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. 2017 Aug 15;56(32):4177-4190.
doi: 10.1021/acs.biochem.7b00389. Epub 2017 Aug 3.

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

Rhesa N Ledbetter  1 , Amaya M Garcia Costas  2 , Carolyn E Lubner  3 , David W Mulder  3 , Monika Tokmina-Lukaszewska  2 , Jacob H Artz  4 , Angela Patterson  2 , Timothy S Magnuson  5 , Zackary J Jay  6 , H Diessel Duan  7 , Jacquelyn Miller  2 , Mary H Plunkett  8 , John P Hoben  7 , Brett M Barney  8 , Ross P Carlson  6 , Anne-Frances Miller  7 , Brian Bothner  2 , Paul W King  3 , John W Peters  2   4 , Lance C Seefeldt  1
Affiliations

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

Rhesa N Ledbetter et al. Biochemistry. .

Abstract

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 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.

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Figures

Figure 1.
Figure 1.
Phenotype of A. vinelandii DJ wt (A), Δfix (B), Δrnf1 (C), ΔfixΔrnf1 (D), Δrnf2 (E), and ΔfixΔrnf2 (F) strains. Wild type and mutant strains were cultivated in Burk’s medium supplemented with ammonium acetate (+N) or with no added fixed nitrogen (−N). All samples were grown aerobically.
Figure 2.
Figure 2.
(A) Proposed mechanism of FixABCX electron bifurcation in A. vinelandii, operating within the framework of the known oxidation-reduction potentials of NADH, Fld/Fd, and CoQ. It is hypothesized that crossed potentials of the bifurcating flavin (a-FAD) promote electron bifurcation based on the appearance of a short-lived ASQ. Once the first electron transfers into the exergonic branch, the second electron, sitting at a very low reduction potential, is thermodynamically unstable and immediately transfers into the endergonic branch composed of the low potential iron-sulfur clusters of FixX. The exergonic branch is set up to ensure electron delivery to c-FAD where electrons accumulate before being transferred to CoQ. The specific oxidation-reduction potential levels are qualitative unless otherwise noted. (B) Electron transfer pathways illustrated in FixABCX structural model generated by docking (ClusPro2) four homology models (Phyre2) (Figure S8).
Figure 3.
Figure 3.
SDS-PAGE gel of purified (~80%) FixABCX complex from A. vinelandii (extra lanes were removed). All Fix protein bands were verified by mass spectrometry (Figure S3) and according to densitometry, the subunit stoichiometry of FixABCX is 1:1:1:1.
Figure 4.
Figure 4.
A. vinelandii FixABCX complex composition. (A) Native mass spectrum of FixAB containing two FAD cofactors (71,414.8 Da; calculated MW = 71,413.7 Da) generated during FixABCX complex activation in the gas phase. Red diamonds signify charge state envelope centered around charge 18+. The unmarked masses are the charge state envelop of the molecular chaperone DnaK. (B) Sub-complexes obtained during FixABCX complex activation. (C) SDS-PAGE gel of FixABCX complex (right) cross-linked with BS3 reagent (left). The most distinguished bands in cross-linking reaction, migrating around the 60 kDa, 70 kDa, and 88 kDa molecular weight marker, were identified as FixCX, FixAB and FixBC dimers respectively. (D) Protein-protein interaction map based on highest scoring cross-links (score 7 and higher; red and blue lines indicate intra and inter cross-links, respectively). A complete list of generated cross-links can be found in Table S3.
Figure 5.
Figure 5.
UV-visible spectrum of the FixABCX complex from A. vinelandii demonstrating the presence of flavin. Black: as-purified FixABCX, red: cofactors released upon denaturation of FixABCX, and blue: flavins as observed in the spectrum of FixAB from R. palustris, vertically offset by −0.05 AU for clarity. The two spectra representing FixABCX were corrected for Raleigh scattering by fitting the baseline to the equation for scattering and then subtracting the fit from the measured spectrum to obtain a corrected spectrum. A. vinelandii FixABCX was prepared in 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.02% (w/v) DDM. R. palustris FixAB was prepared in 20 mM Bis-Tris propane, pH 9, 200 mM KCl, 10% (w/v) glycerol and 1 mM tris(2-carboxyethyl)phosphine (TCEP).
Figure 6.
Figure 6.
EPR spectra of FixABCX from A. vinelandii prepared in 50 mM HEPES pH 7.5, 150 mM NaCl, and 5% glycerol. Black: As-prepared FixABCX (100 μM); Blue: FixABCX (100 μM) reduced with NADH (1 mM); Red: FixABCX (100 μM) reduced with sodium dithionite (10 mM). Simulations of spectra of NADH- and Na-dithionite-reduced FixABCX are shown in lighter colored traces. Microwave frequency, 9.38 GHz; microwave power, 1 mW; modulation frequency, 100 kHz; modulation amplitude, 10.0 G; sample temperature, 10 K.
Figure 7.
Figure 7.
Electron bifurcation by the FixABCX complex of A. vinelandii under anaerobic conditions. (A) Overview of electron bifurcation by the FixABCX complex showing the NADH bifurcating an electron to the high-potential acceptor, CoQ1 and the other to the low-potential acceptor, FldSq. Evidence of electron bifurcation was obtained using UV-visible spectrophotometry. NADH oxidation as well as FldSq reduction and oxidation were monitored over time as signatures of the bifurcation reaction. (B) NADH oxidation at 340 nm, C) FldSq reduction/oxidation at 580 nm, and D) formation of FldOx at 450 nm. Final concentrations of components added to the reactions were as follows: 0.8 μM FixABCX, 85 μM FldSq, 200 μM NADH, and 300 μM CoQ1. Data were normalized and demonstrate the overall change in absorbance.
Figure 8.
Figure 8.
Transient absorption spectra of as-prepared R. rubrum FixAB (red traces) and A. vinelandii FixABCX (black traces). (A) Kinetic traces of ASQ signal (dots) at 365 nm. ASQ decay shows half-lives of ~15 ps and ~1000 ps when fit with a double exponential function (solid lines). (B) An NSQ absorption peak (~565–650 nm) is only observed in the A. vinelandii FixABCX complex.
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