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

Journal Article · · Biochemistry
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)
+ Show Author Affiliations

The biological reduction of dinitrogen (N2) to ammonia (NH3) 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 (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 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.

Research Organization:
National Renewable Energy Laboratory (NREL), Golden, CO (United States); Energy Frontier Research Centers (EFRC) (United States). Center for Biological Electron Transfer and Catalysis (BETCy)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
AC36-08GO28308; SC0012518
OSTI ID:
1376663
Report Number(s):
NREL/JA-2700-68237
Journal Information:
Biochemistry, Vol. 56, Issue 32; ISSN 0006-2960
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 90 works
Citation information provided by
Web of Science

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Cited By (22)

Control of nitrogen fixation in bacteria that associate with cereals journal December 2019
Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review journal March 2018
The oxygen reduction reaction catalyzed by Synechocystis sp. PCC 6803 flavodiiron proteins journal January 2019
Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation text January 2019
Rhizobia: from saprophytes to endosymbionts journal January 2018
Origin and Evolution of Flavin-Based Electron Bifurcating Enzymes journal August 2018
Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein journal January 2019
Aerobic nitrogen-fixing bacteria for hydrogen and ammonium production: current state and perspectives journal December 2019
Structural and Functional Characterization of an Electron Transfer Flavoprotein Involved in Toluene Degradation in Strictly Anaerobic Bacteria journal August 2019
Manipulating nitrogen regulation in diazotrophic bacteria for agronomic benefit journal April 2019
Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics journal August 2019
Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives journal August 2019
The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD + journal December 2018
Concerted Metabolic Shifts Give New Insights Into the Syntrophic Mechanism Between Propionate-Fermenting Pelotomaculum thermopropionicum and Hydrogenotrophic Methanocella conradii journal July 2018
Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds journal February 2018
Influence of Energy and Electron Availability on In Vivo Methane and Hydrogen Production by a Variant Molybdenum Nitrogenase journal March 2019
Electron bifurcation: progress and grand challenges journal January 2019
On the nature of organic and inorganic centers that bifurcate electrons, coupling exergonic and endergonic oxidation–reduction reactions journal January 2018
The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium journal July 2018
One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window journal January 2019
Organohalide-respiring Desulfoluna species isolated from marine environments journal January 2020
The Role of Mass Spectrometry in Structural Studies of Flavin-Based Electron Bifurcating Enzymes journal July 2018

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Related Subjects

09 BIOMASS FUELS
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
electron paramagnetic resonance
EPR
electron transfer
flavoprotein
nitrogen fixation
oxidation-reduction
redox