Molecular forms of aconitase and their interconversions.
Aconitase, as isolated from mammalian mitochondria by traditional methods, is virtually inactive and contains an oxidized [3Fe-4S]+ cluster. The activation of the enzyme and attendant conformational change have been studied by monitoring the changes in activity, in tryptophan fluorescence, and in the electron paramagnetic resonance of the cluster on incubation with dithionite, with and without added Fe2+. Restoration of the full activity is achieved with one electron per 3Fe cluster and at least 0.6 g-atoms of Fe2+ per mol. The process involves building up of [4Fe-4S]2+ clusters. Other metal ions do not substitute for Fe2+. Reduction alone, in the absence of added Fe2+, yields up to 70% of the maximum activity, but requires approx. 1.8 electrons of reductant per cluster. The results presented are consistent with the view that activation without added Fe2+ involves the destruction of some of the [3Fe-4S] clusters and the incorporation of the Fe so liberated into other clusters to yield a tetra-nuclear one. In particular, the effect of EDTA and of other iron chelators in inhibiting activation by dithionite alone is in accord with this view, although recent magnetic-circular-dichroism studies do not support this interpretation. The rates of increase in activity and tryptophan fluorescence are the same when Fe2+ is present, but in its absence, activation is very much slower than the increase in fluorescence, suggesting that the protein conformational change triggered by reduction of the Fe-S clusters precedes the insertion of the iron. Consistent with this view is the observation that iron chelators inhibit activation by dithionite, but not the increase in fluorescence and, hence, the conformational change. The results are discussed in light of data in the literature on the forms of the cluster and its possible function in catalysis.
D000154:Aconitate Hydratase / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004227:Dithionite / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005296:Ferrous Compounds / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013440:Sulfides / Q000494:pharmacology
D002413:Cations, Divalent; D005296:Ferrous Compounds; D007527:Isoenzymes; D013440:Sulfides; D004227:Dithionite; D004492:Edetic Acid; D000154:Aconitate Hydratase