome c increase tyrosine nitration yields and site-specificity.
      ,
      • Surmeli N.B.
      • Litterman N.K.
      • Miller A.F.
      • Groves J.T.
      Peroxynitrite mediates active site tyrosine nitration in manganese superoxide dismutase. Evidence of a role for the carbonate radical anion.
      ). As biologically relevant examples, protein tyrosine nitration in HSP90 (heat shock protein 90) (
      • Franco M.C.
      • Ye Y.
      • Refakis C.A.
      • Feldman J.L.
      • Stokes A.L.
      • Basso M.
      • et al.
      Nitration of Hsp90 induces cell death.
      ,
      • Jandy M.
      • Noor A.
      • Nelson P.
      • Dennys C.N.
      • Karabinas I.M.
      • Pestoni J.C.
      • et al.
      Peroxynitrite nitration of Tyr 56 in Hsp90 induces PC12 cell death through P2X7R-dependent PTEN activation.
      ) and NGF (nerve growth factor) (
      • Pehar M.
      • Vargas M.R.
      • Robinson K.M.
      • Cassina P.
      • England P.
      • Beckman J.S.
      • et al.
      Peroxynitrite transforms nerve growth factor into an apoptotic factor for motor neurons.
      ) triggers cell death pathways, causes PP2A (protein serine phosphatase) inactivation and enhanced cellular phosphorylation (
      • Low I.C.C.
      • Loh T.
      • Huang Y.
      • Virshup D.M.
      • Pervaiz S.
      Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56 δ stabilizes its antiapoptotic activity.
      ,
      • Yee Y.H.
      • Chong S.J.F.
      • Kong L.R.
      • Goh B.C.
      • Pervaiz S.
      Sustained IKKβ phosphorylation and NF-κB activation by superoxide-induced peroxynitrite-mediated nitrotyrosine modification of B56γ3 and PP2A inactivation.
      ), affects the ability of the cytokine CCL2 (C–C motif chemokine ligand 2) to exert its normal chemoattractant activity for immune cells (
      • Molon B.
      • Ugel S.
      • del Pozzo F.
      • Soldani C.
      • Zilio S.
      • Avella D.
      • et al.
      Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells.
      ), and disrupts insulin receptor substrate−1 (IRS−1)–dependent signaling (
      • Pilon G.
      • Charbonneau A.
      • White P.J.
      • Dallaire P.
      • Perreault M.
      • Kapur S.
      • et al.
      Endotoxin mediated-INOS induction causes insulin resistance via ONOO- induced tyrosine nitration of IRS-1 in skeletal muscle.
      ,
      • André D.M.
      • Calixto M.C.
      • Sollon C.
      • Alexandre E.C.
      • Tavares E.B.G.
      • Naime A.C.A.
      • et al.
      High-fat diet-induced obesity impairs insulin signaling in lungs of allergen-challenged mice: improvement by resveratrol.
      ). In these contexts, information on how CO2 levels modulate tyrosine nitration and signaling events is basically lacking.
      On the other hand, CO2 may also affect tyrosine nitration and signaling cascades by changes in gene expression patterns. Early work showed that hypercapnic exposure to lung cells lead to enhanced protein nitrotyrosine levels, which were associated to increased NOS expression and activity (
      • Zhu S.
      • Basiouny K.F.
      • Crow J.P.
      • Matalon S.
      Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages.
      ,
      • Lang J.D.
      • Chumley P.
      • Eiserich J.P.
      • Estevez A.
      • Bamberg T.
      • Adhami A.
      • et al.
      Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway.
      ). Thus, cellular tyrosine nitration stimulated by high CO2 levels may be a combination of increased NO/peroxynitrite formation together with the favored formation of ONOOCO2-, although precise disclosure is lacking.
      The effects of hypercapnia in vivo in terms of protein tyrosine nitration and inflammatory oxidative stress are yet to be defined. For example, while it was indicated that long term exposure to hypercapnia may exert anti-inflammatory actions and potentially decrease extents of protein tyrosine nitration (
      • Masood A.
      • Yi M.
      • Lau M.
      • Belcastro R.
      • Shek S.
      • Pan J.
      • et al.
      Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats.
      ), other works show that CO2 can promote NO and peroxynitrite production, protein nitrotyrosine formation, and oxidative injury (
      • Honoré J.C.
      • Kooli A.
      • Hou X.
      • Hamel D.
      • Rivera J.C.
      • Picard É.
      • et al.
      Sustained hypercapnia induces cerebral microvascular degeneration in the immature brain through induction of nitrative stress.
      ). A complex relationship between cell/animal CO2 exposure and inflammatory mediators exists with an overall anti-inflammatory effect of CO2, in part through downregulation of the NF-κB signaling pathways (
      • Taylor C.T.
      • Cummins E.P.
      Regulation of gene expression by carbon dioxide.
      ). Nonetheless, recent works indicated that selective tyrosine nitration in protein serine phosphatase PP2A regulatory subunits leads to enzyme inactivation and enhanced cellular phosphorylation events that result in upregulation of the NF-κB pathway (
      • Low I.C.C.
      • Loh T.
      • Huang Y.
      • Virshup D.M.
      • Pervaiz S.
      Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56 δ stabilizes its antiapoptotic activity.
      ,
      • Yee Y.H.
      • Chong S.J.F.
      • Kong L.R.
      • Goh B.C.
      • Pervaiz S.
      Sustained IKKβ phosphorylation and NF-κB activation by superoxide-induced peroxynitrite-mediated nitrotyrosine modification of B56γ3 and PP2A inactivation.
      ); these processes may promote proinflammatory phenotypes. No information is available as to what extent CO2 regulates tyrosine nitration (and activity) in PP2A. Thus, as inflammation, cytokines and NF-κB are intertwined with the NO and redox pathways; the effect of different CO2 exposure regimes on peroxynitrite-dependent signaling cascades involving tyrosine nitration require specific future studies.

