Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation.
Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.
Tórtora
Verónica
V
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
Quijano
Celia
C
Freeman
Bruce
B
Radi
Rafael
R
Castro
Laura
L
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013552:Swine
D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D000154:Aconitate Hydratase