Characterization of a fourth tungsten-containing enzyme from the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

Pyrococcus furiosus grows optimally near 100 degrees C using peptides and carbohydrates as carbon sources, and it reduces elemental sulfur (S(0)), if present, to H(2)S. Tungsten (W), an element rarely used in biology, is required for optimal growth, and three different tungsten-containing enzymes have been previously purified from this organism. They all oxidize aldehydes of various types and are thought to play primary roles in the catabolism of sugars or amino acids. Here, the purification of a fourth tungsten-containing enzyme, termed WOR 4, from cell extracts of P. furiosus grown with S(0) is described. This was achieved by monitoring through multiple chromatography steps the W that is not associated with the three characterized tungstoenzymes. The N-terminal sequence of WOR 4 and the approximate molecular weight of its subunit determined electrophoretically (69,000) correspond to the product of an ORF (PF1961, wor4) present in the complete genome sequence of P. furiosus. WOR 4 is a homodimer and contains approximately one W, three Fe, three or four acid-labile sulfide, and one Ca atom per subunit. The visible and electron paramagnetic resonance spectra of the oxidized and reduced enzyme indicate the presence of an unusual iron-sulfur chromophore. WOR 4 does not oxidize aliphatic or aromatic aldehydes or hydroxy acids, nor does it reduce keto acids. Consistent with prior microarray data, the protein could not be purified from P. furiosus cells grown in the absence of S(0), suggesting that it may have a role in S(0) metabolism.

FIG. 1.

DEAE-Sepharose column profile during purification of WOR 4 from P. furiosus cell extracts. The peaks indicate either the amount of tungsten (open circles) or the specific activity of GAPOR (closed circles), FOR (closed squares), or AOR (closed triangles) in the various fractions. The diagonal line across the graph indicates the salt gradient (from 50 to 500 mM NaCl) that was applied to the column.

FIG. 2.

EPR spectrum of oxidized WOR 4 from P. furiosus. The enzyme sample (2.5 mg/ml in 50 mM Tris-HCl, pH 8.0) was oxidized with a fivefold excess of potassium hexachloroiridate (E_m + 350 mV). The temperature was 5.4 K, and the spectrometer settings were as follows: microwave power, 1.0 mW; gain, 5 × 10⁴; time constant, 0.0409 s; sweep time, 168 s; modulation amplitude, 6.37 G, microwave frequency, 9.60 GHz.