Mössbauer and EPR studies of activated aconitase: development of a localized valence state at a subsite of the [4Fe-4S] cluster on binding of citrate.

Abstract

During activation of aconitase a Fe2+ ion is incorporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or 56Fe for activation the beef heart enzyme was studied with Mossbauer spectroscopy in the presence of citrate. The environment of 1 Fe site (Fea) of the [4Fe-4S] cluster is drastically altered in the presence of citrate. Fea is the Fe acquired during activation of aconitase. In the oxidized [4Fe-4S]2+ state 2 species with enzyme-bound substrate are observed, whereas only one is observed in the reduced [4Fe-4S]+ state. The Mossbauer parameters of Fea reveal that the site has acquired substantial high-spin ferrous character. This is most pronounced in the 1+ state where at Fea the cluster exhibits a localized valence state. The dramatic increase of the isomer shift upon substrate binding strongly suggests that the ligand environment of Fea hs become at least 5-coordinate and that the cluster may function as a Lewis acid. In the absence of citrate the EPR spectra of the active [4Fe-4S]+ enzyme show no hyperfine broadening in the presence of H217O. However, in the presence of citrate sizable transferred hyperfine interactions are observed; under the experimental conditions the hydroxyl groups of citrate and isocitrate as well as water are labeled with 17O. No broadening by 17O-labeled carboxyl groups of citrate in H216O was detected. Implications for the mechanism of aconitase are discussed.