title: The role of iron in the activation-inactivation of aconitase.
abstract: Methods are described for the convenient preparation of aconitase from beef heart mitochondria in its inactive [3Fe-4S] form and largely in its active [4Fe-4S] form. Inactive aconitase can be activated anaerobically by various reducing agents without addition of iron. Under these conditions, maximally 70-80% of the activity attainable in the presence of added iron can be reached. It is concluded that during activation without added iron, [4Fe-4S] clusters are built from [3Fe-4S] clusters at the expense of a fraction of the 3Fe clusters present. This explains the approximately 75% maximal activation observed and concomitant loss of approximately 25% of total clusters as quantitated by EPR. Time course plots of aconitase activation appear to be second order but are not amenable to simple kinetic analysis because of the requirements of both reduction and Fe2+ for activation. Activation of aconitase with 59Fe leads to rapid (minutes) incorporation of 1 iron atom/cluster, which on subsequent inactivation is readily lost again. With longer incubation times (hour), 59Fe is found in more than a single site/cluster. It is concluded that, in analogy to cluster loss during activation in absence of added iron, the appearance of 59Fe in more than one cluster site can be due to complete breakdown and rebuilding of clusters. However, exchange into intact clusters cannot be ruled out. Ferric iron can be bound nonspecifically to active and inactive aconitase but can be readily removed by chelating agents. Sulfide is not required for activation of aconitase in keeping with the proposal that inactive aconitase, as isolated, contains a [3Fe-4S] cluster. It is demonstrated that oxidation initiates the inactivation of aconitase with concomitant release of iron and formation of 3Fe clusters as determined by EPR.
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism*; D000693:Anaerobiosis; D000818:Animals; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D007501:Iron / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D004492:Edetic Acid; D007501:Iron; D000154:Aconitate Hydratase