Wolinella succinogenes grows by oxidative phosphorylation with polysulfide as terminal electron acceptor and either H2 or formate as electron donor (polysulfide respiration). The function of the respiratory chains catalyzing these reactions was investigated. Proteoliposomes containing polysulfide reductase (Psr) and either hydrogenase or formate dehydrogenase isolated from the membrane fraction of Wolinella succinogenes catalyzed polysulfide respiration, provided that methyl-menaquinone-6 isolated from W. succinogenes was also present. The specific activities of electron transport were commensurate with those of the bacterial membrane fraction. Using site-directed mutagenesis, certain residues were substituted in PsrC, the membrane anchor of polysulfide reductase. Replacement of Y23, D76, Y159, D218, E225 or R305 caused nearly full inhibition of polysulfide respiration without affecting the activity of Psr, which was still bound to the membrane. These residues are predicted to be located in hydrophobic helices of PsrC, or next to them. Substitution of 13 other residues of PsrC either caused partial inhibition ofblankpolysulfide respiration or had no effect. The function of methyl-menaquinone-6, which is thought to be bound to PsrC, is discussed.