2-Oxoacid:ferredoxin oxidoreductase

Luo J, Fukuda E, Takase H et al (2009) Identification of the lysine residue responsible for coenzyme A binding in the heterodimeric 2-oxoacid ferredoxin oxidoreductase from Sulfo-lobus tokodaii, a thermoacidophilic archaeon, using 4-fluoro-7-nitrobenzofurazan as an affinity label. Biochim Biophys Acta 1794 335-340... 

Ferredoxins (Fds) are widespread in the three domains of life and an abundance of sequence data and structural information are available for Fds isolated from several sources. In particular, the bacterial type Fds are small electron-transfer proteins that posses cubane xFe-yS clusters attached to the protein matrix by Fe ligation of Cys via a conserved consensus ligating sequence. The archaeal type ferredoxins are water-soluble electron acceptors for the acyl-coenzyme A forming 2-oxoacid/ferredoxin oxidoreductase, a key enzyme involved in the central archaeal metabolic pathways. Fds have been distinguished according to the number of iron and inorganic sulphur atoms, 2Fe-2S, 4Fe-4S/3Fe-4S (Fig. Ib-d) and Zn-containing Fds. 

The molar masses of the 2-oxoacid ferredoxin oxidoreductases are 200,000-300,000 g/mol and they are composed of four subunits of the kind a2p2. It has been shown that halobacteria have only these systems of 2-oxoacid ferredoxin oxidoreductases. The two enzymes of H. halobium (pyruvate and oxoglutarate) were isolated and characterized by Kerscher and Oesterhelt (1981a). These systems proved to be thiamin diphosphate-containing iron-sulfur proteins. The relative stability of the halobacterial enzymes enabled detailed analysis of the various steps of the catalytic cycles (Kerscher and Oesterhelt, 1981b), demonstrating two distinct steps of one-electron transfer reactions. 

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. 

The 2-oxoacid ferredoxin oxidoreductase-containing fraction is dialyzed against 20 mAf potassium phosphate buffer, pH 6.8, and applied to a Bio-Rad hydroxylapatite HTP column (1.0 x 23 cm) equilibrated with 10 vaM potassium phosphate buffer, pH 6.8. The enzyme adsorbed onto the column is eluted with a 90-ml linear gradient of potassium phosphate buffer, pH 6.8 (10-300 mAf), and the peak fraction is collected and concentrated by pressure filtration through an Amicon YMIO membrane at 4°. 

Although 2-oxoacid ferredoxin oxidoreductase activity in the cytosol fraction is not very stable upon storage at room temperature, at 4° or —80°, the purified enzyme is quite resistant to oxygen and can be stored aerobically at —80° after dialysis against 20 mAf potassium phosphate buffer, pH 6.8. Approximately 6-8 mg of purified enzyme is obtained from about 100 g (wet weight) of cells. 

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... 

The molecular biological studies have suggested that 2-oxoacid ferredoxin oxidoreductases of archaea, bacteria, and anaerobic amitochondrial eukarya with different sizes and subunit compositions are a phylogenetically closely related... 

Based on the biochemical and sequence data, we have proposed that 2-oxoacid ferredoxin oxidoreductases with different subunit compositions have an essentially similar catalytic mechanism with one TPP and at least one [4Fe-4S] cluster as the minimal set of redox centers, which will include the intermediacy of a hydroxyalkyl-TPP radical, and that the 8 subunit/domain carrying two [4Fe-4S] clusters may serve as (accessory) intramolecular electron transmitter from the [4Fe-4S] cluster in the p subunit/domain to the physiological electron acceptor... 

Purification of Red Iron-Sulfur Flavoprotein. The red iron-sulfur flavoproteinis eluted from the DEAE-Sephacel column shortly after (or sometimes together with) the cognate 2-oxoacid ferredoxin oxidoreductase activity, as described above. The red-colored fractions are combined and made to 1 A/ ammonium sulfate solution by addition of solid ammonium sulfate at 4°, with stirring. 

The suspension is gently degassed by aspirator at room temperature, and directly adsorbed onto aPhenyl-Toyopearl 650M column (1.2 x 25 cm Tosoh Corp) equilibrated with 20 mAf potassium phosphate buffer, pH 6.8, containing 1 M ammonium sulfate. The column is washed with 100 ml of the equilibration buffer, and a 200-ml linear gradient is run between 1 M and 0 Af ammonium sulfate in 20 mM potassium phosphate buffer, pH 6.8. At this step, the red-colored fractions eluted at the end of the linear gradient are clearly separated from the 2-oxoacid ferredoxin oxidoreductase and diaphorase [NAD(P)H dye oxidoreductase] activities. When required, the column is further washed by 20 mAf potassium phosphate buffer, pH 6.8, to complete the elution of the red protein. 

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... 

FdR ferredoxin/flavodoxin reductase, Fdx OFOR 2-oxoacid-ferredoxin oxidoreductase. 

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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