Steric hindrance controls pyridine nucleotide specificity of a flavin-dependent NADH:quinone oxidoreductase
Abstract not provided
Ball, Jacob↗
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Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus
NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Alternative binding modes in abortive NADH-alcohol complexes of horse liver alcohol dehydrogenase
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Plapp, Bryce V.↗
The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex
ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.
Zhang, Bo [Department of Chemical Engineering, Uni↗
Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism
Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. In this work, we investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.
36 MATERIALS SCIENCE↗
DFT Mechanism Studies: Biomimetic 1,4-NADH Chemoselective, Co-factor Regeneration with [Cp*Rh(bpy)H] + , in Tandem with the Biocatalysis Pathways of a Core Model of the (HLADH)-Zn(II) Mediated Enzyme, in the Enantioselective Reduction of Achiral Ketones to Chiral S-Alcohols
In this study, Quantum Chemical (QC) calculations, utilizing Density Functional Theory (DFT), were performed to investigate the mechanistic aspects of the chemoselective catalyzed reaction of [Cp*Rh(bpy)H] + with the biomimetic NAD + analogues, N-benzylnicotinamide triflate, 1, and β-nicotinamide ribose-5'-methyl phosphate, 2, in the conversion to their 1,4-NADH analogues, 1,4-dihydro-N-benzylnicotinamide, 4, and β-1,4-dihydronicotinamide-5'-ribose methyl phosphate, 5. This reaction was in tandem with the 1,4-NADH dependent HLADH-Zn(II)- catalyzed reduction of achiral ketones to chiral S-alcohols. The [Cp*Rh(bpy)H] + complex, and not its equilibrium tautomer, [η 4 -Cp*HRh(bpy)] + , was found to control the hydride transfer during the biomimetic NAD + /1,4-NADH conversion, through the non-covalent interactions of the biomimetic co-factors with [Cp*Rh(bpy)H] + . The thermodynamics and kinetics for the chiral reduction of the Zn(II) bound ketones, 2-pentanone and 4-phenyl-2-butanone, with co-factor, 4, catalyzed by Zn(SCH 3 ) 2 (Imidazole), a core model of the Zn(II)-based catalytic center of HLADH, was also investigated by the evaluation of two possible reaction pathways: (1) formation of a ZnH from the C4-H hydride transfer of co-factor, 4, followed by reaction of the postulated ZnH with the bound 2-pentanone or 4-phenyl-2-butanone substrate, and (2), the direct C4-H transfer to the bound achiral ketone substrates, to provide the dominant chiral alcohols, S-2-pentanol or S-4-phenyl-2-butanol. The latter pathway was found most viable, and DFT calculations also revealed an essential η 2 -coordination of the 5,6 double bond of co-factor, 4, to the HLADH-Zn(II) metal ion center, upon imidazole decomplexation, providing an asymmetric differentiation of S-η 2 -5,6-1,4-NADH-Zn(II) binding. A proposed new paradigm for the Zn(II)'s non-innocent role in the HLADH-Zn(II) biocatalysis reduction mechanism, for enantioselective hydride transfer to a Zn(II) bound ketone, providing S-alcohols.
1,4 NADH co-factors↗
Improving methyl ketone production in Escherichia coli by heterologous expression of NADH‐dependent FabG
Abstract We previously engineered Escherichia coli to overproduce medium‐ to long‐chain saturated and monounsaturated methyl ketones, which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry. Recent efforts at strain optimization have focused on cofactor balance, as fatty acid‐derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β‐ketoacyl‐ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance by heterologously expressing NADH‐dependent, rather than NADPH‐dependent, versions of FabG identified in previous studies. Of the four NADH‐dependent versions of FabG tested in our previously best‐reported methyl ketone‐producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed the greatest increase in methyl ketone yield in shake flasks (35–75% higher than for an RFP negative‐control strain, depending on sugar loading). An improved strain (EGS2920) attained methyl ketone titers during fed‐batch fermentation of 5.4 ± 0.5 g/L, which were, on average, ca. 40% greater than those for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed‐batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain EGS2920 significantly upregulated glucose‐6‐phosphate isomerase (directing flux into glycolysis rather than the NADPH‐producing pentose phosphate pathway) and downregulated MaeB (a NADP + ‐dependent malate dehydrogenase). Overall, the results suggest that heterologous expression of NADH‐dependent FabG in E. coli may improve sustained production of fatty acid‐derived renewable fuels and chemicals.
