Zinc-containing ferredoxin

The archaeal zinc-containing ferredoxin and several related metalloproteins can be purified from the same batch of the cells, as described below. ... 

Purification of Zinc-Containing Ferredoxin from Sulfolobus sp. Strain 7. The isolation of ferredoxin from the aerobic thermoacidophilic crenarchaeote, Sulfolobus acidocaldarius, was described by Kerscher et al. This procedure was adapted to purify ferredoxin and related metalloproteins from the Sulfolobus sp. strain 7 cells. 

The zinc-containing ferredoxin fraction, recognized by the brown color, is diluted twofold with distilled water. This is applied onto a Bio-Rad (Hercules, CA) hydroxylapatite HTP column (equilibrated with Milli-Q water), is equilibrated with Milli-Q-purified distilled water, and is eluted by 2 mM potassium phosphate buffer, pH 6.8 (alternatively, a Toyopearl HW-55C hydrophobic column chromatography (Tosoh Corp, Japan) may be used at this step ). Finally, the pooled fraction is concentrated by pressure filtration through an Amicon (Danvers, MA) YM3 membrane, and passed through a preparative Sephadex G-50 gel filtration column (2.5 X 75 cm Amersham Pharmacia Biotech) equilibrated with 80 mM potassium phosphate buffer, pH 6.8. 

Zinc-containing ferredoxin thus obtained gives a single band on 20% analytical polyacrylamide gel electrophoresis (PAGE) and has a purity index (A408/A280) of 0.70. Approximately 40 mg of purified ferredoxin is routinely obtained from about 150 g (wet weight) of the cells, and this is stored in aliquots at —80°. 

General Properties ofArchaeal Zinc-Containing Ferredoxins... 

Zanc-containing ferredoxin is the most abundant ferredoxin in chemohetero-trophically grown Sulfolobus sp. strain and T. acidophilum. The types and spectroscopic properties of the iron-sulfur clusters and the isolated zinc center are very similar in archaeal zinc-containing ferredoxins (Table I). Zinc-containing ferredoxin from Sulfolobus sp. strain 7 (103 amino acids, 7 cysteines) contains one [3Fe-4S] cluster (cluster I) with a midpoint redox potential of —280 mV, one [4Fe-4S] cluster (cluster II) with a midpoint redox potential of —530 mV, and a tetragonal zinc center. j acidophilum zinc-containing ferredoxin... 

Zinc-Containing Ferredoxins from Thermoacidophilic Archaea... 

Zinc-containing ferredoxin Number of amino acids in mature protein Cys Cluster type Redox potential (mV) Coordination environment of isolated zinc site... 

The three-dimensional structure of Sulfolobus sp. ferredoxin contains two [3Fe-4S] clusters and corresponds to the 6Fe form. Determined with the as-isolated zinc-containing ferredoxin which contains one [3Fe-4S] cluster, one [4Fe-4S] cluster, and one isolated zinc center. ... 

Fig. 3. Schematic illustration of the oxidative conversion of the [4Fe-4S] cluster II in Sulfolobus sp. zinc-containing ferredoxin, with a concomitant formation of a stable 6Fe-containing intermediate.

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... 

In anaerobic archaea, ferredoxin functions as an intermediate electron acceptor of a variety of key steps in the central metabolic pathways involved in saccharolytic and peptide fermentation, and reduced ferredoxin thus formed donates its reducing equivalent to ferredoxiniNADP" oxidoreductase and hydrogenase. - In aerobic and thermoacidophilic archaea, the reoxidation steps of reduced zinc-containing ferredoxin are poorly characterized. The soluble fraction of Sulfolobus sp. strain 7 also contains an NADPH ferredoxin oxidoreductase activity, but this enzyme has not been purified and characterized. The following section describes the purification and partial characterization of a red iron-sulfur flavoprotein with a weak ferredoxin-reoxidizing activity fi om Sulfolobus sp. strain 7. ... 

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