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 (KI = 35 μM). 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 × 105 M− 1 s− 1). Carbon dioxide enhanced ONOO–-dependent inactivation via reaction of CO3− with the [4Fe-4S] cluster (3 × 108 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 O2− 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 O2− production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O2− 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-derived species.