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Irreversible cross‐linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H 2 O 2
Author(s) -
Kitajima Sakihito,
Shimaoka Taise,
Kurioka Miyo,
Yokota Akiho
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.05829.x
Subject(s) - peroxidase , heme , tryptophan , chemistry , stromal cell , biochemistry , biology , enzyme , amino acid , cancer research
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 H 2 O 2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H 2 O 2 , 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 H 2 O 2 ‐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 H 2 O 2 .