The Rnf complex from the acetogenic bacterium Acetobacterium woodii: Purification and characterization of RnfC and RnfB

• Published in: Biochimica et biophysica acta. Bioenergetics Vol. 1861; no. 11; p. 148263
• Main Authors: Kuhns, Martin, Schuchmann, Verena, Schmidt, Silke, Friedrich, Thorsten, Wiechmann, Anja, Müller, Volker
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2020
• Subjects: Acetobacterium - enzymology; Anaerobic bacteria; ATP synthesis; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Electron Transport; Energy conservation; Ferredoxins - metabolism; Flavin Mononucleotide - metabolism; NAD - metabolism; Oxidation-Reduction; Respiratory chain; Anaerobic bacteria; Respiratory chain; Energy conservation; EPR; CN PAGE; ATP synthesis; MTT; DDM; Electron transport
• ISSN: 0005-2728; 1879-2650
• DOI: 10.1016/j.bbabio.2020.148263

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex. •The Rnf complex is an ancient respiratory enzyme present in many anaerobic bacteria.•The complex as well as subunits were purified.•RnfC binds NADH and transfers electrons from NADH via FMN into the complex.•RnfB contains FeS centers that are reduced by reduced ferredoxin.•RnfB and RnfC are the entry and exit points for electrons in the Rnf complex.