Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues.
Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.
MacMillan-Crow
L A
LA
Department of Surgery, University of Alabama at Birmingham 35294, USA.
Crow
J P
JP
Thompson
J A
JA
D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D008345:Manganese; D008958:Models, Molecular; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013482:Superoxide Dismutase / Q000032:analysis / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D009566:Nitrates; C039642:peroxynitric acid; D014443:Tyrosine; D008345:Manganese; C007543:dityrosine; D013482:Superoxide Dismutase; D003545:Cysteine