Ascorbate peroxidase

Ascorbate peroxidase (or L-ascorbate peroxidase, APX or APEX) (EC 1.11.1.11) is an enzyme that catalyzes the chemical reaction

L-ascorbate + H₂O₂ ${\displaystyle \rightleftharpoons }$ 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]

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]

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]

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]

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, Fe^IV) 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]

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.

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]

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]

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]

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]

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: 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.

• Cytochrome c peroxidase
• Manganese peroxidase

• EC 1.11.1.11 Archived 2011-05-16 at the Wayback Machine
• L-ascorbate peroxidase (EC-Number 1.11.1.11 ) Archived 2012-02-04 at the Wayback Machine