Properties of the iron-sulfur cluster electron transfer relay in an [FeFe]-hydrogenase that is tuned for H2 oxidation catalysis.

[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays amongst the [FeFe]-hydrogenases, however it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of F-cluster relays. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII), exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric EPR, we resolved two EPR signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 ± 18 mV and -406 ± 12 mV vs. NHE, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.

Hole-scavenging in photo-driven N2 reduction catalyzed by a CdS-nitrogenase MoFe protein biohybrid system.

The light-driven reduction of dinitrogen (N2) to ammonia (NH3) catalyzed by a cadmium sulfide (CdS) nanocrystal­nitrogenase MoFe protein biohybrid is dependent on a range of different factors, including an appropriate hole-scavenging sacrificial electron donor (SED). Here, the impact of different SEDs on the overall rate of N2 reduction catalyzed by a CdS quantum dot (QD)-MoFe protein system was determined. The selection of SED was guided by several goals: (i) molecules with standard reduction potentials sufficient to reduce the oxidized CdS QD, (ii) molecules that do not absorb the excitation wavelength of the CdS QD, and (iii) molecules that could be readily reduced by sustainable processes. Earlier studies utilized buffer molecules or ascorbic acid as the SED. The effectiveness of ascorbic acid as SED was compared to dithionite (DT), triethanolamine (TEOA), and hydroquinone (HQ) across a range of concentrations in supporting N2 reduction to NH3 in a CdS QD-MoFe protein photocatalytic system. It was found that TEOA supported N2 reduction rates comparable to those observed for dithionite and ascorbic acid. HQ was found to support significantly higher rates of N2 reduction compared to the other SEDs at a concentration of 50 mM. A comparison of the rates of N2 reduction by the biohybrid complex to the standard reduction potential (Eo) of the SEDs reveals that Eo is not the only factor impacting the efficiency of hole-scavenging. These findings reveal the importance of the SED properties for improving the efficiency of hole-scavenging in the light-driven N2 reduction reaction catalyzed by a CdS QD-MoFe protein hybrid.

Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor.

Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Low-temperature trapping of N2 reduction reaction intermediates in nitrogenase MoFe protein-CdS quantum dot complexes.

The biological reduction of N2 to ammonia requires the ATP-dependent, sequential delivery of electrons from the Fe protein to the MoFe protein of nitrogenase. It has been demonstrated that CdS nanocrystals can replace the Fe protein to deliver photoexcited electrons to the MoFe protein. Herein, light-activated electron delivery within the CdS:MoFe protein complex was achieved in the frozen state, revealing that all the electron paramagnetic resonance (EPR) active E-state intermediates in the catalytic cycle can be trapped and characterized by EPR spectroscopy. Prior to illumination, the CdS:MoFe protein complex EPR spectrum was composed of a S = 3/2 rhombic signal (g = 4.33, 3.63, and 2.01) consistent with the FeMo-cofactor in the resting state, E0. Illumination for sequential 1-h periods at 233 K under 1 atm of N2 led to a cumulative attenuation of E0 by 75%. This coincided with the appearance of S = 3/2 and S = 1/2 signals assigned to two-electron (E2) and four-electron (E4) reduced states of the FeMo-cofactor, together with additional S = 1/2 signals consistent with the formation of E6 and E8 states. Simulations of EPR spectra allowed quantification of the different E-state populations, along with mapping of these populations onto the Lowe-Thorneley kinetic scheme. The outcome of this work demonstrates that the photochemical delivery of electrons to the MoFe protein can be used to populate all of the EPR active E-state intermediates of the nitrogenase MoFe protein cycle.

Fe protein docking transduces conformational changes to MoFe nitrogenase active site in a nucleotide-dependent manner.

The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N2 reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site.

High Affinity Electrostatic Interactions Support the Formation of CdS Quantum Dot:Nitrogenase MoFe Protein Complexes.

