The biological reduction of dinitrogen (N<
sub>
2<
/sub>
) to ammonia (NH<
sub>
3<
/sub>
) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP
however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from <
i>
Azotobacter vinelandii<
/i>
was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E<
sub>
m<
/sub>
= -320 mV) coupled to reduction of flavodoxin semiquinone (E<
sub>
m<
/sub>
= -460 mV) and reduction of coenzyme Q (E<
sub>
m<
/sub>
= 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.