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Role of iron in the activation inactivation of aconitase

Role of iron in the activation inactivation of aconitase

Journal of Biological Chemistry 258(18): 11098-11105

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 Fe. Under these conditions, maximally 70-80% of the activity attainable in the presence of added Fe can be reached. During activation without added Fe, [4Fe-4S] clusters are built from [3Fe-4S] clusters at the expense of a fraction of the 3Fe clusters present. This explains the .apprx. 75% maximal activation observed and concomitant loss of .apprx. 25% of total clusters as quantitated by EPR. Time course plots of aconitase activation appear to be 2nd 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 Fe 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. In analogy to cluster loss during activation in absence of added Fe, the appearance of 59Fe in more than one cluster site can be due to complete breakdown and rebuilding of clusters. 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. Oxidation apparently initiates the inactivation of aconitase with concomitant release of Fe and formation of 3Fe clusters as determined by EPR.

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