Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy.

Abstract

The active form of aconitase has a diamagnetic [4Fe-4S]21 cluster. A specific iron ion (Fea, which is lost during inactivation) is the binding site for substrate, as shown by Mossbauer spectroscopy. We have studied the mode of substrate and analogue binding at equilibrium to the paramagnetic [4Fe-4S]+ cluster of the reduced active form by 170 and 13C electron-nuclear double resonance spectroscopy with specifically labeled substrates. The data show that with substrate, only the carboxyl at C-2 of the propane backbone is strongly bound in addition to H20 or OH-(HxO) from the solvent, whereas in an isocitrate analogue that has a nitro group at C-2, the carboxyl and hydroxyl at C-1 are bound along with solvent HxO. We conclude from these data that, on addition of any one of the three substrates, cis-aconitate is the predominant species bound to Fe. of the cluster along with solvent HxO and that cis-aconitate is bound in the citrate mode (carboxyl at C-2). The results with the nitro analogue show that the enzyme can also bind a substrate-like ligand to the cluster in the alternative isocitrate mode (carboxyl at C-1), as is implicit in models proposed for the aconitase reaction.

Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

“…Inasmuch as we have been unable to prepare an enzyme that contains more than one-half of a [4Fe-4S] cluster per subunit (31), it is possible that only two of the cysteine residues are ligated to the cluster, which is shared between two subunits. Another explanation would suggest a cluster ligated to the protein with only three cysteine residues, the fourth ligation site being occupied by a water or hydroxide ion, as in the case of aconitase (23,43), or by another amino acid side chain.…”

In Vitro Serial Passage of Staphylococcus aureus : Changes in Physiology, Virulence Factor Production, and agr Nucleotide Sequence

“…Aconitase activity (i.e., the conversion of citrate to isocitrate) is mediated by an essential redox inactive iron-sulfur center. The [4Fe-4S] center is necessary for the binding of the C-2 carboxyl of citrate or cis-aconitate and for the binding of the C-1 carboxyl of isocitrate (18). This coordination of binding occurs through the labile fourth iron atom designated Fe a .…”

Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli

“…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 paramagnetic electron density in the reduced [4Fe-4S] ' cluster than are those of the other two (Werst et al, 1990b).…”