1. Protein 3-Nitrotyrosine: Formation, Evaluation and Biological Consequences Dr. José M. Souza Departamento de Bioquímica Centro de Radicales Libres Facultad de Medicina, Universidad de la República Av. Gral. Flores 2125, Montevideo, Uruguay E-mail: jsouza@fmed.edu.uy

2. Nitrating agent Formation of 3-nitro-tyrosine (NO 2 ) 4 C pH  8 (NO 2 ) 3 C - + 2H +

3. Mechanism of 3-nitroTyrosine Formation Two major pathways have been established: PeroxynitritePeroxynitrite Peroxidases or MPO/H 2 O 2 /NO 2 -Peroxidases or MPO/H 2 O 2 /NO 2 - NO. ?NO. ? All pathways for 3-nitroTyr formation depend on nitric oxide formation

4. Oxidation state (n)2345. NO 2 -. 2 ONOO - Intermediates/ catalysts Tyr. H + /HNO 2 H 2 O 2,HOCl Myeloperoxidase Hemeproteins Tyr. CO 2 Me n+ ROH,RCO 2 Myeloperoxidase Eosinophil peroxidase In search of the in vivo nitrating agents Tyrosyl Radical: Prostaglandin H Synthase-2, Ribonucleotide Reductase Peroxidases: Catalysts of both nitrite and peroxynitrite-mediated nitration Hypochlorous acid: Likely not involved in peroxidase-mediated nitration Nitrogen Dioxide: Inefficient in the absence of tyrosyl radical ONO(O)CO 2 - : More efficient nitrating agent than peroxynitrite

5. Reactive Pathways ONOO - O 2 -. NO + RC-NO 2 NitrativeStress OxFe-S, Carbonyls OxidativeStress RS-NO, RN-NO NitrosativeStress O 2 Me n+ RSH CO 2 2 ON-OCO 2 - H 2 O 2 Me n+ MPOEPO

6. Radical mechanism of nitration

7. Peroxynitrite free radical-independent nitration mechanism This mechanism may ocurre within protein metal centers ONOO - + Me n X ONOO-Me n X NO 2 -O-Me n X NO 2 + + O=Me n X Tyr NO 2 -Tyr + O=Me n X + H + O=Me n X + 2H + Me n X + H 2 O NO 2 + + H 2 O NO 3 - + 2H +

8. MPO-catalyzed Cl - -mediated oxidation Fe III Fe IV+. H2O2H2O2H2O2H2O2 H2OH2OH2OH2O MPO Compound I MPO Ground State Cl - HOCl

9. NO 2 Tyr formation by MPO Fe IV Fe III Fe IV+. H2O2H2O2H2O2H2O2 H2OH2OH2OH2O MPO Compound I MPO Compound II NO 2 -. NO 2 Tyr Tyr. MPO Ground State

10. Tyrosine Nitration by Nitric Oxide Nitric oxide may react with stable tyrosyl radical residue that are involved in the catalytic mechanism of ribonucleotide reductase or prostaglandin H synthase, or cytochrome c-H 2 O 2

11. A Tale of Two Controversies: Defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species Brennan M-L et al (2001) J.B.C. 277, 17415-17427 Peroxidases Knockout Model 3-Nitrotyrosine Formation from Lung Tissue after Aeroallergen Challenge 3-Nitrotyrosine Formation from Zymosan-induced Peritonitis Lavage protein after 20h thioglycollate and 4h zymosan

12. Peroxynitrite Pharmacology. NO + O 2.- ONOO - Oxidations and Nitrations NOS inhibitors NOX and XO inhibitors NO scavengers SOD or SOD-mimics Scavengers Decomposition catalysts Repair

13. Peroxidases Pharmacology NO 2 - + H 2 O 2 + MPO / EPO. NO O 2.- SOD NO 3 - HbO 2 Decomposition catalysts (catalase or catalase mimics) Oxidations and Nitrations Peroxidase Inhibitors Peroxidase knockout

14. Consequences of 3-nitrotyrosine in proteins

15. Identification of nitrated proteins in plasma of ARDS patients -Ceruloplasmin -Transferrin -  1antichimotrypsine -  1protease inhibitor -Fibrinogen

16. How could we look at protein 3- nitrotyrosine formation? J.B.C. (2000) 275, 21409 Cytochrome c control Cytochrome c + 0.5 mM ONOO - Cytochrome c + 2 mM ONOO -

17. 3-nitroTyrosine changes the pI of protein J.B.C. (2000) 275, 21409 Native poliacrylamide electrophoresis Cytochrome c 1- Control 2- one bolus ONOO - 3 mM 3- two bolus “ 4- four bolus “ 5- six bolus “ 6- reverse order addition

18. Purification of nitrated forms of cytochrome c by cation-exchange chromatrography Biochemistry (2005) 44, 8038

19. Mapping of 3-nitroTyr in cytochrome c

20. Three-D view of Tyrosines in cytochrome c Biochemistry (2005) 44, 8038

21. 3-nitroTyrosine may induce a “gain of function” Two examples: Nitration of Cytochrome c Nitration of Fibrinogen

22. Nitrated cytochome c shows an increase in its peroxidase activity J.B.C. (2000) 275, 21409 Biochemistry (2005) 44, 8038

23. Nitrated Fibrinogen shows an increase in its pro-thrombotic properties J.B.C. (2004) 279, 8820

24. Scanning EM of: A- Fibrinogen control B- + MPO/H 2 O 2 /NO 2 - C- + SIN-1 D- + MPO/H 2 O 2 J.B.C. (2004) 279, 8820

25. Why is protein tyrosine nitration important in vivo? 4Selective, not all proteins are modified 4Alter function in some but not all proteins 4 Structural alteration, accelerate protein turn-over 4Increase antigenicity and induce immune responses

26. O 2 -. NO + SO D MPO/H 2 O 2 /NO 2 - ONOO - / CO 2 Y Y NO 2 Enzymatic Activity Signal Cascades Immunological Responds Repair Activity ? Proteosome Tyrosine Decarboxilase 3-Nitro-hydroxy-fenilacetaldehyde Y NO 2

27. Controversial and Challengers “3-nitrotyrosine is produced in vivo; there is an increase in 3-nitrotyrosine concentration in many pathological situations” Some controversies remain: 1- The biological significance of nitration. 2- The mechanisms of 3-nitrotyrosine formation. 3- Is there a repair mechanism for 3-nitrotyrosine? Is it a signal pathway? 4- Where is nitration produced? Which are the preferential targets?