The function of the periplasmic Sud protein in polysulfide respiration of Wolinella succinogenes

The periplasmic Sud protein was previously isolated as a sulfide dehydrogenase from Wolinella succinogenes. Sud modified by a C-terminal His-tag (Sud-His6) was produced in Escherichia coli by expression of the sud gene. Sud-His6 catalyzed thiocyanate formation from cyanide and polysulfide. The Vmax of this activity was more than one order of magnitude higher than that of sulfide oxidation by dimethyl-naphthoquinone and that of polysulfide reduction by BH4-. The apparent Km was less than 20 microM polysulfide. Polysulfide and not elemental sulfur was found to be the product of sulfide oxidation by dimethyl-naphthoquinone, in contrast to the earlier view [Kreis-Kleinschmidt, V., Fahrenholz, F., Kojro, E. & Kröger. A. (1995) Arch. Microbiol. 165, 65-68]. Sud-His6 did not contain metal ions or other prosthetic groups. Replacement by site-directed mutagenesis of the single cysteine residue of the Sud monomer caused complete loss of activity, while the exchange of the single histidine residue or of the lysine residue situated next to cysteine did not affect activity. In equilibrium dialysis, the Sud-His6 monomer bound up to ten polysulfide sulfur atoms with a dissociation constant of 0.2 mM. Sud-His6 loaded with polysulfide sulfur showed an absorption spectrum in the range of 350-400 nm; this spectrum differed from that of free polysulfide. Electron transport from H2 to polysulfide catalyzed by the membrane fraction of W. succinogenes was stimulated by the presence of small amounts of Sud-His6. The apparent Km for polysulfide decreased sevenfold in the presence of saturating amounts of Sud-His6 (1 microM Sud-His6 dimer). Similar results were obtained with intact W. succinogenes cells containing low and high amounts of Sud. Sud appears to function as a polysulfide binding protein and probably binds polysulfide sulfur to its cysteine residue and transfers it to the substrate site of the membraneous polysulfide reductase.