Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry

Rapid responses of biological 4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that 3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two 4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from 3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal 3Fe-4S] formation (in excess over 4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The 3Fe-4S]:4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that 3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of 4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with 3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of 3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to 4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the 3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts.