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Fig. 5. Optical spectra of [3Fe—4S] and [4Fe—4S] m-acon and 4Fe m-acon inactivated by spermine NONOate. The concentrations of 3Fe and 4Fe m-acon were 30 ^M. The spectrum of 4Fe + NO was normalized to the absorbance at 280 nm of the 3Fe and 4Fe forms. All spectra were run against a buffer blank of 0.1 M HEPES, pH 7.5.

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Wavelength (nm)

Fig. 5. Optical spectra of [3Fe—4S] and [4Fe—4S] m-acon and 4Fe m-acon inactivated by spermine NONOate. The concentrations of 3Fe and 4Fe m-acon were 30 ^M. The spectrum of 4Fe + NO was normalized to the absorbance at 280 nm of the 3Fe and 4Fe forms. All spectra were run against a buffer blank of 0.1 M HEPES, pH 7.5.

the reduced sample (curve B) from the original spectrum (curve A) gives the signal of the d7 imidazole complex with g values of 2.050 and 2.17. The transient signal observed in the reaction of 3Fe m-acon and NO, Fig. 3, (curve A — B), most closely resembles that of the d9 state of the imidazole complex, with g values of 2.032 and 2.004. Thus, it was concluded that the new signal formed in this reaction is most likely due to d7 and d9 forms of a histidyl-nitrosyl-acon complex. Reaction of NO with 3Fe c-acon did not reveal species other than g ~ 2.04 DNI-acon. Changes in the optical spectra of both the 3Fe and 4Fe forms of aconitase upon addition of NO are shown in Fig. 5. The optical spectrum of the product formed is identical for each, which upon analysis by EPR gave evidence for only the g ~ 2.04 species present.

The data presented above leave no doubt that the Fe—S clusters of mammalian aconitases are attacked by NO leading to the formation of dinitrosyl-iron complexes.

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