tailieunhanh - Báo cáo khoa học: 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 thyla-koid 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 deple-ted. | ỊFEBS Journal Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2 Sakihito Kitajima1 Taise Shimaoka2 Miyo Kurioka1 and Akiho Yokota3 1 Graduate Schoolof Science and Technology Kyoto Institute of Technology Japan 2 Research Institute of Innovative Technology for the Earth RITE Kyoto Japan 3 Graduate Schoolof BiologicalScience Nara Institute of Science and Technology NAIST Nara Japan Keywords ascorbate peroxidase chloroplast cross-link hydrogen peroxide inactivation Correspondence S. Kitajima Graduate Schoolof Science and Technology Kyoto Institute of Technology Sakyo-ku Kyoto 606-8585 Japan Fax 81 75 724 7762 Tel 81 75 724 7791 E-mail sakito@ Received 1 February 2007 revised 22 March 2007 accepted 16 April 2007 doi 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. Ascorbate peroxidase APX EC isoforms of chloroplasts play a central role in scavenging reactive oxygen species such as 02 and H2O2 which are generated in large amounts by photosystems when there is an energy .