VOLTAMMETRIC STUDIES OF THE REACTIONS OF IRON-SULFUR CLUSTERS ([3FE-4S] OR [M3FE-4S]) FORMED IN PYROCOCCUS-FURIOSUS FERREDOXIN

Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] add ucts co-ordinated in the ferredoxin from the hyperthermophile Pyrococc us furiosus have been studied by protein-film voltammetry, bulk-soluti on voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S](+/0) and [3Fe-4S](0/2-). Film v oltammetry is possible over a wide pH range (2-8), revealing that the [3Fe-4S](+/0) couple shows a complex pH dependence with pK(red1) = 2.8 , pK(ox) = 4.9 and pK(red2) = 6.7. From MCD, pK(red1) corresponds with protonation of [3Fe-4S](0) to give a spectroscopically distinct speci es, as reported for ferredoxins from Azotobacter and Sulfolobus. The s tatus of the disulphide/disulphydryl entity makes no significant diffe rence to the data (given for the -S-S-form). Formation of the hyper-re duced [3Fe-4S](2-) state is observed, requiring 3H(+) for the overall 3e(-) reduction of [3Fe-4S](+), the change therefore being electroneut ral. By comparison with the ferredoxin from Desulfovibrio africanus, u ptake of Fe(II) and other M(II) by [3Fe-4S](0) to give [M3Fe-4S] clust ers is slow (t(1/2) > 10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t(1/2) much less than 1 s), suggesti ng that coordination of Tl does not require reorganization of the prot ein structure. Rates of formation of [3 Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involv es a labile 'super-oxidized' [M3Fe-4S](3+) state.