Open AccessReconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes
Author(s) -
Biel Simone,
Simon Jörg,
Gross Roland,
Ruiz Teresa,
Ruitenberg Maarten,
Kröger Achim
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.02842.x
Subject(s) - fumarate reductase , formate dehydrogenase , formate , chemistry , succinate dehydrogenase , reductase , dehydrogenase , electron transport chain , enzyme , hydrogenase , fumarase , dimethyl fumarate , biochemistry , stereochemistry , catalysis , biology , multiple sclerosis , immunology
Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H 2 which generated an electrical proton potential (Δψ = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes . The H + /e ratio brought about by fumarate reduction with H 2 in proteoliposomes in the presence of valinomycin and external K + was approximately 1. The same Δψ and H + /e ratio was associated with the reduction of 2,3‐dimethyl‐1,4‐naphthoquinone (DMN) by H 2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH 2 which did not generate a Δψ. Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Δψ of 0.13 V (negative inside). The H + /e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes . The results support the view that Δψ generation is coupled to menaquinone reduction by H 2 or formate, but not to menaquinol oxidation by fumarate. Δψ generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H 2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.