Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, sulfolobus solfataricus PI

Abstract

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreduetase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus PI (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180 ± 50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S|, type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa α and 34 kD β subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55°C, pH 7.0. Maximum activity was observed at 70°C and the optimum pH for enzymatic activity was 7.0 - 8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with K m and kcat values of 163 μM and 452 min-11, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.