DIRECT SQUARE-WAVE VOLTAMMETRY OF SUPEROXIDIZED [4FE-4S](3+) ACONITASE AND ASSOCIATED 3FE 4FE CLUSTER INTERCONVERSIONS/

We report a direct square-wave voltammetric study of the iron-surfur e nzyme, aconitase, at the pyrolytic graphite edge electrode. New and es tablished redox driven reactions were observed and the equilibrium red uction potential for each couple was determined: E([3Fe-4S]1+/0)(0') = -268 mV, E([4Fe-4S]2+/1+) = -450 mV, E([4Fe-4S]3+/2+) = +100 mV, E(Li near Form) (0') = -281 mV, and putatively, E([3Fe-4S]0/2-)(0') similar or equal to -1000 mV, all versus normal hydrogen electrode. Most impo rtantly we have directly observed the superoxidized [4Fe-4S](3+) form of aconitase (originally proposed by Emptage, M. H., Dreyer, J. L., Ke nnedy, M. C., and Beinert, H. (1983) J. Biol. Chem. 258, 11106-11111) and directly followed its conversion to the [3Fe-4S](1+) form; this in termediate is required for the deactivation of aconitase, Without exog enous ferrous iron, [3Fe-4S](0) aconitase is apparently super-reduced at very negative potentials to the [3Fe-4S](2-) form and the concomita nt formation of [4Fe-4S](2+) aconitase was followed over time. It is t he apparent decomposition of super-reduced [3Fe-4S](2-) aconitase that provides the source of ferrous iron for the interconversion of [3Fe-4 S](0) aconitase to the [4Fe-4S](2+) form. Voltammetry of free and subs trate bound [4Fe-4S]2+ aconitase showed that the latter is less suscep tible to oxidation but, surprisingly, has the same E([4Fe-4S]3+/2+)(0' ).