      Other possible effects of CO2 on peroxynitrite-dependent redox signaling

      In addition to the effects of peroxynitrite on signaling pathways through protein tyrosine nitration, PTPs can be inactivated by peroxynitrite via thiol oxidation (
      • Takakura K.
      • Beckman J.S.
      • MacMillan-Crow L.A.
      • Crow J.P.
      Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.
      ,
      • Metere A.
      • Iorio E.
      • Pietraforte D.
      • Podo F.
      • Minetti M.
      Peroxynitrite signaling in human erythrocytes: synergistic role of hemoglobin oxidation and band 3 tyrosine phosphorylation.
      ). In this regard, low concentrations of peroxynitrite promote a cellular tyrosine hyperphosphorylated state (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Brito C.
      • Naviliat M.
      • Tiscornia A.C.
      • Vuillier F.
      • Gualco G.
      • Dighiero G.
      • et al.
      Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death.
      ), while high concentrations impair phosphorylation and trigger apoptotic cell death. The thiol oxidation process is also expected to be influenced by changing CO2 levels. Indeed, the presence of CO2 shifts the mechanism of peroxynitrite-mediated thiol oxidation and favors the initiation of oxygen-dependent radical chain reactions (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • et al.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). Moreover, enhanced levels of CO2 partially outcompete peroxynitrite detoxification pathways and promote free radical–dependent oxidation and hyperoxidation of thiol-containing proteins, which may end up influencing signaling pathways. In this regard, it is interesting to consider that low levels of peroxynitrite facilitate mitochondrial biogenesis (
      • Marine A.
      • Krager K.J.
      • Aykin-Burns N.
      • MacMillan-Crow L.A.
      Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells.
      ) and, potentially, mitophagy (
      • Geldon S.
      • Fernández-Vizarra E.
      • Tokatlidis K.
      Redox-mediated regulation of mitochondrial biogenesis, dynamics, and respiratory chain assembly in yeast and human cells.
      ). Albeit speculative, it would be worth to explore how the CO2 and peroxynitrite interplay in mitochondria may influence redox-dependent cascades related to mitochondrial turnover and cell signaling. The potential connection among redox homeostasis, mitochondrial oxidant formation, and mitochondrial biogenesis have been reviewed recently (
      • Geldon S.
      • Fernández-Vizarra E.
      • Tokatlidis K.
      Redox-mediated regulation of mitochondrial biogenesis, dynamics, and respiratory chain assembly in yeast and human cells.
      ) and opens possibilities to understand how peroxide-mediated signaling (e.g., via thiol oxidation) in the presence of CO2 contribute to the process.

      Conclusions

      The elements presented in this review lead to the conclusion that metabolically derived CO2 participates in the modulation of redox reactions that range from signaling to toxicity. In this regard, CO2 assists shaping the cellular redox landscape. Indeed, CO2 is well suited to couple intermediary metabolism with redox signaling both in mitochondrial and extramitochondrial sites (Fig. 1). Moreover, the concomitant metabolic formation of reactive species such as O2•-, H2O2, and peroxynitrite during the oxidative burst of activated phagocytes together with CO2 (formed by the PPP) may play important roles on oxidative killing of invading pathogens via formation of HCO4- and/or ONOOCO2- (Fig. 6). Obviously, changes in CO2 levels also lead to pH changes and specific efforts are required to dissect their relative influence in cellular responses. But, it has become clear that CO2 per se participates in the modulation of cell physiology and pathology by a series of mechanisms that include (1) direct posttranslational protein modifications (e.g., carbamylation, (
      • Linthwaite V.L.
      • Janus J.M.
      • Brown A.P.
      • Wong-Pascua D.
      • O’Donoghue A.M.C.
      • Porter A.
      • et al.
      The identification of carbon dioxide mediated protein post-translational modifications.
      ,
      • Linthwaite V.L.
      • Pawloski W.
      • Pegg H.B.
      • Townsend P.D.
      • Thomas M.J.
      • H So V.K.
      • et al.
      Ubiquitin is a carbon dioxide-binding protein.