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キーワード:
flavin-based electron bifurcation; Hydrogen–Deuterium Exchange Mass Spectrometry
要旨:
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.
