PURIFICATION AND CHARACTERIZATION OF A BENZYLVIOLOGEN-LINKED, TUNGSTEN-CONTAINING ALDEHYDE OXIDOREDUCTASE FROM DESULFOVIBRIO-GIGAS

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.