Ascorbate peroxidase

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L-ascorbate peroxidase
APX-ascorbate.png
Structure of ascorbate peroxidase in complex with ascorbate (in blue); a histidine ligand (in red) coordinates to the iron of the heme group (also in red). Image taken from PDB 1OAF and created using Pymol
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EC no. 1.11.1.11
CAS no. 72906-87-7
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Ascorbate peroxidase (or L-ascorbate peroxidase, APX or APEX) (EC 1.11.1.11) is an enzyme that catalyzes the chemical reaction

Contents

L-ascorbate + H2O2 dehydroascorbate + 2 H2O

It is a member of the family of heme-containing peroxidases. Heme peroxidases catalyse the H2O2-dependent oxidation of a wide range of different, usually organic, substrates in biology.

This enzyme belongs to the family of oxidoreductases, specifically those acting on a peroxide as acceptor (peroxidases). The systematic name of this enzyme class is L-ascorbate:hydrogen-peroxide oxidoreductase. Other names in common use include L-ascorbic acid peroxidase, L-ascorbic acid-specific peroxidase, ascorbate peroxidase, and ascorbic acid peroxidase. This enzyme participates in the ascorbate and aldarate metabolism. APXs are important in cellular antioxidant networks in photosynthetic organisms; they are the primary component of the ascorbate-glutathione cycle and are important for peroxide scavenging and redox signaling [1] .

Reaction

In the catalytic cycle, the immediate one-electron oxidized product is monodehydroascorbate (MDHA). MDHA is either enzymatically reduced back to ascorbate by monodehydroascorbate reductase (MDAR) or two MDHA molecules disproportionate to ascorbate and dehydroascorbate (DHA). [2]

Overview

Ascorbate-dependent peroxidase activity was first reported in 1979, [3] [4] more than 150 years after the first observation of peroxidase activity in horseradish plants [5] and almost 40 years after the discovery of the closely related cytochrome c peroxidase enzyme. [6]

Peroxidases have been classified into three types (class I, class II and class III): ascorbate peroxidases is a class I peroxidase enzyme. [7] APXs catalyze the H2O2-dependent oxidation of ascorbate in plants, algae and certain cyanobacteria. [8] APX has high sequence identity to cytochrome c peroxidase, which is also a class I peroxidase enzyme. Under physiological conditions, the immediate product of the reaction, the monodehydroascorbate radical, is reduced back to ascorbate by a monodehydroascorbate reductase (monodehydroascorbate reductase (NADH)) enzyme. In the absence of a reductase, two monodehydroascorbate radicals disproportionate rapidly to dehydroascorbic acid and ascorbate. APX is an integral component of the glutathione-ascorbate cycle. [9]

Substrate specificity

APX enzymes show high specificity for ascorbate as an electron donor, but most APXs will also oxidise other organic substrates that are more characteristic of the class III peroxidases (such as horseradish peroxidase), in some cases at rates comparable to that of ascorbate itself. This means that defining an enzyme as an APX is not straightforward, but is usually applied when the specific activity for ascorbate is higher than that for other substrates. Some proteins from the APX family lack the ascorbate-binding amino acid residues suggesting that they might oxidize other molecules than ascorbate. [10]

Mechanism

Most of the information on mechanism comes from work on the pea cytosolic and soybean cytosolic enzymes. The mechanism of oxidation of ascorbate is achieved by means of an oxidized Compound I intermediate, which is subsequently reduced by substrate in two, sequential single electron transfer steps (equations [1]–[3], where HS = substrate and S• = one electron oxidized form of substrate).

APX follows the typical heme-peroxidase mechanism with high-valent iron intermediates [11] :

  1. Formation of Compound I: APX reacts with H2O2 to form Compound I - where the heme is oxidized to Fe4+ = O (oxyferryl). This produces a porphyrin pi-organic cation radical [12] .
    1. APX + H2O2 → Compound I + H2O [1]
  2. Formation of Compound II: Through an one electron reduction, Compound I is reduced by substrate (HS) to form Compound II; Compound II accepts a second electron from ascorbate to regenerate the ferric resting state [13] . This is s sequential single-electron transfer steps [14] .
    1. Compound I + HS → Compound II + S• [2]
    2. Compound II + HS → APX + S• + H2O [3]

In ascorbate peroxidase, Compound I is a transient (green) species and contains a high-valent iron species (known as ferryl heme, FeIV) and a porphyrin pi-cation radical, [15] [16] as found in horseradish peroxidase. Compound II contains only the ferryl heme. Spectroscopic and kinetic work on plant APXs supports these intermediates and sequential one electron transfers [17] .

