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

Abstract

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.

Elemental sulfur and acetate can support life of a novel strictly anaerobic haloarchaeon

“…Indeed, HSR2 genome harbors three operons encoding membrane-bound polysulfide reductases PsrABC and, according with the gene expression studies, all of them are expressed during this type of respiration. Solubility of elemental sulfur in water at neutral pH is extremely low (less than 160 nmol l − 1 ) and, therefore, S 0 can hardly be used as the terminal electron acceptor without any adaptive implementations (Klimmek et al, 1998). However, in the presence of sulfide, a significant amount of S 0 is transformed into soluble inorganic polysulfide (Hedderich et al, 1999).…”

“…However, in the presence of sulfide, a significant amount of S 0 is transformed into soluble inorganic polysulfide (Hedderich et al, 1999). One of the mechanisms of its utilization as a terminal acceptor was described for bacterial sulfur reducers Wolinella succinogenes and Nautilia profundicola (Klimmek et al, 1998;Campbell et al, 2009). The process relies on the activity of a periplasmic sulfurtransferase/ rhodanese-like protein (Sud), which acts as a polysulfide-binding carrier and represents the actual substrate for the catalytic molybdenum (Mo)-containing subunit PsrA.…”

“…The process relies on the activity of a periplasmic sulfurtransferase/ rhodanese-like protein (Sud), which acts as a polysulfide-binding carrier and represents the actual substrate for the catalytic molybdenum (Mo)-containing subunit PsrA. The Sud of W. succinogenes binds up to 10 mol of polysulfide sulfur per 1 mol of enzyme and drastically increases the activity of polysulfide reductase (Klimmek et al, 1998;Hedderich et al, 1999). As the supernatant of a stationary phase HSR2 culture contained 1.3 mmol l − 1 of polysulfide (S 4 2 − ) sulfur, we suggested that HSR2 could use a similar strategy.…”

“…Compared with the Sud protein of W. succinogenes, which has one sulfur-binding domain and needs dimerization to achieve high substrate affinity (Klimmek et al, 1998;Rabus et al, 2006), the HSR2 sulfurtransferase contains two homologous domains. This feature might be especially important during the early growth phase of the HSR2 cells when sulfide concentration is in the micromolar range.…”

“…Bacteria are highlighted in black, Crenarchaeota in blue, Euryarchaeota in green and Halobacteria in pink (see Supplementary Figure S6 for details). Strictly anaerobic acetate-oxidizing sulfur-reducing haloarchaeon DY Sorokin et al inorganic polysulfide molecules to PsrA subunit (Klimmek et al, 1998) (Figure 5). Table S8).…”