Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H2O2) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H2O2 by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP, were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H2O2. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamydomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena. The enzyme had a molecular mass of 34 kDa. The K-m values of the enzyme for ascorbate and H2O2 were 5.2+/-0.3 and 25+/-3.4 mu M, respectively. Hydrogen peroxide was generated at a;ate of 6.1+/-0.8 mu mol mg(-1) chlorophyll h(-1) in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.