Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli

Abstract

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53 % identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S] +-containing, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal from c-aconitase. This is perhaps related to the greater similarity of the AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but not identical to that of m-aconitase. Whilst the 13Fe-4SJ clusters from both of these enzymes display some features in their MCD spectra common to cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [4Fe-4SI2+-containing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S = 1/2 EPR signal with g=2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction.Keywords: aconitase; Escherichia coli; EPR; magnetic circular dichroism.The dehydratase-hydratase aconitase [citrate (isocitrate) hydro-lyase] catalyses the isomerisation of citrate and isocitrate via the dehydration product cis-aconitate. The application of spectroscopic techniques to the enzyme, including EPR (Emptage et al., 1983a), EXAFS (Beinert et al., 1983), Mossbauer (Kent et al., 1982; Emptage et al., 1983b) and magnetic circular dichroism (MCD; Johnson et al., 1984), revealed the presence of an iron-sulphur cluster interconvertible between the [4Fe-4S] and the [3Fe-4S] form. The oxidative loss of a specific iron atom, denoted Fe,, leads to deactivation of the enzyme. However, this process is reversible: under reducing conditions iron is re-incorporated into the Fe, site in a two-stage process and the [4Fe-4S] cluster reformed with concomitant appearance of enzyme activity (Kennedy et al., 1983;Faridoon et al., 1991). X-ray crystallography (Robbins and Stout, 1989) protein. Mossbauer (Kent et al., 1982; Emptage et al., 1983b) and electron-nuclear double resonance (ENDOR; Telser et al., 1986;Kennedy et al., 1987;Werst et al., 1990a) spectroscopic studies have revealed that Fe;, has, in addition to three inorganic sulphur bridging ligands, a hydroxyl anion ligand which persists but becomes protonated upon binding of the C2 carboxylate anion of cis-aconitate to Fe,. The inequivalence of the iron sites in the cluster has been highlighted by 57Fe-ENDOR and "S-ENDOR and it has been further shown that the nuclei of two of the inorganic sulphide bridging ligands are considerably more strongly coupled to the paramagneti...

Contrasting Sensitivities of Escherichia coli Aconitases A and B to Oxidation and Iron Depletion

“…The difficulty with this idea, however, is that the primary amino acid sequence of aconitase A indicated that it possesses a [4Fe-4S] cluster. Electron paramagnetic resonance studies of the purified enzyme confirmed that this is so (3). Because other [4Fe-4S] dehydratases are oxidant sensitive, it seemed peculiar that SoxRS would induce AcnA during periods of oxidative stress.…”

The role of the Fe‐S cluster in the sensory domain of nitrogenase transcriptional activator VnfA from Azotobacter vinelandii

“…The differing conditions during biosynthetic assembly in vivo and artificial reconstitution in vitro may affect the spectra. For example, the signal intensity ratio of the EPR spectra of [Fe 3 S 4 ] + changes in response to the buffer composition, such as the concentration of glycerol [43]. In ferredoxin II from Desulfovibrio gigas , differing purification conditions cause variation in the shape of the EPR spectrum of [Fe 3 S 4 ] + [44].…”

The second aconitase (AcnB) of Escherichia coli

“…Overproduction to 50% of soluble cell protein allowed AcnB to be purified essentially to homogeneity. The specific activity of pure reactivated AcnB [38 U (mg protein)-'] is somewhat lower than that of reactivated AcnA [59 U (mg protein)-'] purified from a comparably amplified source under similar conditions (Bennett et al, 1995). However, such comparisons must be interpreted with care because a significant proportion of both the amplified-crude and purified AcnA and AcnB proteins could not be reactivated by incubating with ferrous ions under reducing conditions (Bennett e t al., 1995; M. J. Gruer & J. R. Guest, unpublished observations).…”