Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: Mechanisms and relative contributions to aconitase inactivation

作者：Tortora Veronica; Quijano Celia; Freeman Bruce; Radi Rafael; Castro Laura^*

来源：Free Radical Biology and Medicine, 2007, 42(7): 1075-1088.

DOI：10.1016/j.freeradbiomed.2007.01.007

• aconitase
• mitochondria
• superoxide
• nitric oxide
• S-nitrosoglutathione
• peroxynitrite

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide ((NO)-N-.), nitrosoglutathione (GSNO), and peroxynitrite (ONOO-). High (NO)-N-. concentrations are required to inhibit resting aconitase. Brief. NO exposures led to a reversible inhibition competitive with isocitrate (K-I=35 mu M). 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 CO3.- With the [4Fe-4S] cluster (3 x 10(8) 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)-N-. revealed that, when (NO)-N-. 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 CO3.-. which also mediate m-aconitase tyrosine nitration, a footprint of reactive (NO)-N-.-derived species.

• 出版日期2007-4-1