Original Contribution Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: Mechanisms and relative contributions to aconitase inactivation Verónica Tórtora a , Celia Quijano a , Bruce Freeman b , Rafael Radi a , Laura Castro a, ⁎ a Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay b Department of Pharmacology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15260, USA Received 19 May 2006; revised 22 December 2006; accepted 4 January 2007 Available online 8 January 2007 Abstract Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide ( U NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO – ). High U NO concentrations are required to inhibit resting aconitase. Brief U NO exposures led to a reversible inhibition competitive with isocitrate (K I =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 × 10 5 M - 1 s - 1 ). Carbon dioxide enhanced ONOO – - dependent inactivation via reaction of CO 3 U - with the [4Fe-4S] cluster (3 × 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 U - and U NO revealed that, when U NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O 2 U - production in cells but, when cells are exposed to high concentrations of U NO, aconitase inactivation does not exclusively reflect changes in rates of O 2 U - production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO - and CO 3 U - , which also mediate m-aconitase tyrosine nitration, a footprint of reactive U NO-derived species. © 2007 Elsevier Inc. All rights reserved. Keywords: Aconitase; Mitochondria; Superoxide; Nitric oxide; S-nitrosoglutathione; Peroxynitrite Introduction Mitochondria are principal intracellular sources of super- oxide (O 2 U- ) and hydrogen peroxide (H 2 O 2 ) under both physiological and pathological conditions and therefore primary loci for reactions of these species and those derived from them within cells [1–7]. Mitochondrial aconitase (m-aconitase), an enzyme that catalyzes the reversible isomerization of citrate and isocitrate via cis-aconitate in the Krebs cycle, contains an [4Fe- 4S] prosthetic group in which one of the irons, Fe α , is not ligated to a protein residue and thus can bind to hydroxyl groups of substrates or water [8–10]. M-aconitase is very sensitive to O 2 U– mediated inactivation. Superoxide selectively reacts at ∼ 10 7 M - 1 s - 1 with the iron–sulfur cluster leading to the release of the Fe α . Reactivation of m-aconitase is achieved by reincorporation of Fe 2+ , which is facilitated by reductants such as glutathione (GSH). The ratio between [4Fe-4S]-aconitase and [3Fe-4S]-aconitase has been used to calculate the steady state concentration of O 2 U- in cells [5,11–14]. The exquisite Free Radical Biology & Medicine 42 (2007) 1075 – 1088 www.elsevier.com/locate/freeradbiomed Abbreviations: O 2 U - , superoxide; H 2 O 2 , hydrogen peroxide; m-aconitase, mitochondrial aconitase; U NO, nitric oxide; NOS, nitric oxide synthases; ONOO – , peroxynitrite; RSNO, S-nitrosothiols; GSNO, nitrosoglutathione; U OH, hydroxyl radical; U NO 2 , nitrogen dioxide; NO + , nitrosonium cation; CO 3 U - , carbonate radical; NODs, nitric oxide dioxygenases; SOD, superoxide dismutase; IPTG, isopropyl β-D-thiogalactoside; PMSF, phenylmethylsulfonyl fluoride; HPA, para-hydroxyphenylacetic acid; DTT, dithiothreitol; BSA, bovine serum albumin; TBS, Tris-buffered saline. ⁎ Corresponding author. Fax: +598 2 924 9563. E-mail address: lcastro@fmed.edu.uy (L. Castro). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.01.007