Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1.
The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (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 alpha and 34 kDa beta 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 degrees C, pH 7.0. Maximum activity was observed at 70 degrees 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 Km and kcat values of 163 microM and 452 min(-1), 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.
Park
Young-Jun
YJ
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, 200-701, Korea.
Yoo
Chul-Bae
CB
Choi
Soo Young
SY
Lee
Hee-Bong
HB
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005288:Ferredoxins; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050743:Pyruvate Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000737:chemistry / Q000201:enzymology; D013696:Temperature
D019843:Archaeal Proteins; D005288:Ferredoxins; D050743:Pyruvate Synthase