Structural information

The structure of pea cytosolic APX was reported in 1995. [18] The binding interaction of soybean cytosolic APX with its physiological substrate, ascorbate [19] [20] and with a number of other substrates [21] are also known.

As of late 2007, 12 structures have been solved for this class of enzymes, with PDB accession codes 1APX, 1IYN, 1OAF, 1OAG, 1V0H, 2CL4, 2GGN, 2GHC, 2GHD, 2GHE, 2GHH, and 2GHK.

Cellular context and pathways

APX participates in the ascorbate-glutathione cycle, an integrated pathway that couples H2O2 detoxification to regeneration of ascorbate using NAD(P)H and glutathione [22] . This cycle operates in multiple cellular compartments such as cytosol, chloroplast stroma and thylakoid, mitochondria and peroxisomes, enabling compartment specific control of peroxide homeostasis and redox signaling [23] .

Regulation and isoenzymes

Plants typically express multiple APX isoenzymes with distinct sub-cellular localizations (cytosolic, chloroplastic stromal, chloroplastic thylakoid/peripheral, mitochondrial, peroxisomal) and different biochemical properties (pH optima, stability, stress responsiveness) [24] . Expression and activity of APX isoforms are regulated transcriptionally and post-translationally in response to light, development and abiotic stresses (drought, salinity, high light, temperature) [25] .

Increased activity of APX also occurs alongside other antioxidant enzymes responsible for protection mechanisms such as catalase, superoxide dismutase and glutathione [26] . These isoform differences allow fine tuned H2O2 detoxification and localized redox signaling [27] .

Active sites, binding and residues

Structural and mutagenesis studies have identified key residues that form an ascorbate-binding pocket adjacent to the heme [28] . Conserved residues (for example Arg-172 in many plant APXs - numbering varies by sequence) contribute critical hydrogen bonds and electrostatic interactions that position ascorbate for efficient electron transfer to the ferryl heme. mutation on Arg-172 (and neighboring residues) recedes ascorbate binding and catalytic efficiency while sometimes preserving generic peroxidase activity with alternative small substrates [29] [30] .

Known crystal and structural studies

X-ray and neutron crystallography studies, along with spectroscopic/kinetic analysis have characterized APX active site geometry, electron transfer sites and intermediate states [31] . Representative structural and mechanistic studies include crystallographic identification of electron-transfer sites, mutagenesis of active-sites residues and comparisons between APX and other peroxidases (e.g., cytochrome c peroxidase) [32] .

Organismal distribution and representative examples

APX is widely distributed among photosynthetic organisms - higher plants, green algae, dinoflagellates and many cyanobacteria - though gene family size and isoform composition vary. A commonly studied plant enzyme is APX1 from Arabidopsis thaliana (common name mouse-ear cress), which has been extensively used for biochemical, genetic and strutural characterization; other model species with characterized APXs include pea, spinach and sorghum [33] . In some unicellular photosynthetic taxa (e.g., Euglena gracilis [34] ) APX plays a particularly central H2O2 detoxifying role where catalase is reduced or absent [35] [36] .

Physiological significance and applications

Physiological: APX contributes to protection from oxidative damage during photosynthesis [37] , respiration and stress conditions; by controlling local H2O2 concentrations APX also shapes redox signaling networks that affect gene expression, programmed cell death an acclimation responses [38] .

Cellular imaging: Both pea APX [39] and soybean APX and their mutants (APEX, APEX2) [40] have been used in electron microscopy studies for cellular imaging.

