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.

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.

Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.

Heme oxygenases (HOs) 3 are enzymes that oxidatively liberate iron from the heme tetrapyrrole (1-3). In the well characterized HOs from animals and many bacteria, the same heme molecule acts as both the O 2 -activating cofactor and substrate. Three successive monooxygenation steps yield Fe(II), CO, and biliverdin IX␣ as the end products of the reaction (Fig. 1) (2, 3). Animals use HOs to maintain cellular heme homeostasis as part of a constant cycle of heme synthesis and breakdown. The products report on the status of this cycle and serve as antioxidants and signaling agents (4, 5). Many bacteria also use HO homologs, both to control heme homeostasis and to liberate iron from host-derived heme (6, 7). Heme, found primarily in hemoglobin, can therefore be used as a rich nutritional source of iron. Because of the intriguing nature of the reaction, which uses heme as both cofactor and substrate (8 -10), as well as the acute biological importance of HO-mediated processes, HOs from several species have been exceptionally well characterized (2, 3).By the early 2000s, however, it was apparent that many important Gram-positive pathogens that degrade host heme did not possess an HO-encoding gene in their genomes. A new family of heme-degrading proteins known as IsdGs was subsequently discovered, with representatives found in bacteria from both Gram-positive and Gram-negative phyla (11). IsdG family proteins are evolutionarily and structurally distinct from the well studied HOs (12, 13), and they yield different end products. Instead of biliverdin IX␣ and CO, the IsdG protein from Mycobacterium tuberculosis (known as MhuD) generates triply-oxygenated linear tetrapyrroles called mycobilins (Fig. 1) (14, 15). A formyl group remains appended to pyrrole ring A or B at the site of macrocycle cleavage, and an oxo group is generated on the pyrrole ring on the opposite side. Notably, no C1 product is released (16).Although homologous to MhuD, the IsdG from Staphylococcus aureus degrades heme to yet a third set of products. The macrocycle is not cleaved at the ␣-meso-but rather at either the ␤-or ␦-meso-carbon. Oxo groups are generated on both the carbon backbone and the pyrrole rings at the cleavage site, generating tetrapyrrole products known as staphylobilins (Fig. 1) (17). It was recently shown that a C1 product is indeed released by the S. aureus IsdG; however, quite unexpectedly, the major C1 product was determined to be formaldehyde (CH 2 O) instead of CO (18). Unlike CO, formaldehyde may be undetectable by animal immune systems, offering a potential selective advantage for heme-feeding pathogens that use IsdG-type enzymes (5,19,20). Mechanistically, the observation that CH 2 O instead of CO implies that verdoheme, the green inter-* This work was supported in part by National Institutes of Health Grants GM090260 and 5P20RR02437 of the CoBRE Program (to J. L. D.) and Grant RO1 AI069233 (to E. P. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the resp...