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Peroxiredoxin hyperoxidation increases in the presence of bicarbonate/carbon dioxide

Submitted by redoxoma on
peroxidase cycle, hyperoxidation pathway and interaction and oxidation of other thiol proteins, signaling

Highlights by Daniela R. Truzzi and Ohara Augusto, from Instituto de Química da USP
Corresponding author e-mail: dtruz-I-am-here-zi@hotmail.com@iq.usp.br

Peroxiredoxins (Prx) are abundant thiol peroxidases that react rapidly with H2O2, constituting an important antioxidant defense and acting as sensors and transmitters of H2O2 signals in cells. Eukariotic 2-Cys Prxs lose their peroxidase activity at high hydroperoxide levels in a process called hyperoxidation, which constitutes a pathway to Prx functions beyond the antioxidant activity (Fig. 1) [1]. Since the biologically ubiquitous HCO3–/CO2 pair accelerates the reaction of H2O2 with several biothiols [2, 3], we examined whether physiological concentrations of the HCO3–/CO2 pair (25 mM) could increase recombinant human peroxiredoxin 1 (Prx1) hyperoxidation by H2O2 [4]. Immunoblotting, kinetic and MS/MS experiments revealed that HCO3–/CO2 increases Prx1 hyperoxidation and inactivation both in the presence of excess H2O2 and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (dithiothreitol) turnover. Based on previous studies, we hypothesized that the stimulating effect of HCO3–/CO2 was due to HCO4– (peroxymonocarbonate), a peroxide present in equilibrated solutions of H2O2 and HCO3–/CO2 [2, 3]. Indeed, additional experiments and calculations uncovered that HCO4– oxidizes CPSOH to CPSO2– with a second-order rate constant two orders of magnitude higher than H2O2 ((1.5 ± 0.1) × 10⁵ and (2.9 ± 0.2) × 10³ M⁻¹.s⁻¹, respectively) and that HCO4– is 250 times more efficient than H2O2 at inactivating 1% Prx1 per turnover [4]. The fact that the biologically ubiquitous HCO3–/CO2 pair stimulates Prx1 hyperoxidation may be quite relevant to cell homeostasis because the antioxidant and redox relay functions of the enzyme decline but other actions may rise, such as the chaperone-like activity, the redox signaling pathways mediated by Cys-based proteins that are poorly reactive towards H2O2 and the maintenance of Trx-dependent activities (Fig. 1). Relevantly, parallel studies led by Christine Winterbourn and co-workers reported that the HCO3–/CO2 pair also stimulates Prx2 and Prx3 hyperoxidation [5] and protein tyrosine phosphatase 1B inactivation involved in epidermal growth factormediated signaling [6]. Taking together, these recent studies confirm that HCO4– deserves further investigation as a biological oxidant [3] and point to a possible role of HCO3–/CO2 levels in H2O2mediated signaling.

Peroxidase cycle, hyperoxidation pathway


Figure 1. Simplified scheme of the catalytic cycle and the hyperoxidation pathway of 2-Cys Prxs and the activities related to them.

References

  1. E. A. Veal, Z. E. Underwood, L. E. Tomalin, B. A. Morgan, C. S. Pillay. Hyperoxidation of Peroxiredoxins: Gain or Loss of Function? Antioxidants & Redox Signaling, 28(7): 574–90, 2018. | doi: 10.1089/ars.2017.7214
  2. D. F. Trindade, G. Cerchiaro, O. Augusto. A Role for Peroxymonocarbonate in the Stimulation of Biothiol Peroxidation by the Bicarbonate/Carbon Dioxide Pair Chemical Research in Toxicology, 19(11): 1475–82, 2006. | doi: 10.1021/tx060146x
  3. D. R. Truzzi, O. Augusto. Influence of CO2 on Hydroperoxide Metabolism Hydrogen Peroxide Metabolism in Health and Disease, (Vissers, M.C.M., Hampton, M., Kettle, A.J. eds) 81–99, Oxidative stress and disease, Taylor & Francis/CRC Press, Boca Raton, 2017. | doi: 10.1201/9781315154831-4
  4. D. R. Truzzi, F. R. Coelho, V. Paviani, S. V. Alves, L. E. S. Netto, O. Augusto. The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1 Journal of Biological Chemistry, 294(38): 14055–67, 2019. | doi: 10.1074/jbc.ra119.008825
  5. A. V. Peskin, P. E. Pace, C. C. Winterbourn. Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO2 Free Radical Biology and Medicine, 145: 1–7, 2019. | doi: 10.1016/j.freeradbiomed.2019.09.010
  6. M. Dagnell, Q. Cheng, S. H. M. Rizvi, P. E. Pace, B. Boivin, C. C. Winterbourn, E. S. J. Arnér. Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades Journal of Biological Chemistry, 294(33): 12330–8, 2019. | doi: 10.1074/jbc.ra119.009001

