skip to main content

DOE PAGESDOE PAGES

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

The biological reduction of dinitrogen (N 2) to ammonia (NH 3) 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 m = -320 mV) coupled to reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 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 themore » 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.« less
Authors:
ORCiD logo [1] ;  [2] ;  [3] ;  [3] ;  [2] ;  [4] ;  [2] ;  [5] ;  [2] ;  [6] ;  [2] ;  [7] ;  [6] ;  [7] ;  [2] ; ORCiD logo [6] ;  [2] ; ORCiD logo [3] ;  [8] ; ORCiD logo [1]
  1. Utah State Univ., Logan, UT (United States)
  2. Montana State Univ., Bozeman, MT (United States)
  3. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  4. Washington State Univ., Pullman, WA (United States)
  5. Idaho State Univ., Pocatello, ID (United States)
  6. Univ. of Kentucky, Lexington, KY (United States)
  7. Univ. of Minnesota, St. Paul, MN (United States)
  8. Montana State Univ., Bozeman, MT (United States); Washington State Univ., Pullman, WA (United States)
Publication Date:
Report Number(s):
NREL/JA-2700-68237
Journal ID: ISSN 0006-2960
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 56; Journal Issue: 32; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; electron paramagnetic resonance; EPR; electron transfer; flavoprotein; nitrogen fixation; oxidation-reduction; redox
OSTI Identifier:
1376663