Relationship of the oxidation state of the iron sulfur cluster of aconitase to activity and substrate binding

Abstract

Aconitase from mammalian [beef heart] mitochondria is only partially active as isolated but may be activated by incubation with Fe, ascorbate and a thiol, or with dithionite. It has been suggested that the added Fe in the activation mixture is essential for activation and that it is incorporated in the enzyme. However, when the enzyme has a full complement of 3Fe and 3S, full activation is reached coulometrically, without iron or other chemical reducing agents. It is clear, therefore, that the role of activators is to reduce the Fe-S cluster of the enzyme. The appearance of catalytic activity on reduction of the cluster shows a pronounced lag, as does the decay of activity after reoxidizing the cluster. This suggests that catalytic activity requires a conformational change in the protein which is inititated by reduction of the cluster and that, following reoxidation, activity disappears only after the inactive conformation is assumed. Citrate and the competitive inhibitor trans-aconitate are bound to a comparable extent to the active and inactive forms but only the active form can bind 1-hydroxy-2-nitro-1,3-propanedicarboxylic acid, a transition-state analog. This is interpreted to show that in the inactive state aconitase cannot enter the conformation it assumes in the transition state during catalysis.