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A radical connection: Peroxynitrite and carbon dioxide

Submitted by redoxoma on
A radical connection

Redoxoma Highlights by by Ohara Augusto
Corresponding author e-mail: oag-I-am-here-usto@hotmail.com@iq.usp.br

In 2002, my laboratory published a review with the title “Nitrogen dioxide and carbonate radical anion: Two emerging radicals in Biology” [1] to draw attention to these radicals, which had received limited (nitrogen dioxide) or practically no (carbonate radical) attention in the biological literature by then. However, the times were changing due to the work of several researchers around the world. These investigators established that carbon dioxide (produced continuously by cell metabolism) and peroxynitrite (the strong oxidant produced by the extremely rapid reaction between superoxide radical and nitric oxide) react with a considerable second-order rate constant to produce an unstable adduct, nitrosoperoxocarbonate, which undergoes O–O bond homolysis, producing solvent-caged nitrogen dioxide and carbonate radical pairs. Part of these radicals escapes the cage as nitrogen dioxide and carbonate radicals (determined as 0.33 each/peroxynitrite) while the remaining caged radicals decayed to nitrate and carbon dioxide (determined as 0.67 each/peroxynitrite) (Figure 1). Since part of the carbon dioxide is recycled, it can be viewed as a catalyst of peroxynitrite decomposition to radicals. Therefore, nitrogen dioxide and carbonate radicals were likely to play important roles in the biological actions of peroxynitrite. This radical mechanism has passed the test of the years and has attained widespread acceptance, despite occasional attacks by a few researchers, particularly by W H Koppenol and co-workers. The last attack was published in 2018 [2], predicting a yield of nitrogen dioxide/carbonate radical of less than 0.01 (per peroxynitrite) under physiological conditions and, in contrast to the widely accepted view, suggesting that radical generation is inconsequential to peroxynitrite-induced oxidative damage. To avoid the eventual confusion provoked by such claims, a number of researchers actively involved in establishing the radical connection between peroxynitrite and carbon dioxide reviewed the experimental and theoretical evidence for the radical mechanism and critically analyzed the experiments that have led to erroneous conclusions [3]. The authors conclusively show that all confirmed research by several independent groups on the structural and dynamic properties of peroxynitrite support the radical mechanism. Therefore, the radical mechanism, which invokes the intermediacy of nitrogen dioxide and carbonate radical in all carbon dioxide-catalyzed oxidations/nitrations/nitrosations by peroxynitrite, can be confidently used to interpret peroxynitrite biochemistry.

Schematic reaction of peroxynitrite and carbon dioxide

Figure 1. Schematic representation of the reaction between peroxynitrite and carbon dioxide to produce nitrogen dioxide and carbonate radicals, which are quite reactive towards biomolecules (BM).

References

  1. O. Augusto, M. G. Bonini, A. M. Amanso, E. Linares, C. C. Santos, S. L. De Menezes. Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology Free Radical Biology and Medicine, 32(9): 841–59, 2002. | doi: 10.1016/s0891-5849(02)00786-4
  2. S. Serrano-Luginbuehl, R. Kissner, W. H. Koppenol. Reaction of CO2 with ONOO–: One Molecule of CO2 Is Not Enough Chemical Research in Toxicology, 31(8): 721–30, 2018. | doi: 10.1021/acs.chemrestox.8b00068
  3. O. Augusto, S. Goldstein, J. K. Hurst, J. Lind, S. V. Lymar, G. Merenyi, R. Radi. Carbon dioxide-catalyzed peroxynitrite reactivity – The resilience of the radical mechanism after two decades of research Free Radical Biology and Medicine, 135: 210–5, 2019. | doi: 10.1016/j.freeradbiomed.2019.02.026

Ohara Augusto, (oag-I-am-here-usto@hotmail.com@iq.usp.br) Director of CEPID Redoxoma, PhD Professor at Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil

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