Nebraska Redox Biology Center Educational Portal


Hydrogen peroxyde (H2O2) is a strong oxidant which also acts as messenger in cell signaling pathways and used as antimicrobial agent in innate immune defence system. Intracellulat H2O2 concentration is a subject of strict control in all types of cells [ 1, 2, 3, 4 ]. H2O2 can be dagraded enzymatically by its reduction to water with by thiol peroxidases (Peroxiredoxins and Glutatione Peroxidases) and metalloenzyme-mediated disproportionation to O2 and H2O. Such metalloenzymes are known as catalases. Peroxiredoxins and glutathione peroxidases are known to eliminate low concentrations of H2O2, whereas catalases are more efficient at higher concentrations of H2O2. The catalase was discovered by Loew as part of tobacco plant chemistry project at US Department of Agriculture in 1901. Catalase is a one of major antioxidant enzyme in the defense against oxidative stress [ 5, 6, 7, 8 ]. There are three catalase protein families that are able to catalyze H2O2 disproportionation: (I) typical or monofunctional heme containing catalases, (II) heme containing catalase-peroxidases and (III) non-heme manganese catalases [ 7, 9, 10, 11 ]. There are 188 solved Catalase structures in Protein Data Bank at this time (September 2015).

2H2O2 → 2H2O + O2 ;

Typical monofunctional catalases and catalase-peroxidases are heme containing enzymes. The most abundant typical catalases are present in bacteria, archaea and eukaryotes. Catalase-peroxidases are distributed among bacteria and some eukaryotes. Manganese catalases are typical for bacteria and archaea. All three type of catalase are significantly differ in their overall and active-site architecture and the reaction mechanisms [ 7, 11 ].

The reaction of typical monofunctional heme-catalases consist two steps. In the first step, H2O2 serves as a two-electron oxidant to generate a ferryl porphyrin cation radical (O=Fe(IV)Por .+). In the second step O=Fe(IV)Por .+ serves as a two-electron oxidant of H2O2, resulting in release of O2 and regeneration of the initial Fe(III) form of catalase [ 5, 6, 8, 9, 11 ].

Catalase Fe(III) + H2O2 → O=Fe(IV)Por .+ + H2O ;
O=Fe(IV)Por .+ + H2O2 → Catalase Fe(III) + 2H2O + O2 ;

Crystal structure of heme containing bacterial catalase. NDP is shown in blue color and heme is shown in pink color.

The reaction rates and substrate affinities can differ significantly among typical catalases. Catalatically active enzymes do not follow Michaelis-Menten kinetics except at very low substrate concentrations, and different enzymes are affected differently at higher substrate concentrations. Typical catalases are pH independent and active in pH range fron 5 to 10. [ 11, 12 ]. Some typical catalases can utilize NADPH in addition to heme, however, the role of NADPH in catalitic cycle is under discussion. NADPH may serves as an electron source by directly converting O=Fe(IV)Por .+ radical to initial Fe(III), thereby circumventing enzyme inactivation by H2O2 [ 11, 13 ].

In addition to catalatic reaction, catalase-peroxidases can also catalyze a peroxidatic reaction in which electron donors (AH2) are oxidized via one electron mechanism with releasing radicals (AH.). Thus, a peroxidase cycle includes generation of ferryl porphyrin cation radical (O=Fe(IV)Por .+), subsequent formation of oxoiron(IV) species (O=Fe(IV)Por), and reduction of O=Fe(IV)Por back to the initial state Fe(III) by another electron donor molecule AH2. The peroxidatic reaction of typical monofunctional catalases is weak, but in bifunctional catalase- peroxidases peroxidatic function is important [ 11, 14, 15, 16, 17, 18, 19 ].

Catalase Fe(III) + H2O2 → Catalase O=Fe(IV)Por .+ + H2O ;
Catalase O=Fe(IV)Por .+ + AH2 → Catalase O=Fe(IV)Por + AH. ;
Catalase O=Fe(IV)Por + AH2 → Catalase Fe(III) + AH. + H2O ;

Crystal structure of catalase-peroxidase from Synechococcus PCC. Heme is shown in pink color.

