The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

Abstract

The biological reduction of dinitrogen (N) to ammonia (NH) 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 Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E = -320 mV) coupled to reduction of flavodoxin semiquinone (E = -460 mV) and reduction of coenzyme Q (E = 10 mV). Knocking out fix genes rendered Δrnf 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. Overall, 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.

Molecular basis of the flavin‐based electron‐bifurcating caffeyl‐CoA reductase reaction

“…A further significant resemblance was found between CarE of A. woodii and the family of FixB proteins (28–30% identity) also composed of members with and without a Fd‐like domain. FixB proteins are involved in the anaerobic carnitine and amine/polyamine metabolism and presumably also in a FBEB event . Altogether, no significant common features between EtfAB sequences with and without a Fd‐like domain and between the metabolisms of the corresponding microorganisms were deducible.…”

The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium

“…In vitro studies with FixABCX from Azotobacter vinelandii showed that this complex bifurcates electrons from NADH to generate reduced quinone and reduced Fd or Fld as the primary electron donor to nitrogenase (Ledbetter et al ., ). R. palustris has three Fd genes in its nif gene cluster ( rpa4602‐rpa4634 ): fdxB ( rpa4612 ), ferN ( rpa4629 ) and fer1 ( rpa4631 ), all of which are upregulated under nitrogen‐fixing conditions (Oda et al ., ) (Supporting Information Fig.…”

“…Known electron transfer pathways to nitrogenase include pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR/NifJ), the Rhodobacter nitrogen fixation (Rnf) complex, hydrogenases and FixABCX, and a number of nitrogen‐fixing organisms can utilize more than one pathway (Edgren and Nordlund, ; Herrmann et al ., ; Khanna and Lindblad, ; Poudel et al ., ; Ledbetter et al ., ). R. palustris does not encode Rnf, but it encodes genes that are annotated as PFOR as well as FixABCX.…”

“…The reduced Fd 2‐ generated via electron transfer from NADH serves as the low potential electron donor to nitrogenase. In vitro , Fd can be replaced by the Fld semiquinone during a FBEB event (Chowdhury et al ., ; Ledbetter et al ., ). Here, we show in vivo evidence that FixABCX from R. palustris can also donate electrons to Fd and Fld (Fig.…”

“…R. palustris has served as a model organism for in vivo studies of production of energy‐rich compounds including H 2 and more recently, methane, by nitrogenase (Gosse et al ., ; Fixen et al ., ; Zheng et al ., ). This organism encodes the electron‐transfer complex FixABCX, which bifurcates electrons from NADH to generate reduced quinone and reduced Fd or Fld (Edgren and Nordlund, ; Ledbetter et al ., ). Mutants with deletions in fixC or fixX are unable to grow under nitrogen‐fixing conditions, suggesting that FixABCX is required for electron transfer to nitrogenase (Huang et al ., ).…”