Sulfolobus 2-Oxoacid:ferredoxin oxidoreductase

Purification. Purification of the 2-oxoacid ferredoxin oxidoreductase of Sulfolobus sp. strain 7 is carried out by following the 2-oxoacid ferredoxin oxidoreductase activity (as described below) and the absorption bands at 280, 408, and 450 nm of each fraction at different steps. 

Assay Methods. 2-Oxoacid ferredoxin oxidoreductase activity is determined by following the absorbance at 550 nm, due to the ferredoxin-dependent reduction of horse heart cytochrome c (Sigma Chemicals, St. Louis, MO) in the presence of 2-oxoacid substrates, essentially as described by Kerscher et al. The assay is conducted at 50°, in 10 mAf potassium phosphate buffer, pH 6.8, in the presence of 2-4 mAf 2-oxoacids (2-oxoglutarate purchased from Nacalai Tesque, Japan, was mainly used), 50-100 p,Af coenzyme A (Kohjin, Japan), 17 pg of the Sulfolobus zinc-containing ferredoxin (purified as described above), 50 pAf horse heart cytochrome c (Sigma Chemicals), and an appropriate amount of enzyme, in a total volume of 1 ml. The reaction is initiated by addition of the enzyme, and nonenzymatic reduction of cytochrome c by coenzyme A at this temperature is... 

Fukuda E, Kino H, Matsuzawa H, Wakagi T (2001) Role of a highly conserved YPITP motif in 2-oxoacid ferredoxin oxidoreductase heterologous expression of the gene from Sulfolobus sp strain 7, characterization of the recombinant and variant enzymes. Eur J Biochem 268 5639-5646... 

Zhang Q, Iwasaki T, Wakagi T, Oshima T. 1996. 2-oxoacid ferredoxin oxidoreductase from the thermoacidophilic archaeon Sulfolobus sp strain 7. J Biochem 120 587-599. 

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