Resonance Raman and electron paramagnetic resonance studies on oxidized and ferricyanide-treated Clostridium pasteurianum ferredoxin. Vibrational assignments from 34S shifts and evidence for conversion of 4 to 3 iron-sulfur clusters via oxidative damage. Vibrational assignments from 34S shifts and evidence for conversion of 4 to 3 iron-sulfur clusters via oxidative damage.

Resonance Raman spectra are reported for oxidized ferredoxin from Clostridium pasteurianum and for protein reconstituted with 34S2-, using 4579 A laser excitation. The spectra are of much higher quality than that previously reported, and the 34S shifts provide assignments of the Fe-S modes. After treatment with ferricyanide, the resonance Raman spectrum closely resembles that of the [3Fe-3S] protein, ferredoxin II from D. gigas; the 34S shifts aid in assignments of the [3Fe-3S] modes. The epr signal associated with the [3Fe-3S] cluster (g = 2.01) corresponds maximally to 0.80 spin/molecule. Anaerobic addition of excess sulfide to the reduced, ferricyanide-treated protein regenerates a [4Fe-4S]1+ epr spectrum, equivalent in intensity to the [3Fe-3S] signal. The ubiquitous occurrence of a g = 2.01 signal in preparations of [4Fe-4S] proteins can be attributed to variable amounts of [3Fe-3S] cluster generated by adventitious oxidation. The ready conversion of [4Fe-4S] to [3Fe-3S] clusters in C. pasterianum raises the possibility that some [3Fe-3S] proteins may actually arise by oxidative damage of [4Fe-4S] proteins during aerobic isolation.