AN EPR INVESTIGATION OF THE PRODUCTS OF THE REACTION OF CYTOSOLIC ANDMITOCHONDRIAL ACONITASES WITH NITRIC-OXIDE

Cellular studies have indicated that some Fe-S proteins, and the aconi tases in particular, are targets for nitric oxide, Specifically, NO ha s been implicated in the intracellular process of the conversion of ac tive cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-for m which functions as an iron-regulatory protein, We have undertaken th e in vitro study of the reaction of NO with purified forms of both mit ochondrial and cytosolic aconitases by following enzyme activity and b y observing the formation of EPR signals not shown by the original rea ctants, Inactivation by either NO solutions or NO-producing NONOates u nder anaerobic conditions is seen for both enzyme isoforms, This inact ivation, which occurs in the presence or absence of substrate, is acco mpanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clus ters and the g approximate to 2.04 signal of a protein-bound dinitrosy l-iron-dithiol complex in the d(7) state, In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g (parallel to) = 2.11 and g(perpendicular to) = 2.03, is observed, Disa ssembly 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 approximate to 2.04 species and in the case of mitochondrial aconitase, a transient signal atg approximate to 2.032 appeared. This signal is tentatively assigned to the d(9) form of an iron-nitrosylhi stidyl complex of the mitochondrial protein. Inactivation of the [4Fe- 4S] forms of both aconitases by either superoxide anion or peroxynitri te produces the g = 2.02 [3Fe-4S] proteins.