Abstract
Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O-2 gateway in Mn-SOD.
Highlights
Peroxynitrite has recently been implicated in the inactivation of many enzymes
This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite
Each subpeak had an increase in molecular mass (38 Da) compared with the respective main peaks that corresponds to binding of potassium ion to each enzyme preparation
Summary
Peroxynitrite has recently been implicated in the inactivation of many enzymes. little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite This residue is located near manganese and in a substrate O2. If mitochondria are exposed to a relatively large amount of NO, a sufficient amount of peroxynitrite could be formed even in the presence of Mn-SOD. MacMillan-Crow et al [16] reported inactivation of Mn-SOD activity and concomitant increase of 3-nitrotyrosine in a tissue homogenate of transplanted allogripha during chronic rejection.
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