Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione.
Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.
Han
Derick
D
Research Center for Liver Disease, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90089-9121, USA.
Canali
Raffaella
R
Garcia
Jerome
J
Aguilera
Rodrigo
R
Gallaher
Timothy K
TK
Cadenas
Enrique
E
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019343:Citric Acid / Q000494:pharmacology*; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000494:pharmacology*; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D030421:Peroxynitrous Acid; D019343:Citric Acid; D014443:Tyrosine; D000154:Aconitate Hydratase; D005978:Glutathione; D003545:Cysteine