Nitrogenase MoFe protein can be coupled with CdS nanocrystals (NCs) to enable photocatalytic N2 reduction. The nature of interactions that support complex formation is of paramount importance in intermolecular electron transfer that supports catalysis. In this work we have employed microscale thermophoresis to examine binding interactions between 3-mercaptopropionate capped CdS quantum dots (QDs) and MoFe protein over a range of QD diameters (3.4-4.3 nm). The results indicate that the interactions are largely electrostatic, with the strength of interactions similar to that observed for the physiological electron donor. In addition, the strength of interactions is sensitive to the QD diameter, and the binding interactions are significantly stronger for QDs with smaller diameters. The ability to quantitatively assess NC protein interactions in biohybrid systems supports strategies for understanding properties and reaction parameters that are important for obtaining optimal rates of catalysis in biohybrid systems.

Cryo-annealing of Photoreduced CdS Quantum Dot-Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E4(2N2H) Intermediate.

A critical step in the mechanism of N2 reduction to 2NH3 catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E4(4H)). This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H2 or going forward with N2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H2 to form the E4(2N2H) state. E4(2N2H) can relax to E4(4H) by the oxidative addition (oa) of H2 and release of N2 or can be further reduced in a series of catalytic steps to release 2NH3. If the H2 re/oa mechanism is correct, it requires that oa of H2 be associative with E4(2N2H). In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E4(2N2H). Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates. Electron paramagnetic resonance spectroscopy identified E-state signals including E2 and E4(2N2H), as well as signals suggesting the formation of E6 or E8. It is shown that in the frozen state when pN2 is much greater than pH2, the E4(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H2 to the E4(2N2H) state follows an associative reaction mechanism.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Mechanisms for Generating Low Potential Electrons across the Metabolic Diversity of Nitrogen-Fixing Bacteria.

The availability of fixed nitrogen is a limiting factor in the net primary production of all ecosystems. Diazotrophs overcome this limit through the conversion of atmospheric dinitrogen to ammonia. Diazotrophs are phylogenetically diverse bacteria and archaea that exhibit a wide range of lifestyles and metabolisms, including obligate anaerobes and aerobes that generate energy through heterotrophic or autotrophic metabolisms. Despite the diversity of metabolisms, all diazotrophs use the same enzyme, nitrogenase, to reduce N2. Nitrogenase is an O2-sensitive enzyme that requires a high amount of energy in the form of ATP and low potential electrons carried by ferredoxin (Fd) or flavodoxin (Fld). This review summarizes how the diverse metabolisms of diazotrophs utilize different enzymes to generate low potential reducing equivalents for nitrogenase catalysis. These enzymes include substrate-level Fd oxidoreductases, hydrogenases, photosystem I or other light-driven reaction centers, electron bifurcating Fix complexes, proton motive force-driven Rnf complexes, and Fd:NAD(P)H oxidoreductases. Each of these enzymes is critical for generating low potential electrons while simultaneously integrating the native metabolism to balance nitrogenase's overall energy needs. Understanding the diversity of electron transport systems to nitrogenase in various diazotrophs will be essential to guide future engineering strategies aimed at expanding the contributions of biological nitrogen fixation in agriculture.

A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H2 formation.

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e-/H+] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e-/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e-/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e-/H+] generates E4(4H) with Fe2-H-Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by slower HP of Fe3-H-Fe7, which leads to transient accumulation of E2(2H) containing Fe3-H-Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3-H-Fe7 occurs first and the resulting E2(2H) contains Fe2-H-Fe6. It is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.

Rnf and Fix Have Specific Roles during Aerobic Nitrogen Fixation in Azotobacter vinelandii.

Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, but deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADH's endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the chemiosmotic potential, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different reaction stoichiometries and condition-specific differential gene expression indicate specific roles for the two reactions. This work's complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf is presumed to maintain reduced Fd/Fld production for nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions. IMPORTANCE The availability of fixed nitrogen is critical for life in many ecosystems, from extreme environments to agriculture. Due to the energy demands of biological nitrogen fixation, organisms must tailor their metabolism during diazotrophic growth to deliver the energy requirements to nitrogenase in the form of ATP and low potential electrons. Therefore, a complete understanding of diazotrophic energy metabolism and redox homeostasis is required to understand the impact on ecological communities or to promote crop growth in agriculture through engineered diazotrophs.

The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

The Unfolded-Protein Response Triggers the Arthropod Immune Deficiency Pathway.

