title: Ligand binding and conformational dynamics in a flavin-based electron-bifurcating enzyme complex revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.
abstract: Flavin-based electron bifurcation (FBEB) is a novel mechanism of energy coupling used by anaerobic microorganisms to optimize their energy metabolism efficiency. The first high-resolution structure of a complete FBEB enzyme complex, the NADH-dependent reduced ferredoxin: NADP+-oxidoreductase (NfnAB) of Thermotoga maritima, was recently solved. However, no experimental evidence for the NADPH-binding site and conformational changes during the FBEB reaction are available. Here we analyzed ligand binding and the conformational dynamics of oxygen-sensitive NfnAB using Hydrogen-Deuterium Exchange Mass-Spectrometry, including a customized anaerobic workflow. We confirmed the NADH and the previously postulated NADPH-binding site. Furthermore, we observed an NfnA-NfnB rearrangement upon NADPH binding which supports the proposed FBEB mechanism.
authors.affiliation: Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Julius K
authors.initials: JK
authors.lastname: Demmer
authors.affiliation: Max Planck Institute of Biophysics, Frankfurt am Main, Germany.|Max Planck Institute for Brain Research, Frankfurt am Main, Germany.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Rupprecht
authors.affiliation: Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Eisinger
authors.affiliation: Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Ermler
authors.affiliation: Max Planck Institute of Biophysics, Frankfurt am Main, Germany.|Max Planck Institute for Brain Research, Frankfurt am Main, Germany.
authors.forename: Julian D
authors.initials: JD
authors.lastname: Langer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D041961:Deuterium Exchange Measurement; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D008024:Ligands; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D008024:Ligands; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases
keywords: Hydrogen-Deuterium Exchange Mass Spectrometry; flavin-based electron bifurcation