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An EPR investigation of the reaction products of cytosolic and mitochondrial aconitases with nitric oxide
Journal of Inorganic Biochemistry
IRON-SULFUR AND OTHER METAL-SULFUR CLUSTERS 257
AN EPR INVESTIGATION OF REACTION PRODUCTS OF CYTOSOLIC AND MITOCHONDRIAL ACONITASES WITH NITRIC OXIDE
J12
M. Claire Kennedy ~, William E. Antholine b, and Helmut Beinert c
aDeparOnent of Biochemistry and bBiophysics Research Institute, Medical College of Wisconsin, Milwaukee W153226; qnstitute for Enzyme Research, University of Wisconsin, Madison W153705, USA Mitochondrial (m-acon) and cytosolic (c-acon) aconitases are Fe-S enzymes that catalyze the interconversion of citrate and isocitrate via the obligatory intermdeiate cis-acorfitate. __~ CO0- ~ ~1~ ~ C O 0 +H20 H O - - ~ CO0HO CO0" _ coocooC O O - + S 20 (Y) C O 0 - - H 20 COOCitrate cis-Aconitate Isocitrate 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 shown that the aconitases can be inactivated by oxidants through the loss of a specific iron, Fe~, from the [4Fe-4S] cluster with the formation of the EPR detectable, g =2.02 [3Fe-4S] form [1].
•%-'-'|~ .,,.
o..,q'F--V ---: y ' Recent cellular studies [2] by a number of groups have indicated that production of NO inactivates both enzymes, and furthermore, in the case of c-acon results in an increase in the RNA binding form, IRP1. We have studied the anaerobic reaction of NO with the various purified ( > 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-thiolcomplex of the protein which on reduction with dithionite yields the d9 species [3]. During inactivation of c-acon with NO, a transient thiyl radical, gl =2.11 and g± =2.03, is observed [4]. The rate of inactivation of either m-acon or cacon 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 ironnitrosyl histidine complex [5]. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g=2.02 [3Fe-4S] enzymes. Acknowlegement: Research supported by NIH grants GM38412(HB) and GM518310VICK). 1. 2. 3. 4. 5.
H. Beinert, M. C. Kennedy and C. D. Stout Chem. Rev., 96, 2335 (1996). J.-C. Drapier and J. B. Hibbs Methods Enz., 269, 26 (1996). A. F.Vanin Biochemistry(Moscow), 60, 225 (1995). D. Nelson and M. C. R. Symons, Chem Phys. Lett. 36, 340 (1975). M. Lee, P. Arosio, A. Cozzi and N. D. Chasteen Biochemistry 33, 3679 (1994).
IRON-SULFUR AND OTHER METAL-SULFUR CLUSTERS 257
AN EPR INVESTIGATION OF REACTION PRODUCTS OF CYTOSOLIC AND MITOCHONDRIAL ACONITASES WITH NITRIC OXIDE
J12
M. Claire Kennedy ~, William E. Antholine b, and Helmut Beinert c
aDeparOnent of Biochemistry and bBiophysics Research Institute, Medical College of Wisconsin, Milwaukee W153226; qnstitute for Enzyme Research, University of Wisconsin, Madison W153705, USA Mitochondrial (m-acon) and cytosolic (c-acon) aconitases are Fe-S enzymes that catalyze the interconversion of citrate and isocitrate via the obligatory intermdeiate cis-acorfitate. __~ CO0- ~ ~1~ ~ C O 0 +H20 H O - - ~ CO0HO CO0" _ coocooC O O - + S 20 (Y) C O 0 - - H 20 COOCitrate cis-Aconitate Isocitrate 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 shown that the aconitases can be inactivated by oxidants through the loss of a specific iron, Fe~, from the [4Fe-4S] cluster with the formation of the EPR detectable, g =2.02 [3Fe-4S] form [1].
•%-'-'|~ .,,.
o..,q'F--V ---: y ' Recent cellular studies [2] by a number of groups have indicated that production of NO inactivates both enzymes, and furthermore, in the case of c-acon results in an increase in the RNA binding form, IRP1. We have studied the anaerobic reaction of NO with the various purified ( > 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-thiolcomplex of the protein which on reduction with dithionite yields the d9 species [3]. During inactivation of c-acon with NO, a transient thiyl radical, gl =2.11 and g± =2.03, is observed [4]. The rate of inactivation of either m-acon or cacon 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 ironnitrosyl histidine complex [5]. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g=2.02 [3Fe-4S] enzymes. Acknowlegement: Research supported by NIH grants GM38412(HB) and GM518310VICK). 1. 2. 3. 4. 5.
H. Beinert, M. C. Kennedy and C. D. Stout Chem. Rev., 96, 2335 (1996). J.-C. Drapier and J. B. Hibbs Methods Enz., 269, 26 (1996). A. F.Vanin Biochemistry(Moscow), 60, 225 (1995). D. Nelson and M. C. R. Symons, Chem Phys. Lett. 36, 340 (1975). M. Lee, P. Arosio, A. Cozzi and N. D. Chasteen Biochemistry 33, 3679 (1994).