SPECTROSCOPIC CHARACTERIZATION OF AN ACONITASE (ACNA) OF ESCHERICHIA-COLI

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mamma lian cytosolic aconitase (c-aconitase) which is the translational regu lator known as iron regulatory factor (IRF). In the [3Fe-4S](+)-contai ning, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aco nitase) but is even more similar to the signal from c-aconitase. This is perhaps related to the greater similarity of the AcnA amino acid se quence with c-aconitase. Magnetic circular dichroism (MCD) spectroscop y has revealed that the electronic structure of the [3Fe-4S] cluster o f AcnA must be similar to, but not identical to that of m-aconitase. W hilst the [3Fe-4S] clusters from both of these enzymes display some fe atures in their MCD spectra common to [3Fe-4S] clusters in general, th eir spectra overall are unique and indicate that the Fe-a atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters o f aconitases. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S](+) cluster which is indistinguishable from the signals from the [4Fe-4S](+) cluster in the mammalian enzymes . However, in contrast to the mammalian enzymes, the EPR signals of th e cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [4Fe-4S](+)-contain ing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S = 1/2 EPR signal w ith g approximate to 2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermedia te in cluster deconstruction.