Tyrosine nitration catalyzed by the metalloproteins copper-zinc superoxide dismutase, horseradish peroxidase, and myeloperoxidase.

摘要

Nitrotyrosine has been found in many inflammatory and neurodegenerative diseases, but the identity of the nitrating agents remains controversial. This dissertation project evaluated nitration catalyzed by Cu,Zn superoxide dismutase (Cu,ZnSOD) and by peroxidases. Both enzymes utilized peroxynitrite (ONOO-) or hydrogen peroxide (H2O2) plus nitrite (NO2-) as substrates. Hydrogen peroxide is a product of superoxide dismutation. Peroxynitrite is formed by the reaction of nitric oxide (NO·) with superoxide (O 2·-), while nitrite is formed from reactions of either nitric oxide or peroxynitrite.;Mutations in Cu,ZnSOD have been linked to familial amyotrophic lateral sclerosis (ALS). Tyrosine nitration has been detected in both sporadic and familial ALS. Since Cu,ZnSOD is a known catalyst of peroxynitrite nitration of tyrosines, we tested Cu,ZnALS-SOD for this activity. We found peroxynitrite nitration catalysis by Cu,ZnALS-SOD was identical to wild-type Cu,ZnSOD. However, ALS-SODs have lower zinc affinity than wild-type SOD. Zinc-deficient SOD (Cu,ESOD) is a better catalyst of peroxynitrite nitration than Cu,ZnSOD. Hydrogen peroxide inactivated the binding of zinc to zinc-deficient SOD six times faster than it inactivated dismutation activity. Cu,ESOD dismutation activity was not protected by ascorbate, urate, or xanthine, which were all protective for Cu,ZnSOD. Consequently, oxidants can render zinc loss from ALS-SOD irreversible.;Myeloperoxidase (MPO) and horseradish peroxidase (HRP) were better peroxynitrite nitration catalysts than Cu,ZnSOD. Furthermore, MPO and HRP also catalyze nitration from hydrogen peroxide plus nitrite. This mechanism was independent of chloride and hypochlorous, acid. MPO was capable of catalyzing nitration of proteins in a tissue homogenate where competing targets of oxidation were also present. The peroxidase-generated nitrating agent was capable of crossing erythrocyte ghost membranes to nitrate internal proteins. Peroxidases could possibly form the nitrating species by oxidation of nitrite to peroxynitrous acid (ONOOH) by the addition of an OH group. Hydrogen peroxide and nitrite are more stable products of superoxide and nitric oxide that eluded formation of peroxynitrite. Since superoxide and nitric oxide are often co-induced in neutrophils, MPO could recycle accumulating hydrogen peroxide and nitrite into an additional nitrating agent.