The bare bones of bifurcation: small flavoproteins as artificial electron bifurcases

Abstract

Flavin‐based electron bifurcation (FBEB) is an essential mode of energy conservation in bacteria and archaea wherein specific flavoenzymes couple exergonic and endergonic metabolic electron flux from a single electron donor. While our understanding of the role of FBEB in metabolism continues to grow, the mechanism by which the flavin cofactor and the protein scaffold coordinate electron bifurcation remains elusive, largely due to the complexity of native bifurcases. We have leveraged the light‐oxygen‐voltage (LOV) domain flavoprotein as a robust and straight‐forward scaffold to interrogate the operant principles of electron bifurcation, including the role of the flavin binding pocket on tuning the flavin redox potential, proton‐coupled electron transfer, and potential “inversion,” as well as the role of conformational gating. Using bioinformatics and structural homology, we have remodeled the LOV flavin binding site to mimic that of native bifurcases and demonstrated key residues stabilize the anionic hydroquinone and increases potential inversion by 60 mV relative to wild‐type LOV. Furthermore, reactions with single electron donors/acceptors implicates a reactive neutral semiquinone, and lay the groundwork for kinetic electron bifurcation studies. Using cytochrome c as a model one‐electron acceptor, we can model the conformational gating mechanism by controlling donor‐acceptor concentrations, and have demonstrated the facile inter‐protein electron transfer, and photochemical crosslinking indicates a unique electron transfer interface. Kinetic modeling, including short‐circuit pathways, suggests that a bifurcation prone state should be long enough lived for direct observation, and studies are ongoing to spectroscopically identify this species. These results illustrate the utility of this reductionist approach in gaining insight into the physical principles that dictate FBEB and inform novel bifurcase design.