REACTION PRODUCTS OF CYTOSOLIC & MITOCHONDRIAL ACONITASES W/ NITRICOXIDE

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Mitochondrial (m-acon) and cytosolic (c-acon) aconitases are Fe-S enzymes that catalyze the interconversion of citrate and isocitrate via the obligatory intermediate cis-aconitase. In addition, the apo-form of c-acon functions as an iron-regulatory protein (IRP1) by binding to conserved stem-loop structures, iron-regulatory elements (IREs) found in the untranslated regions in the mRNA of a number of proteins, including ferritin and transferrin receptor. It has been previously shown that the aconitases can be inactivated by oxidants through the loss of a specific iron, Fe2, from the [4Fe-4S] cluster with the formation of the EPR-detectable, g=2.02 [3Fe-4S] form. Recent cellular studies by a number of groups have indicated that production of NO inactivates both enzymes, and furthermore, in the case of a-con results in an increase in the RNA binding form, IRP1. We have studied the anaerobic reaction of NO with the various purified (less than 95%) forms of both aconitases by EPR spectroscopy and for the active 4Fe forms by following the loss of activity. The principal end product observed upon reaction of NO with the various forms of both aconitases is the "g=2.04," d7 dinitrosyl-iron-thiol complex of the protein which on reduction with dithionite yields the d9 species. During inactivation of c-acon with NO, a transient thiyl radical, g-parallel=2.11 and g-perpendicular=2.03, is observed. The rate of inactivation of either m-acon or c-acon was not retarded by the presence of substrate when spermineNONOate was used as the source of NO. Reaction of NO with [3Fe-4S] m-acon yields a species, g=2.032, that is observed only during the early part of the reaction, which is tentatively assigned to the d9 form of an iron-nitrosyl histidine complex. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g=2.02 [3Fe-4S] enzymes.