Elucidation of the Azotobacter vinelandii FixABCX Energy Landscape Reveals a Conformationally Gated Flavin-Based Electron Bifurcation

Abstract Electron bifurcation was first used to describe the electron-transfer events associated with the quinone-cycle (Q-cycle), which occurs in complex III of the electron transport chain. Decades later, flavin-based electron bifurcation (FBEB) was discovered, where flavin cofactors act in analogy to Q to drive the endergonic reduction of ferredoxin (Fd) or flavodoxin (Fld) by NAD(P)H. The soil microbeAzotobacter vinelandii is known to generate low-potential electrons in the form of reduced Fd or Fld using FBEB via the enzyme complex FixABCX. FixABCX in A. vinelandii (AvFixABCX) couples the exergonic reduction of coenzyme Q (E = +10 mV) to the endergonic reduction of Fd/Fld (E = ∼−450 mV) during NADH oxidation (E = −320 mV). In this work, we successfully optimized the expression of AvFixABCX in E. coli. We have employed protein thin-film electrochemistry, UV–vis spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy, and transient absorption (TA) spectroscopy to elucidate the complete energy landscape, and structural characterization methods, cryo-electron microscopy (cryo-EM), small-angle X-ray scattering (SAXS), and cross-linking mass spectrometry (XL-MS) to show the dynamics of the complex energy landscape during AvFixABCX electron-transfer catalysis. Our results show that AvFixABCX contains two [4Fe-4S] clusters with disparate reduction potentials: the higher-potential cluster participates in the exergonic electron-transfer pathway, whereas the lower-potential cluster is off-pathway. We experimentally determined that AvFixABCX undergoes a conformationally gated, reversible FBEB mechanism to simultaneously reduce coenzyme Q and the semiquinone state of Fld during NADH oxidation.