See also

References

  1. ↑ Shigeoka, S. (2002-05-15). "Regulation and function of ascorbate peroxidase isoenzymes". Journal of Experimental Botany. 53 (372): 1305–1319. doi:10.1093/jexbot/53.372.1305.
  2. ↑ Pang, Cai-Hong; Wang, Bao-Shan (2010), Anjum, Naser A.; Chan, Ming-Tsair; Umar, Shahid (eds.), "Role of Ascorbate Peroxidase and Glutathione Reductase in Ascorbate–Glutathione Cycle and Stress Tolerance in Plants" , Ascorbate-Glutathione Pathway and Stress Tolerance in Plants, Dordrecht: Springer Netherlands, pp. 91–113, doi:10.1007/978-90-481-9404-9_3, ISBN   978-90-481-9403-2 , retrieved 2025-10-21
  3. ↑ Kelly GJ, Latzko E (December 1979). "Soluble ascorbate peroxidase: detection in plants and use in vitamim C estimation". Die Naturwissenschaften. 66 (12): 617–9. doi:10.1007/bf00405128. PMID   537642. S2CID   12729653.
  4. ↑ Groden D, Beck E (June 1979). "H2O2 destruction by ascorbate-dependent systems from chloroplasts". Biochimica et Biophysica Acta (BBA) - Bioenergetics. 546 (3): 426–35. doi:10.1016/0005-2728(79)90078-1. PMID   454577.
  5. ↑ Planche LA (1810). "Note sur la sophistication de la résine de jalap et sur les moyens de la reconnaître". Bull Pharm. 2: 578–80.
  6. ↑ Altschul AM, Abrams R, Hogness TR (1940). "Cytochrome c Peroxidase" (PDF). Journal of Biological Chemistry. 136 (3): 777–794. doi: 10.1016/S0021-9258(18)73036-6 .
  7. ↑ Welinder KG (1992). "Superfamily of plant, fungal and bacterial peroxidases". Curr. Opin. Chem. Biol. 2 (3): 388–393. doi:10.1016/0959-440x(92)90230-5.
  8. ↑ Raven EL (August 2003). "Understanding functional diversity and substrate specificity in haem peroxidases: what can we learn from ascorbate peroxidase?". Natural Product Reports. 20 (4): 367–81. doi:10.1039/B210426C. PMID   12964833.
  9. ↑ Noctor G, Foyer CH (June 1998). "Ascorbate and Glutahione: Keeping Active Oxygen Under Control". Annual Review of Plant Physiology and Plant Molecular Biology. 49: 249–279. doi:10.1146/annurev.arplant.49.1.249. PMID   15012235.
  10. ↑ Lazzarotto F, Menguer PK, Del-Bem LE, Zámocký M, Margis-Pinheiro M (April 2021). "Ascorbate Peroxidase Neofunctionalization at the Origin of APX-R and APX-L: Evidence from Basal Archaeplastida". Antioxidants. 10 (4): 597. doi: 10.3390/antiox10040597 . PMC   8069737 . PMID   33924520.
  11. ↑ Battistuzzi, Gianantonio; Bellei, Marzia; Bortolotti, Carlo Augusto; Sola, Marco (2010-08-01). "Redox properties of heme peroxidases". Archives of Biochemistry and Biophysics. Heme Peroxidases. 500 (1): 21–36. doi:10.1016/j.abb.2010.03.002. hdl: 11380/637520 . ISSN   0003-9861.
  12. ↑ Anjum, Naser A.; Sharma, Pallavi; Gill, Sarvajeet S.; Hasanuzzaman, Mirza; Khan, Ekhlaque A.; Kachhap, Kiran; Mohamed, Amal A.; Thangavel, Palaniswamy; Devi, Gurumayum Devmanjuri; Vasudhevan, Palanisamy; Sofo, Adriano; Khan, Nafees A.; Misra, Amarendra Narayan; Lukatkin, Alexander S.; Singh, Harminder Pal (2016-10-01). "Catalase and ascorbate peroxidase—representative H2O2-detoxifying heme enzymes in plants". Environmental Science and Pollution Research. 23 (19): 19002–19029. doi:10.1007/s11356-016-7309-6. hdl: 11563/122159 . ISSN   1614-7499.
  13. ↑ Shigeoka, S. (2002-05-15). "Regulation and function of ascorbate peroxidase isoenzymes". Journal of Experimental Botany. 53 (372): 1305–1319. doi:10.1093/jexbot/53.372.1305.
