An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide.
Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approximately 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7 state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g parallel = 2.11 and g perpendicular = 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approximately 2.04 species and in the case of mitochondrial aconitase, a transient signal at g approximately 2. 032 appeared. This signal is tentatively assigned to the d9 form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.
Kennedy
M C
MC
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. ckennedy@mcw.edu
Antholine
W E
WE
Beinert
H
H
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology; D009569:Nitric Oxide / Q000737:chemistry*
D009569:Nitric Oxide; D000154:Aconitate Hydratase