The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed "vector competency." Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease (Borrelia burgdorferi) and anaplasmosis (Anaplasma phagocytophilum). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.

A site-differentiated [4Fe-4S] cluster controls electron transfer reactivity of Clostridium acetobutylicum [FeFe]-hydrogenase I.

One of the many functions of reduction-oxidation (redox) cofactors is to mediate electron transfer in biological enzymes catalyzing redox-based chemical transformation reactions. There are numerous examples of enzymes that utilize redox cofactors to form electron transfer relays to connect catalytic sites to external electron donors and acceptors. The compositions of relays are diverse and tune transfer thermodynamics and kinetics towards the chemical reactivity of the enzyme. Diversity in relay design is exemplified among different members of hydrogenases, enzymes which catalyze reversible H2 activation, which also couple to diverse types of donor and acceptor molecules. The [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is a member of a large family of structurally related enzymes where interfacial electron transfer is mediated by a terminal, non-canonical, His-coordinated, [4Fe-4S] cluster. The function of His coordination was examined by comparing the biophysical properties and reactivity to a Cys substituted variant of CaI. This demonstrated that His coordination strongly affected the distal [4Fe-4S] cluster spin state, spin pairing, and spatial orientations of molecular orbitals, with a minor effect on reduction potential. The deviations in these properties by substituting His for Cys in CaI, correlated with pronounced changes in electron transfer and reactivity with the native electron donor-acceptor ferredoxin. The results demonstrate that differential coordination of the surface localized [4Fe-4S]His cluster in CaI is utilized to control intermolecular and intramolecular electron transfer where His coordination creates a physical and electronic environment that enables facile electron exchange between electron carrier molecules and the iron-sulfur cluster relay for coupling to reversible H2 activation at the catalytic site.

Phosphorus containing analogues of SAHA as inhibitors of HDACs.

Histone deacetylases (HDACs) are a family of enzymes responsible for regulating DNA transcription by modulating its binding to histone proteins. HDACs are overexpressed in several types of cancers and are recognised as drug targets. Vorinostat, or suberanilohydroxamic acid (SAHA), is an histone deacetylase (HDAC) inhibitor with a hydroxamic acid as a zinc-binding group (ZBG), and it has been FDA approved for the treatment of T-cell lymphoma. In this work, phosphorus-based SAHA analogues were synthesised to assess their zinc-binding effectiveness compared to the hydroxamic acid of SAHA. Specifically, we examined phosphate, phosphoramidate and phosphorothiolate groups as isosteres of the canonical hydroxamic acid motif of conventional HDAC inhibitors. The compounds were screened for binding to HDAC enzymes from HeLa cell lysate. The most potent derivatives were then screened against HDAC3 and HDAC8 isoforms. HDAC inhibition assays demonstrated that these phosphorus-based SAHA analogs exhibited slow binding to HDACs but with greater potency than phosphonate SAHA analogs examined previously. All compounds inhibited HDACs, the most potent having an IC50 of 50 µM.

A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H+ and CO2 to favor the metabolically preferred carboxylation product, acetoacetate.

Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction.

The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, PN, P+, P2+ of which PN↔P+ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P+ during electron transfer, photochemical reduction of MoFe protein at 231-263 K was used to trap formation of P+ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P2+ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P+ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation.

Electron bifurcation, an energy-conserving process utilized extensively throughout all domains of life, represents an elegant means of generating high-energy products from substrates with less reducing potential. The coordinated coupling of exergonic and endergonic reactions has been shown to operate over an electrochemical potential of ∼1.3 V through the activity of a unique flavin cofactor in the enzyme NADH-dependent ferredoxin-NADP+ oxidoreductase I. The inferred energy landscape has features unprecedented in biochemistry and presents novel energetic challenges, the most intriguing being a large thermodynamically uphill step for the first electron transfer of the bifurcation reaction. However, ambiguities in the energy landscape at the bifurcating site deriving from overlapping flavin spectral signatures have impeded a comprehensive understanding of the specific mechanistic contributions afforded by thermodynamic and kinetic factors. Here, we elucidate an uncharacteristically low two-electron potential of the bifurcating flavin, resolving the energetic challenge of the first bifurcation event.