  14. ↑ Lad, Latesh; Mewies, Martin; Raven, Emma Lloyd (2002-11-01). "Substrate Binding and Catalytic Mechanism in Ascorbate Peroxidase:  Evidence for Two Ascorbate Binding Sites" . Biochemistry. 41 (46): 13774–13781. doi:10.1021/bi0261591. ISSN   0006-2960.
  15. ↑ Patterson WR, Poulos TL, Goodin DB (April 1995). "Identification of a porphyrin pi cation radical in ascorbate peroxidase compound I". Biochemistry. 34 (13): 4342–5. doi:10.1021/bi00013a024. PMID   7703248.
  16. ↑ Jones DK, Dalton DA, Rosell FI, Raven EL (December 1998). "Class I heme peroxidases: characterization of soybean ascorbate peroxidase". Archives of Biochemistry and Biophysics. 360 (2): 173–8. doi:10.1006/abbi.1998.0941. PMID   9851828.
  17. ↑ Meharenna, Yergalem T.; Oertel, Patricia; Bhaskar, B.; Poulos, Thomas L. (2008-09-30). "Engineering Ascorbate Peroxidase Activity into Cytochrome c Peroxidase". Biochemistry. 47 (39): 10324–10332. doi:10.1021/bi8007565. ISSN   0006-2960. PMC   2770236 . PMID   18771292.
  18. ↑ Patterson WR, Poulos TL (April 1995). "Crystal structure of recombinant pea cytosolic ascorbate peroxidase". Biochemistry. 34 (13): 4331–41. doi:10.1021/bi00013a023. PMID   7703247.
  19. ↑ Sharp KH, Mewies M, Moody PC, Raven EL (April 2003). "Crystal structure of the ascorbate peroxidase-ascorbate complex". Nature Structural Biology. 10 (4): 303–7. doi:10.1038/nsb913. PMID   12640445. S2CID   32035409.
  20. ↑ Macdonald IK, Badyal SK, Ghamsari L, Moody PC, Raven EL (June 2006). "Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis". Biochemistry. 45 (25): 7808–17. doi:10.1021/bi0606849. PMID   16784232.
  21. ↑ Gumiero A, Murphy EJ, Metcalfe CL, Moody PC, Raven EL (August 2010). "An analysis of substrate binding interactions in the heme peroxidase enzymes: a structural perspective". Archives of Biochemistry and Biophysics. 500 (1): 13–20. doi:10.1016/j.abb.2010.02.015. PMID   20206594.
  22. ↑ Jiménez, Ana; Hernández, José A.; Pastori, Gabriela; del Rı́o, Luis A.; Sevilla, Francisca (1998-12-01). "Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves" . Plant Physiology. 118 (4): 1327–1335. doi:10.1104/pp.118.4.1327. ISSN   1532-2548.
  23. ↑ Meyer, Andreas J. (September 2008). "The integration of glutathione homeostasis and redox signaling" . Journal of Plant Physiology. 165 (13): 1390–1403. doi:10.1016/j.jplph.2007.10.015. ISSN   0176-1617.
  24. ↑ Pang, Cai-Hong; Wang, Bao-Shan (2010), Anjum, Naser A.; Chan, Ming-Tsair; Umar, Shahid (eds.), "Role of Ascorbate Peroxidase and Glutathione Reductase in Ascorbate–Glutathione Cycle and Stress Tolerance in Plants" , Ascorbate-Glutathione Pathway and Stress Tolerance in Plants, Dordrecht: Springer Netherlands, pp. 91–113, doi:10.1007/978-90-481-9404-9_3, ISBN   978-90-481-9404-9 , retrieved 2025-10-21
  25. ↑ Stevens, Rebecca G.; Creissen, Gary P.; Mullineaux, Philip M. (November 1997). "Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress" . Plant Molecular Biology. 35 (5): 641–654. doi:10.1023/a:1005858120232. ISSN   0167-4412.
  26. ↑ Shigeoka, S. (2002-05-15). "Regulation and function of ascorbate peroxidase isoenzymes". Journal of Experimental Botany. 53 (372): 1305–1319. doi:10.1093/jexbot/53.372.1305.
