Triple mutation of Cys26, Trp35, and Cys126 in stromal ascorbate peroxidase confers H2O2 tolerance comparable to that of the cytosolic isoform.
Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid of the chloroplast play a principle role in detoxifying hydrogen peroxide (H(2)O(2)) generated in photosystem I; however, once the ascorbate is depleted, the enzyme is attacked by H(2)O(2) and rapidly loses its activity. Here, we report that radical transfer across the porphyrin moiety and amino acid residues in the reaction intermediate and H(2)O(2)-mediated enzyme inactivation involve cooperative interactions of the Cys26, Trp35, and Cys126 residues of stromal APX. The wild-type enzyme had a half-time of inactivation of <10s, while the triple mutant of the three residues retained 50% of the initial activity after H(2)O(2) treatment for 3 min. The H(2)O(2) tolerance of this mutant was comparable to that of the H(2)O(2)-tolerant APX isoform localized in the cytosol.
Kitajima
Sakihito
S
Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. sakito@kit.ac.jp
Kitamura
Masahito
M
Koja
Namiko
N
D001059:Apoproteins / Q000378:metabolism; D060387:Ascorbate Peroxidases; D018360:Crystallography, X-Ray; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D010544:Peroxidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D010940:Plant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D011166:Porphyrins / Q000378:metabolism; D011487:Protein Conformation; D014026:Nicotiana / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D001059:Apoproteins; D003497:Cyclic N-Oxides; D007527:Isoenzymes; D010940:Plant Proteins; D011166:Porphyrins; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D003545:Cysteine; C003959:TEMPO