Molecular weight of beef heart aconitase and stoichiometry of the components of its iron-sulfur cluster

Abstract

Existing estimates of the molar content of iron and labile sulfide in aconitase are varying and deviate from integral numbers. The proposed model of the iron-sulfur cluster of inactive aconitase, suggesting it to contain a single [3Fe-4S] cluster, has prompted us to reinvestigate the basic physicochemical data of the enzyme to arrive at a more precise figure of the stoichiometry of Fe and S2-. The molecular weight of aconitase estimated from low speed sedimentation equilibrium was 80,900 +/- 2,200. Gel chromatography in 6 M guanidine HCl showed the presence of a single peptide chain of 710 residues, corresponding to a Mr of 78,400, while gel electrophoresis in presence of sodium dodecyl sulfate gave a value of 83,000. Both values are in reasonable agreement with the value obtained from sedimentation equilibrium. Protein determination by amino acid analyses, together with iron and sulfur analyses of 20 different preparations of greater than or equal to 95% purity, gives values of 2.9 +/- 0.2 Fe/mol and 3.9 +/- 0.2 S2-/mol. The data obtained are thus in agreement with the [3Fe-4S] model of the iron-sulfur cluster of inactive aconitase.