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.

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 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 (° values) to individual flavins, and the ° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-° 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-° 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.

The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases—such as the yeast Drs2 and human ATP8A1—have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the S. cerevisiae Dnf1–Lem3 and Dnf2–Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.

Experiments and computation establish the locations of the two flavins whose contrasting reactivities produce electron bifurcation in ETFs. They confirm the accepted model and support homologies & distinctions between bifurcating and canonical ETFs.

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-E° carrier and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that the CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this may be the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

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P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory β-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a β-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1’s ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

The brain as a neuronal system has very complex structures with a large diversity of neuronal types. The most basic complexity is seen from the structure of neuronal morphology, which usually has a complex tree-like structure with dendritic spines distributed in branches. To simulate a large-scale network with spiking neurons, the simple point neuron, such as the integrate-and-fire neuron, is often used. However, recent experimental evidence suggests that the computational ability of a single neuron is largely enhanced by its morphological structure, in particular, by various types of dendritic dynamics. As the morphology reduction of detailed biophysical models is a classic question in systems neuroscience, much effort has been taken to simulate a neuron with a few compartments to include the interaction between the soma and dendritic spines. Yet, novel reduction methods are still needed to deal with the complex dendritic tree. Here, using 10 individual Purkinje cells of the cerebellum from three species of guinea-pig, mouse and rat, we consider four types of reduction methods and study their effects on the coding capacity of Purkinje cells in terms of firing rate, timing coding, spiking pattern, and modulated firing under different stimulation protocols. We found that there is a variation of reduction performance depending on individual cells and species, however, all reduction methods can preserve, to some degree, firing activity of the full model of Purkinje cell. Therefore, when stimulating large-scale network of neurons, one has to choose a proper type of reduced neuronal model depending on the questions addressed. Among these reduction schemes, Branch method, that preserves the geometrical volume of neurons, can achieve the best balance among different performance measures of accuracy, simplification, and computational efficiency, and reproduce various phenomena shown in the full morphology model of Purkinje cells. Altogether, these results suggest that the Branch reduction scheme seems to provide a general guideline for reducing complex morphology into a few compartments without the loss of basic characteristics of the firing properties of neurons.