4 research outputs found
Distinct Properties Underlie Flavin-Based Electron Bifurcation in a Novel Electron Transfer Flavoprotein FixAB from \u3cem\u3eRhodopseudomonas palustris\u3c/em\u3e
A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV–visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e−) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e−) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins
Structural insight into an Arl1–ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin
Abstract Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1–Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1–Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1–Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1–Gea2 interaction and improves our understanding of molecular events in the membrane trafficking
The Electron Bifurcating FixABCX Protein Complex from <i>Azotobacter vinelandii</i>: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis
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 (<i>E</i><sub>m</sub> = −320 mV) coupled to reduction of
flavodoxin semiquinone (<i>E</i><sub>m</sub> = −460
mV) and reduction of coenzyme Q (<i>E</i><sub>m</sub> =
10 mV). Knocking out <i>fix</i> genes rendered Δ<i>rnf A. vinelandii</i> 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