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Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite
L A MacMillan-Crow1, J A Thompson
1Departments of Surgery and Biochemistry/Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA. lmcrow@ccc.uab.edu
Abstract:
Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.
Insights
Human manganese superoxide dismutase (MnSOD) inactivation by peroxynitrite (ONOO-) involves tyrosine oxidation and dityrosine formation, independent of specific active site tyrosine residues. This finding is crucial for understanding chronic inflammatory diseases.
Area of Science:
- Biochemistry
- Enzymology
- Oxidative Stress
Background:
- Human manganese superoxide dismutase (MnSOD) is a key antioxidant enzyme.
- MnSOD is targeted by tyrosine nitration in chronic inflammatory diseases.
- Peroxynitrite (ONOO-) inactivates MnSOD, nitrates tyrosine residues, and induces dityrosine formation.
Purpose of the Study:
- To differentiate the roles of tyrosine nitration and oxidation in MnSOD inactivation by ONOO-.
- To compare ONOO- treatment effects on wild-type MnSOD (WT-MnSOD) and a Y34F-MnSOD mutant.
Main Methods:
- Treatment of WT-MnSOD and Y34F-MnSOD with ONOO-.
- Assessment of MnSOD enzymatic inactivation.
- Analysis of tyrosine nitration and dityrosine formation.
Main Results:
- Both WT-MnSOD and Y34F-MnSOD showed similar sensitivity to ONOO--mediated inactivation.
- Y34F-MnSOD exhibited reduced tyrosine nitration and increased dityrosine formation compared to WT-MnSOD.
- Complete MnSOD inactivation by ONOO- can occur without nitration of the primary susceptible tyrosine residue.
Conclusions:
- MnSOD inactivation by ONOO- is a complex process involving both tyrosine nitration and oxidation.
- Tyrosine oxidation and dityrosine formation contribute to MnSOD inactivation.
- These findings offer insights into MnSOD's role in chronic inflammation and disease pathogenesis.