  27. ↑ Gupta, Rajeev; Luan, Sheng (2003-07-01). "Redox Control of Protein Tyrosine Phosphatases and Mitogen-Activated Protein Kinases in Plants". Plant Physiology. 132 (3): 1149–1152. doi:10.1104/pp.103.020792. ISSN   1532-2548.
  28. ↑ Lloyd Raven, E.; Çelik, A.; Cullis, P.M.; Sangar, R.; Sutcliffe, M.J. (2001-04-01). "Engineering the active site of ascorbate peroxidase". Biochemical Society Transactions. 29 (2): 105. doi:10.1042/0300-5127:0290105.
  29. ↑ Bursey, Evan H.; Poulos, Thomas L. (2000-06-01). "Two Substrate Binding Sites in Ascorbate Peroxidase:  The Role of Arginine 172" . Biochemistry. 39 (25): 7374–7379. doi:10.1021/bi000446s. ISSN   0006-2960.
  30. ↑ Zhang, Bixia; Lewis, Jacob A; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee (2023-05-02). "A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids". Plant Physiology. 192 (1): 102–118. doi:10.1093/plphys/kiac604. ISSN   0032-0889. PMC   10152656 . PMID   36575825.
  31. ↑ Mandelman, David; Jamal, Joumana; Poulos, Thomas L. (1998-12-01). "Identification of Two Electron-Transfer Sites in Ascorbate Peroxidase Using Chemical Modification, Enzyme Kinetics, and Crystallography" . Biochemistry. 37 (50): 17610–17617. doi:10.1021/bi981958y. ISSN   0006-2960.
  32. ↑ Kono, Fumiaki; Tamada, Taro (December 2021). "Neutron crystallography for the elucidation of enzyme catalysis" . Current Opinion in Structural Biology. 71: 36–42. doi:10.1016/j.sbi.2021.05.007.
  33. ↑ Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem 278:46869–46877
  34. ↑ Ishikawa, Takahiro; Takeda, Toru; Shigeoka, Shigeru; Hirayama, Osamu; Mitsunaga, Toshio (August 1993). "Hydrogen peroxide generation in organelles of Euglena gracilis" . Phytochemistry. 33 (6): 1297–1299. doi:10.1016/0031-9422(93)85078-6. ISSN   0031-9422.
  35. ↑ Dietz, Karl-Josef (January 2016). "Thiol-Based Peroxidases and Ascorbate Peroxidases: Why Plants Rely on Multiple Peroxidase Systems in the Photosynthesizing Chloroplast?". Molecules and Cells. 39 (1): 20–25. doi:10.14348/molcells.2016.2324. PMC   4749869 . PMID   26810073.
  36. ↑ Szabó, Milán, Anthony W. D. Larkum, and Imre Vass. “A Review: The Role of Reactive Oxygen Species in Mass Coral Bleaching.” In Photosynthesis in Algae: Biochemical and Physiological Mechanisms, edited by Anthony W.D. Larkum, Arthur R. Grossman, and John A. Raven. Springer International Publishing, 2020. https://doi.org/10.1007/978-3-030-33397-3_17.
  37. ↑ Pang, Cai-Hong; Wang, Bao-Shan (2008), "Oxidative Stress and Salt Tolerance in Plants", Progress in Botany, Berlin, Heidelberg: Springer Berlin Heidelberg, pp. 231–245, ISBN   978-3-540-72953-2 , retrieved 2025-10-21
  38. ↑ Baek KH, Skinner DZ (2003) Alteration of antioxidant enzyme gene expression during cold acclimation of near-isogenic wheat lines. Plant Sci 165:1221–1227
  39. ↑ Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, et al. (November 2012). "Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy". Nature Biotechnology. 30 (11): 1143–8. doi:10.1038/nbt.2375. PMC   3699407 . PMID   23086203.
  40. ↑ Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY (January 2015). "Directed evolution of APEX2 for electron microscopy and proximity labeling". Nature Methods. 12 (1): 51–4. doi:10.1038/nmeth.3179. PMC   4296904 . PMID   25419960.

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