Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and formation of histidinyl radical.
Human recombinant copper-zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC(50)) was approximately 100 microM at 5 microM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 +/- 1.0) x 10(3) M(-1) s(-1) per monomer at pH 7.5 and 37 degrees C. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical.
Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay. Betariz.Alvarez@fcien.edu.uy
Beatriz
B
Alvarez
Verónica
V
Demicheli
Rosario
R
Durán
Madia
M
Trujillo
Carlos
C
Cerveñansky
Bruce A
BA
Freeman
Rafael
R
Radi
D001639:Bicarbonates / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005609:Free Radicals; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008353:Mannitol / Q000737:chemistry; D008956:Models, Chemical; D009573:Nitrites / Q000737:chemistry; D009603:Nitroso Compounds / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors* / Q000737:chemistry
D001639:Bicarbonates; D004791:Enzyme Inhibitors; D005609:Free Radicals; D009573:Nitrites; D009603:Nitroso Compounds; D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D008353:Mannitol; D006639:Histidine; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; C034526:tert-nitrosobutane