Non-heme manganese catalases are minor group of catalases distributed over bacteria and archaea. This type of enzymes represent an important alternative to heme-containing catalases in antioxidant defense. Manganese catalases contain a binuclear manganese complex as their catalytic active site instead a heme. The reactive mechanism is base on cycling between (Mn2+, Mn2+) and (Mn3+, Mn3+) states during turnover [ 11, 20, 21, 22, 23, 24 ]. The (Mn2+, Mn2+) cluster is involved in polarization of the peroxide O-O bond, resulting in cleavage of the peroxidic bond and release of water. Then, (Mn3+, Mn3+) cluster is catalizing oxidation of hydrogen peroxide and oxyden release. The dimanganese cluster is equally stable in either (Mn2+, Mn2+) or (Mn3+, Mn3+) oxidation states. Hydrogen peroxyde degradation rate for manganese catalases is lower compared with typical catalases and catalase-peroxidases [ 11, 22, 23, 24 ].

Catalase-(Mn2+, Mn2+) + H2O2 + 2H+ → Catalase-(Mn3+, Mn3+) + 2H2O ;
Catalase-(Mn3+, Mn3+) + H2O2 → Catalase-(Mn2+, Mn2+) + O2 ;

Crystal structure of homohexamer of manganese catalase from Lactobacillus plantarum. Mn atoms are shown as blue spheres.

Catalases are not efficien at low concentration of H2O2 and most probably catalases are not a major players in H2O2 mediated signaling. Hovewer, the role of typical catalases in inhibition of cellular redox signaling was proven. [ 25, 26 ]. Hemoglobin is protected by catalase colocalized in the erythrocytes. In catalase defficiency connditions, ferrous hemoglobin is oxidized with hydrogen peroxide to methemoglobin resulting in hemolysis of erythrocytes, polymerization of haemoglobin, and aggregation of malfunctioned erythrocytes [ 27, 28 ]. The human genetic disease related to loss of functional catalase is generally known as acatalasemia, or Takahara disease [ 29, 30 ]. The most frequently occurring clinical features of acatalasemia include oral gangrene, ulceration; altered lipid, saccharide and homocysteine metabolism (homocysteinemia). Catalases deficiency and polimorfism may be associated with impaired glucose tolerance, insulin resistance, hypertension, dyslipidemia and impired bone metabolism, however, all of these observations requires additional verifications and confirmations [ 30, 31, 32, 33, 34 ].

Methods for Measurement of Catalase Activity

Numerous highly sensitive methods for catalase activity estimation were developed and offered by commertial companies. For example, most frequently used and highly sensitive, simple, direct assay for measuring Catalase activity in biological samplesis based on inhibition of OxiRed oxidation by H2O2 in probe. In this assay, catalase reacts with H2O2 to produce water and oxygen, the unconverted H2O2, reacts with OxiRed probe to produce a product, which can be measured at 570 nm (Colorimetric method) or at Ex/Em=535/587nm (fluorometric method). Catalase activity is reversely proportional to the signal. The kit detects high pico-unit of catalase in samples [ 35, 36 ].

Another method is based on the reaction of the catalase with methanol in the presence of an optimal concentration of H2O2. As result, formaldehyde is produced and measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen. 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from color less to a purple color. Such assay can be used to measure CAT activity in plasma, serum, erythrocyte lysates, tissue homogenates, and cell lysates [ 37 ].

Another colorimetric method uses a substituted phenol (3,5-dichloro-2-hydroxybenzene-sulfonic acid), which couples oxidatively to 4-aminoantipyrine in the presence of hydrogen peroxide and horseradish peroxidase to give a red quinoneimine dye (N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinone-monoimine) that absorbs at 520 nm [ 38 ].

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