The periplasmic Sud protein was previously isolated as a sulfide dehydrogenase from Wolinella succinogenes. Sud modified by a C-terminal His-tag (Sud-His 6 ) was produced in Escherichia coli by expression of the sud gene. Sud-His 6 catalyzed thiocyanate formation from cyanide and polysulfide. The V max of this activity was more than one order of magnitude higher than that of sulfide oxidation by dimethylnaphthoquinone and that of polysulfide reduction by BH Ϫ 4 . The apparent K m was less than 20 µM polysulfide. Polysulfide and not elemental sulfur was found to be the product of sulfide oxidation by dimethylnaphthoquinone, in contrast to the earlier view [Kreis-Kleinschmidt, V., Fahrenholz, F., Kojro, E. & Kröger, A. (1995) Arch. Microbiol. 165, 65Ϫ68]. Sud-His 6 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-His 6 monomer bound up to ten polysulfide sulfur atoms with a dissociation constant of 0.2 mM. Sud-His 6 loaded with polysulfide sulfur showed an absorption spectrum in the range of 350Ϫ400 nm ; this spectrum differed from that of free polysulfide. Electron transport from H 2 to polysulfide catalyzed by the membrane fraction of W. succinogenes was stimulated by the presence of small amounts of Sud-His 6. The apparent Km for polysulfide decreased sevenfold in the presence of saturating amounts of Sud-His 6 (1 µM Sud-His 6 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.