title: Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf).
abstract: Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 μm ferredoxin or 0.42 μm flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD(+) reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH + H(+) = butyryl-CoA + NAD(+) with Km = 1.4 μm ferredoxin or 2.0 μm flavodoxin. This reaction requires Na(+) (Km = 0.12 mm) or Li(+) (Km = 0.25 mm) for activity, indicating that Rnf acts as a Na(+) pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation.
authors.affiliation: From the Laboratorium für Mikrobiologie, Fachbereich Biologie and Synmikro and the Max-Plank-Institut für terrestrische Mikrobiologie, 35043 Marburg, Germany.
authors.forename: Nilanjan Pal
authors.initials: NP
authors.lastname: Chowdhury
authors.affiliation: From the Laboratorium für Mikrobiologie, Fachbereich Biologie and Synmikro and.
authors.forename: Katharina
authors.initials: K
authors.lastname: Klomann
authors.affiliation: the Fachbereich Chemie, Philipps-Universität, 35032 Marburg, and.
authors.forename: Andreas
authors.initials: A
authors.lastname: Seubert
authors.affiliation: From the Laboratorium für Mikrobiologie, Fachbereich Biologie and Synmikro and the Max-Plank-Institut für terrestrische Mikrobiologie, 35043 Marburg, Germany buckel@staff.uni-marburg.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
mesh_terms: D045850:Acidaminococcus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D016227:Benzoquinones / Q000378:metabolism; D044926:Butyryl-CoA Dehydrogenase / Q000378:metabolism; D002384:Catalysis; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins / Q000235:genetics / Q000378:metabolism*; D004583:Electrons; D006859:Hydrogen / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D016227:Benzoquinones; D044927:Electron-Transferring Flavoproteins; D006873:Hydroquinones; D011994:Recombinant Proteins; D009243:NAD; C004532:quinone; D006859:Hydrogen; C010701:crotonyl-coenzyme A; D012964:Sodium; D010088:Oxidoreductases; C028878:ferredoxin-NAD+ reductase; D044926:Butyryl-CoA Dehydrogenase; D012256:Riboflavin; C031927:hydroquinone
keywords: bioenergetics; electron bifurcation; electron transport; ferredoxin; flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); flavodoxin; flavoprotein; redox potential; sodium transport