Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and th e ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues wit h a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferr edoxins. The unusual Fe-S cluster type, which was identified in the resonan ce Raman and EPR spectra, has three cysteines and one aspartate as the clus ter ligands, as in the Pyrococcus furiosus 4Fe_ferredoxin. Under aerobic co nditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-termi nal amino acid se-quence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferr edoxin was degraded to 3Fe ferredoxin during aerobic purification. The aero bically-purified ferredoxin was reversibly converted back to the [4Fe-4S] f erredoxin by the addition of ferrous ions under reducing conditions. The an aerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degreesC, while the half-life of the aerobic ally-purified ferredoxin is 10 h at 100 degreesC. Both the anaerobically- a nd aerobically-purified ferredoxins were found to function as electron acce ptors for the pyruvate-ferredoxin oxidoreductase purified from the same arc haeon.