Goh, Ee‐Been↗
Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol
Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. Here, in this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.
2-methyl-1-butanol↗
NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism
Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4’s AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4’s β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4’s redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import. These results establish AIF as an integral component of the metazoan disulfide relay and point to NADH-activated dimeric AIF as an organizational import center for CHCHD4 and its substrates. Importantly, AIF regulation of CHCHD4 directly links AIF’s cellular NAD(H) sensing to CHCHD4 chaperone function, suggesting a mechanism to balance tissue-specific oxidative phosphorylation (OXPHOS) capacity with NADH availability.
Brosey, Chris A↗
The anaerobic fungus Caecomyces churrovis produces H2 via a non-3 bifurcating NADH-dependent enzyme complex
Anaerobic fungi (AF) decompose lignocellulose-based biomass into fermentable sugars through the production of powerful biomass-degrading enzymes. AF are unusual among fungi in that they generate energy via hydrogenosomes, which are also associated with the release of H2 though yet unknown metabolic mechanisms. In particular, it remains unclear how NAD(P)+ is regenerated within hydrogenosomes and how H2 is formed. Here, we reveal the molecular mechanism for hydrogenosomal H2 production in the AF strain C. churrovis by combining genomic search, proteomic analysis, and enzymology. Our enzyme assays on the large organelle fraction of C. churrovis revealed the activity of H2:NAD+ oxidoreductase but not pyruvate:ferredoxin oxidoreductase activity. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis, and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified proteins, we found that the assay system consisting of Hyd-Strep and NuoEF-Strep reduced NAD+ with H2. Furthermore, this system formed H2 directly from NADH independent of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is widely conserved among the early-branching AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could be a target for controlling AF H2 production and altering fungal metabolism.
fungi↗
Mechanisms in the Synthesis of S -Alcohols with 1,4-NADH Biomimetic Co-factor N-Benzyl-1,4-dihydronicotinamide using Horse Liver Alcohol Dehydrogenase: A Hybrid Computational Study
The enantioselective reduction of prochiral ketones catalyzed by horse liver alcohol dehydrogenase (HLADH), was investigated via a hybrid computational approach, for molecular reactions involved in chiral synthesis of S-alcohols, when the natural co-factor, 1,4-dihyronicotinamide adenine dinucleotide, 1,4-NADH, was replaced with biomimetic co-factor, N-benzyl-1,4-dihydronicotinamide, 1. We surmised that different hydride and proton transfer mechanisms were involved using co-factor, 1. An alternative mechanism, where the hydride transfer step occurred, via an η 1 -keto-S-η 2 -5,6-1,4-dihydronicotinamide-Zn(II) complex, was previously investigated with a model of the HLADH-Zn(II) catalytic site (J. Organometal. Chem. 2021, 943, 121810). Presently, we studied canonical and alternative mechanisms compared to models of the entire enzyme structure. We disproved the η 2 -Zn(II) complex, and discovered a canonical hydride transfer from biomimetic 1,4-NADH, 1, to the Zn(II) bound prochiral ketone substrate, followed by a new proton relay, consisting of a water chain connecting His51 to Ser48 that accomplished the S-alkoxy anion's protonation to yield the final S-alcohol product. The HLADH catalysis, with biomimetic co-factor, 1, that replaced the ribose group, the 5'-diphosphate groups, and the adenine nucleotide with a N-benzyl group, has provided a new paradigm for the design of other structures of 1,4-NADH biomimetic co-factors, including their economic value in biocatalysis reactions.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Kinetic Model of HoxEFU reduction by NADH [SWR-26-087]
This repository is used to release code generated for manuscripts on the Photosynthetic Energy Transduction core program. This code simulates the reduction of HoxEFU by NADH. The electro transfer rate constants for the simulation are specified in the .csv files. The two .csv files correspond tot he two models described in Dawson et al. Cell. Rep. Phys. Sci. 2026. The code utilizes a chemical master equation, a set of differential equations, defining the time evolution of the oxidation and reduction kinetics of NAD+, NADH, a FMN flavin, and a set of iron sulfur clusters. The kinetics of HoxEFU reduction by NADH are evaluated by numerical integration of the chemical master equation using a variable-time-step Runge-Kutta algorithm.
