Desulfomonile tiedjei DCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized chemiosmotic process. Respiratory electron transport components were examined in D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation, and sulfate reduction. Reductive dehalogenation was inhibited by the respiratory quinone inhibitor 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that a respiratory quinoid is a component of the electron transport chain coupled to reductive dehalogenation. Moreover, reductive dehalogenation activity was dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinoid. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, a UV-absorbing quinoid which is different from common respiratory quinones in chemical structure according to mass spectrometric and UV absorption spectroscopic analyses was extracted. ATP sulfurylase, adenosine phosphosulfate reductase, and desulfoviridin sulfite reductase, enzymes involved in sulfate reduction, were constitutively expressed in the cytoplasm of D. tiedjei cells grown under all three metabolic conditions. A periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. A membrane-bound, periplasm-oriented formate dehydrogenase was detected only in cells grown with formate as electron donor, while a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with D. tiedjei whole-cell suspensions and cell extracts suggest that the membrane-bound reductive dehalogenase is cytoplasm oriented. The data clearly demonstrate an enzyme topology in D. tiedjei which produces protons directly in the periplasm, generating a proton motive force by a scalar mechanism.