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Updated: Mar 11, 2026

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
Published on: October 21, 2016
Ligand binding and conformational dynamics in a flavin-based electron-bifurcating enzyme complex revealed by
Julius K Demmer1, Fiona A Rupprecht1,2, Martin L Eisinger1
1Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
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.
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