Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2.
Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from <10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.
Kitajima
Sakihito
S
Graduate School of Science and Technology, Kyoto Institute of Technology, Japan. sakito@kit.ac.jp
Shimaoka
Taise
T
Kurioka
Miyo
M
Yokota
Akiho
A
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D060387:Ascorbate Peroxidases; D001665:Binding Sites; D020134:Catalytic Domain; D002736:Chloroplasts / Q000201:enzymology; D003432:Cross-Linking Reagents / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D014026:Nicotiana / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D001059:Apoproteins; D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011994:Recombinant Proteins; D006418:Heme; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases