We report a direct square-wave voltammetric study of the iron-sulfur enzyme, aconitase, at the pyrolytic graphite edge electrode. New and established redox driven reactions were observed and the equilibrium reduction potential for each couple was determined: E0'[3Fe-4S]1+/0 = -268 mV, E0'[4Fe-4S]2+/1+ = -450 mV, E0'[4Fe-4S]3+/2+ = +100 mV, E0'Linear Form = -281 mV, and putatively, E0'[3Fe-4S]0/2- congruent to -1000 mV, all versus normal hydrogen electrode. Most importantly we have directly observed the superoxidized [4Fe-4S]3+ form of aconitase (originally proposed by Emptage, M. H., Dreyer, J.-L., Kennedy, M. C., and Beinert, H. (1983) J. Biol. Chem. 258, 11106-11111) and directly followed its conversion to the [3Fe-4S]1+ form; this intermediate is required for the deactivation of aconitase. Without exogenous ferrous iron, [3Fe-4S]0 aconitase is apparently super-reduced at very negative potentials to the [3Fe-4S]2- form and the concomitant formation of [4Fe-4S]2+ aconitase was followed over time. It is the apparent decomposition of super-reduced [3Fe-4S]2- aconitase that provides the source of ferrous iron for the interconversion of [3Fe-4S]0 aconitase to the [4Fe-4S]2+ form. Voltammetry of free and substrate bound [4Fe-4S]2+ aconitase showed that the latter is less susceptible to oxidation but, surprisingly, has the same E0'[4Fe-4S]3+/2+.