1. Metabolism of Reactive Oxygen Species in Peroxisomes: Rob P. Donaldson Department of Biological Sciences George Washington Univ. robdon@gwu.edu

2. Reactive Oxygen Metabolism in Peroxisomes Peroxisomal oxidases produce Hydrogen Peroxide in Liver, Yeast, Leaves, Germinating oil seeds. Peroxisomal proteins are subject to oxidation. Prevention of protein oxidation in peroxisomes: Catalase, Ascorbate Peroxidase, Ascorbate Free Radical Reductase NADH oxidation cycle within peroxisomes YeastLiverLeaf

3. Malate Synthase Malate Dehydrogenase Citrate Synthase Aconitase Fe Isocitrate lyase Catalase Multifunctional hydratase dehydrogenase Acyl CoA Oxidase H2O2H2O2 H2O2H2O2 H2O2H2O2 H2O2H2O2 H2O2H2O2 Thiolase Acetyl-CoA Fatty Acid Glyoxylate  Oxidation Glyoxylate Cycle Succinate, Glucose O2O2

4. Proteins that are targeted to peroxisomes: Peroxisomal Oxidases bypass Mitochondrial ATP generation  -hydroxyacid Oxidase Glycolate Oxidase (Tolbert) D-amino acid oxidase Xanthine Oxidase Urate Oxidase (de Duve) NADH Oxidase Acyl-CoA Oxidase (Beevers) The peroxisomal oxidases generate Reactive Oxygen Species Acyl-CoA + O 2 ----> Enoyl-CoA + H 2 O 2 Xanthine + O 2 ----> Urate + O 2 Catalase scavenges Hydrogen Peroxide (de Duve) 2H 2 O 2 ----> H 2 O + O 2 Ascorbate Peroxidase, also, in membrane of plant peroxisomes

5. Glyxoysomal proteins, including Catalase CAT and Isocitrate Lyase ICL, are oxidized by H2O2 when CAT is inhibited by amino triazole (AT). Immunoblot detects oxidized proteins dervitized with Dinitrophenyl Hydrazine (DNP). Yanik, 2005

6. Isocitrate Lyase (ICL) is associated with Catalase (CAT) within peroxisomes. ICL and CAT proteins co-purify, The two proteins can be chemically cross-linked ICL co-immunoprecipitates with CAT Catalase Far-western blot indicates affinity for ICL ICL CAT Yanik, 2005

7. Peroxisomal proteins are oxidized by incubation with Cu (10 uM) and Ascorbate (4 mM) which bypasses Catalase protection. Carbonylated proteins were detected as DNP derivitives with anti-DNP antibody (B) The same nitrocellulose membrane was stripped with 3M guanidine thiocyanate. Probed with antibodies against Malate Synthase MS Isocitrate Lyase ICL Catalase CAT. These superimposed on proteins seen with the DNP. Spectophotometric determination of total DNP content indicates ~30 nmol DNP per mg protein at 90 min, ~2 mol carbonlyl per mol protein. METAL CATALYZED PROTEIN OXIDATION Nguyen, 2005

8. Malate Synthase Malate Dehydrogenase Citrate Synthase Aconitase Isocitrate lyase Catalase Multifunctional hydratase dehydrogenase Acyl CoA Oxidase H2O2H2O2 H2O2H2O2 H2O2H2O2 Ascorbate H2O2H2O2 Thiolase Acetyl-CoA Fatty Acid Glyoxylate APx AFR NADH H2O2H2O2 H2O2H2O2 H2O2H2O2 H2O2H2O2