In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320-360 nm range with additional soret peaks around 400-410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer.

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Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell lysates, and intact cells, requires oxygen and utilizes electron donor substrates in the absence of hydrogen peroxide or any added cofactors. Using purified bovine catalase and 10-acetyl-3,7-dihydroxyphenoxazine as the substrate, the oxidase activity was found to be temperature-dependent and displays a pH optimum of 7-9. The K m for the substrate is 2. Mammalian catalase belongs to a family of Fe-protoporphyrin IX containing proteins that include a variety of cytochromes, globins, and peroxidases and is one of the best characterized antioxidant enzymes (1). As a homotetrameric heme-containing enzyme, it is known for its ability to convert hydrogen peroxide into water and oxygen (catalatic activity), and in the presence of low concentrations of hydrogen peroxide to oxidize low molecular weight alcohols (peroxidatic activity). The conversion of hydrogen peroxide to water and oxygen by catalase is a two-step process whereby catalase heme Fe 3ϩ reduces one molecule of hydrogen peroxide to water, generating a covalent Fe 4ϩ ϭO oxyferryl species and a porphyrin cation radical. This reaction intermediate, referred to as compound I, then oxidizes a second hydrogen peroxide molecule forming molecular oxygen and water (1-3) (see Fig. 1). The peroxidatic activity of catalase results from the ability of compound I to oxidize alcohols to aldehydes and water (4 -6) ( Fig. 1). Each catalase monomer binds one molecule of heme; the holoenzyme also binds two molecules of NADPH, although the precise role of this cofactor in enzymatic activity is unclear, because hydrogen peroxide provides both oxidative and reductive potential during catalysis. Recent studies suggest that NADPH may be important in maintaining catalase in an active state (7).In mammalian cells, catalase is found at high concentrations in peroxisomes, along with a variety of oxidases and peroxidases (8). It has been suggested that the enzyme protects cells by removing hydrogen peroxide produced by flavin containing oxidases in the peroxisome, thereby preventing the accumulation of toxic levels of this reactive oxygen intermediate (9). However, hydrogen peroxide is important for an array of activities, including peroxidase-mediated metabolism, in cells, and potentially, without this reactive oxygen intermediate, cellular functioning would be limited. In addition, the K m for the catalatic activity of catalase is Ͼ10 mM, therefore, at low intracellular concentrations of hydrogen peroxide, this reaction is not kinetically favored, and it is assumed that peroxidases such as glutathione peroxidase or the recently discovered l-Cys peroxiredoxins effectively lower intracellular concentrations of hydrogen peroxide (10). In...