Dahl, Peter [National Laboratory of the Rockies (N↗
Photoelectrochemical NADH Regeneration is Highly Sensitive to the Nature of Electrode Surface
(Photo)electrochemistry enables the synthesis of high-value fine chemicals and highly selective activation of molecules that are difficult to prepare using conventional chemical methods. In this study, light-driven NADH (reduced nicotinamide adenine dinucleotide) regeneration is achieved using a molecular Rh(III) mediator on Si photoelectrodes. This process is observed to be highly sensitive to the surface nature of Si photoelectrodes, exhibiting an overpotential reduction up to 600 mV on Si nanowires (SiNWs) as compared to planar Si. The use of a molecular mediator and SiNWs enables 100% selectivity toward NADH synthesis within a broad potential window. The origin of the striking difference is identified as the multifaceted nature of SiNWs.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
The first comprehensive phylogenetic and biochemical analysis of NADH diphosphatases reveals that the enzyme from Tuber melanosporum is highly active towards NAD +
Nudix (for nucleoside diphosphatases linked to other moieties, X) hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD+-capped RNAs. Although all the members of this superfamily share a common conserved catalytic motif, the Nudix box, their substrate specificity lies in specific sequence traits, which give rise to different subfamilies. Among them, NADH pyrophosphatases or diphosphatases (NADDs) are poorly studied and nothing is known about their distribution. To address this, we designed a Prosite-compatible pattern to identify new NADDs sequences. In silico scanning of the UniProtKB database showed that 3% of Nudix proteins were NADDs and displayed 21 different domain architectures, the canonical architecture (NUDIX-like_zf-NADH-PPase_NUDIX) being the most abundant (53%). Interestingly, NADD fungal sequences were prominent among eukaryotes, and were distributed over several Classes, including Pezizomycetes. Unexpectedly, in this last fungal Class, NADDs were found to be present from the most common recent ancestor to Tuberaceae, following a molecular phylogeny distribution similar to that previously described using two thousand single concatenated genes. Finally, when truffle-forming ectomycorrhizal Tuber melanosporum NADD was biochemically characterized, it showed the highest NAD+/NADH catalytic efficiency ratio ever described.
59 BASIC BIOLOGICAL SCIENCES↗
Polarized Fluorescence in NADH Two-Photon Excited by Femtosecond Laser Pulses in the Wavelength Range of 720–780 nm
The decay of polarized fluorescence in coenzyme NADH in aqueous-methanol solution under two-photon excitation by femtosecond laser pulses within the spectral range of 720–780 nm has been studied. Molecular fluorescence parameters: decay times τ{sub 1} and τ{sub 2}, rotational diffusion time τ{sub rot}, and pre-exponential factor ratio a{sub 2}/a{sub 1} have been determined from experiment. The results obtained have been compared with those reported recently in NADH in aqueous solution.
