The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.

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.

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD ؉ , we propose a catalytic cycle involving formation of an intermediary NAD ؉ -bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low-and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP ؉ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD ؉ , the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two twoelectron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.Electron-bifurcating enzymes couple exergonic and endergonic reactions, thus maximizing conservation of free energy available from exergonic reactions (1). In this way, electrochemical energy can be captured for cellular metabolism, lowering the demands on transmembrane gradients or substrate-level phosphorylation. Thus, electron bifurcation provides a unifying explanation for many peculiar fermentative pathways found in anaerobic microorganisms, with important implications for understanding anaerobic microbial physiology in general (2-9).So far, the bifurcating enzymes that have been characterized fall into one of four phylogenetically unrelated groups: electron transfer flavoproteins (EtfAB-containing), [FeFe]-hydrogenase/formate dehydrogenases (HydABC-containing), heterodisulfide reductases (HdrA-containing), and transhydrogenases (NfnAB-containing) (8, 10). These enzymes catalyze more than a dozen different reactions, most involving the oxidation or reduction of ferredoxin, and are found mainly in anaerobic organisms (reviewed in Refs. 3,8,11,and 12). However, some of the EtfAB-containing complexes, such as that described below, can also be found in microaerophiles and aerobes.Bifurcating ETFs 2 are the best-studied bifurcating enzymes, and they form a subset of the large and well-known family of ETFs, which ar...

This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.