Graph showing how the ratio of [3Fe-4S] to [4Fe-4S] clusters depends on the pulse potential. Pulse duration 3 seconds; cluster populations measured on the third cycle.

Before the pulse, the 8Fe protein shows a simple voltammogram; this consists of a single signal that is actually due to both of the [4Fe-4S] clusters, which have similar reduction potentials. Application of a single 3-second pulse at +0.72 V followed by a period at normal potentials produces a form in which a [3Fe-4S] cluster is clearly present (note the characteristic A′+C′ signal pattern) in an amount approximately equal to that of the remaining [4Fe-4S] population. Further examination revealed that immediately after the pulse, there is actually an excess of [3Fe-4S] over [4Fe-4S]; this means that some proteins must contain two [3Fe-4S] clusters, with the second one being rapidly “repaired” to regenerate [4Fe-4S]. By varying the pulse potential and measuring the ratio of 3Fe/4Fe clusters present in the product, a bell-shaped curve was obtained that was analyzed in terms of two competing reactions-formation of a species that is either [3Fe-4S] itself or a precursor, and destruction of [3Fe-4S] (or its precursor). Double-pulse experiments (applying a second pulse after allowing the film to relax to the 7Fe form) showed that the [3Fe-4S] cluster is more stable than [4Fe-4S] with regard to oxidative degradation, consistent with the high-potential side of the bell curve being due to a reaction occurring prior to (and not after) formation of [3Fe-4S]. Accordingly, a reaction sequence has been proposed (Figure 20), which invokes formation of [4Fe-4S]3+ (reduction potential E1), which either expels Fe to give [3Fe-4S]+ or is oxidized further (E2) to give a species that is possibly [4Fe-4S]4+, which is otherwise unknown; this highly oxidized cluster breaks down rapidly to give apoprotein. The [3Fe-4S] cluster thus appears to be a relatively stable product of [4Fe-4S] disassembly in C.p. Fd, and is capable of converting easily back to [4Fe-4S] once reducing conditions are restored and Fe is available. Viewed more generally, this may be important because it would enable Biology to limit the damage due to release of Fe in the presence of oxidants, i.e. from the well-known and dangerous Fenton reaction.