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
Catalytic Bias of NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase (Nfn) and its Relevance to Ethanol Production in Thermoanaerobacterium Saccharolyticum
NADH-dependent reduced ferredoxin: NADP+ oxidoreductase (Nfn) enzyme catalyzes an energy-conserving flavin-based electron bifurcation (FBEB) reaction. In microbial metabolism, Nfn links redox pools of three electron carriers - ferredoxin (Fd), NAD(H), and NADP(H) - through the following FBEB reaction: 2 NADPH + NAD+ + 2 Fdox 2 NADP+ + NADH + 2 Fdred + H+ The forward reaction is termed electron bifurcation, and the reverse reaction is electron confurcation. Catalytic bias describes an enzyme's tendency to favor one direction of a reversible reaction over the other and is expressed as the ratio of activities in the two directions. Thermoanaerobacterium saccharolyticum (Tsac) is a thermophilic, ethanologenic bacterium that ferments hemicellulose to ethanol at yields above 90%. Its Nfn enzyme is known to support ethanol production, presumably by operating in the confurcating direction to balance cellular cofactors, but this has not previously been demonstrated. To investigate the Tsac Nfn further, we heterologously expressed, purified, and reconstituted the proteins NfnS (NfnA), NfnL (NfnB), and the putative partner Fd with iron-sulfur cluster and/or FAD cofactors. Activity assays monitoring the oxidation or reduction of Fd showed that, across pH 5-10, Tsac Nfn is catalytically biased towards the confurcating direction, favoring NADPH generation over NADPH oxidation by at least fivefold. Using protein electrochemistry, we also determined the reduction potentials of the cofactors in NfnL and Fd. These results indicate that the energetic landscape of FBEB in Tsac Nfn is similar to that in an ortholog. However, Tsac Fd has redox properties distinct from previously assayed Fds, suggesting that the identity and redox properties of Fd may help determine the catalytic bias of Nfn. Additionally, we confirmed the standalone ferredoxin: NADP+ oxidoreductase (FNOR) activity of NfnL but found it to be low and likely insignificant for in vivo redox conversion. We also show how the catalytic bias of Nfn integrates with the hydrogen cycling mechanism proposed in Tsac to better explain cofactor balancing for ethanol production. Our work advances the understanding of electron transfer processes within metabolic networks for the generation of valuable bioproducts.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Sub-100-fs energy transfer in coenzyme NADH is a coherent process assisted by a charge-transfer state
Excitation energy transfer (EET) is a key photoinduced process in biological chromophoric assemblies. Here we investigate the factors which can drive EET into efficient ultrafast sub-ps regimes. We demonstrate how a coherent transport of electronic population could facilitate this in water solvated NADH coenzyme and uncover the role of an intermediate dark charge-transfer state. High temporal resolution ultrafast optical spectroscopy gives a 54 ± 11 fs time constant for the EET process. Nonadiabatic quantum dynamical simulations computed through the time-evolution of multidimensional wavepackets suggest that the population transfer is mediated by photoexcited molecular vibrations due to strong coupling between the electronic states. The polar aqueous solvent environment leads to the active participation of a dark charge transfer state, accelerating the vibronically coherent EET process in favorably stacked conformers and solvent cavities. Our work demonstrates how the interplay of structural and environmental factors leads to diverse pathways for the EET process in flexible heterodimers and provides general insights relevant for coherent EET processes in stacked multichromophoric aggregates like DNA strands.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
What Makes a Bifurcase? Insights from a NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase (Nfn) and Homologs
NADH-dependent ferredoxin:NADP+ oxidoreductases (Nfn) is an enzyme family that engage in flavin-based electron bifurcation (FBEB), a mode of energy conservation utilized by life. The protein comprises one large (NfnL) and one small (NfnS) subunits. Thermoanaerobacterium sacchaloryticum (Tsac) is an anaerobic thermophilic bacterium that - with known involvement of Tsac Nfn - can produce ethanol in high, commercially viable concentrations. We sought to investigate the activity and energetic landscape of Tsac Nfn to determine how the enzyme effectuates FBEB. Tsac NfnS, NfnL, and the partner ferredoxin (Tsac Fd) were recombinantly expressed, purified, and reconstituted with iron-sulfur cluster and FAD cofactors. Electron paramagnetic resonance (EPR) was utilized for all proteins. Spectroelectrochemistry was performed with NfnL. Square-wave voltammetry was conducted on NfnL and Fd. Spectrophotometric activity was assayed for NfnL with or without NfnS. Our group continues to investigate the Nfn from Pyrococcus furiosus (Pf Nfn). Unlike Pf NfnS, Tsac NfnS can be stably expressed, purified, and reconstituted in the absence of its partner NfnL, allowing for this subunit to be characterized separately. The energetic profile of FBEB in Tsac Nfn is overall similar to that of Pf NfnL, with some differences: (1) the proximal cluster is at a lower potential (-780 mV vs -711), (2) the bifurcating FAD is at a higher potential (-406 mV vs -436 mV), and (3) Tsac Fd has two [4Fe-4S] clusters at -550 and -410 mV, unlike Pf Fd with a single cluster at approximately 400 mV. Activity assays indicate that the two enzymes perform FBEB in a similar way. Our work continues to build upon the new field of FBEB by demonstrating that the energetic landscape between distantly related archael (Pf) and bacterial (Tsac Nfn) are largely similar. This equips us to understand design principles for FBEB, allowing us to modulate the process in vivo for specific metabolic outcomes.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