Lack of physiological evidence for cytochrome filaments functioning as conduits for extracellular electron transfer.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Targeting SOX13 inhibits assembly of respiratory chain supercomplexes to overcome ferroptosis resistance in gastric cancer.

Therapeutic resistance represents a bottleneck to treatment in advanced gastric cancer (GC). Ferroptosis is an iron-dependent form of non-apoptotic cell death and is associated with anti-cancer therapeutic efficacy. Further investigations are required to clarify the underlying mechanisms. Ferroptosis-resistant GC cell lines are constructed. Dysregulated mRNAs between ferroptosis-resistant and parental cell lines are identified. The expression of SOX13/SCAF1 is manipulated in GC cell lines where relevant biological and molecular analyses are performed. Molecular docking and computational screening are performed to screen potential inhibitors of SOX13. We show that SOX13 boosts protein remodeling of electron transport chain (ETC) complexes by directly transactivating SCAF1. This leads to increased supercomplexes (SCs) assembly, mitochondrial respiration, mitochondrial energetics and chemo- and immune-resistance. Zanamivir, reverts the ferroptosis-resistant phenotype via directly targeting SOX13 and promoting TRIM25-mediated ubiquitination and degradation of SOX13. Here we show, SOX13/SCAF1 are important in ferroptosis-resistance, and targeting SOX13 with zanamivir has therapeutic potential.

Flavonoid Oxidation Potentials and Antioxidant Activities-Theoretical Models Based on Oxidation Mechanisms and Related Changes in Electronic Structure.

Herein, I will review our efforts to develop a comprehensive and robust model for the estimation of the first oxidation potential, Ep1, and antioxidant activity, AA, of flavonoids that would, besides enabling fast and cheap prediction of Ep1 and AA for a flavonoid of interest, help us explain the relationship between Ep1, AA and electronic structure. The model development went forward with enlarging the set of flavonoids and, that way, we had to learn how to deal with the structural peculiarities of some of the 35 flavonoids from the final calibration set, for which the Ep1 measurements were all made in our laboratory. The developed models were simple quadratic models based either on atomic spin densities or differences in the atomic charges of the species involved in any of the three main oxidation mechanisms. The best model takes into account all three mechanisms of oxidation, single electron transfer-proton transfer (SET-PT), sequential proton loss electron transfer (SPLET) and hydrogen atom transfer (HAT), yielding excellent statistics (R2 = 0.970, S.E. = 0.043).

Transcriptomic evidence for an energetically advantageous relationship between Syntrophomonas wolfei and Methanothrix soehngenii.

Syntrophic interactions are key in anaerobic food chains, facilitating the conversion of complex organic matter into methane. A typical example involves acetogenic bacteria converting fatty acids (e.g., butyrate and propionate), a process thermodynamically reliant on H2 consumption by microorganisms such as methanogens. While most studies focus on H2-interspecies transfer between these groups, knowledge on acetate cross-feeding in anaerobic systems is lacking. This study investigated butyrate oxidation by co-cultures of Syntrophomonas wolfei and Methanospirillum hungatei, both with and without the addition of the acetate scavenger Methanothrix soehngenii. Growth and gene expression patterns of S. wolfei and M. hungatei were followed in the two conditions. Although butyrate consumption rates remained constant, genes in the butyrate degradation pathway of S. wolfei were less expressed in the presence of M. soehngenii, including genes involved in reverse electron transport. Higher expression of a type IV-pili operon in S. wolfei hints to the potential for direct interspecies electron transfer between S. wolfei and M. soehngenii and an energetically advantageous relationship between the two microorganisms. Overall, the presence of the acetate scavenger M. soehngenii positively influenced the energy metabolism of S. wolfei and highlighted the relevance of including acetate scavengers when investigating syntrophic fatty acid degradation.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Activating peroxymonosulfate with Fe-doped biochar for efficient removal of tetracycline: Dual action of reactive oxygen species and electron transfer.

If pharmaceutical wastewater is not managed effectively, the presence of residual antibiotics will result in significant environmental contamination. In addition, inadequate utilization of agricultural waste represents a squandering of resources. The objective of this research was to assess the efficacy of iron-doped biochar (Fe-BC) derived from peanut shells in degrading high concentrations of Tetracycline (TC) wastewater through activated peroxymonosulfate. Fe-BC demonstrated significant efficacy, achieving a removal efficiency of 87.5% for TC within 60 min without the need to adjust the initial pH (20 mg/L TC, 2 mM PMS, 0.5 g/L catalyst). The degradation mechanism of TC in this system involved a dual action, namely Reactive Oxygen Species (ROS) and electron transfer. The primary active sites were the Fe species, which facilitated the generation of SO4•-, •OH, O2•-, and 1O2. The presence of Fe species and the C=C structure in the Fe-BC catalyst support the electron transfer. Degradation pathways were elucidated through the identification of intermediate products and calculation of the Fukui index. The Toxicity Estimator Software Tool (T.E.S.T.) suggested that the intermediates exhibited lower levels of toxicity. Furthermore, the system exhibited exceptional capabilities in real water and circulation experiments, offering significant economic advantages. This investigation provides an efficient strategy for resource recycling and the treatment of high-concentration antibiotic wastewater.

Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer.

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Impact of Peripheral Hydrogen Bond on Electronic Properties of the Primary Acceptor Chlorophyll in the Reaction Center of Photosystem I.

Photosystem I (PS I) is a photosynthetic pigment-protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A-1, A0, A1, respectively), converging in a single iron-sulfur complex Fx. While there is a consensus that the ultimate electron donor-acceptor pair is P700+A0-, the involvement of A-1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr → Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr → Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role.

Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

Enhancement of electron transfer via magnetite in nitrite-dependent anaerobic methane oxidation system.

Nitrite-dependent anaerobic methane oxidation (n-DAMO) is a novel denitrification process that simultaneously further removes and utilizes methane from anaerobic effluent from wastewater treatment plants. However, the metabolic activity of n-DAMO bacteria is relative low for practical application. In this study, conductive magnetite was added into lab-scale sequencing batch reactor inoculated with n-DAMO bacteria to study the influence on n-DAMO process. With magnetite amendment, the nitrogen removal rate could reach 34.9 mg N·L-1d-1, nearly 2.5 times more than that of control group. Magnetite significantly facilitated the interspecies electron transfer and built electrically connected community with high capacitance. Enzymatic activities of electron transport chain were significantly elevated. Functional gene expression and enzyme activities associated with nitrogen and methane metabolism had been highly up-regulated. These results not only propose a useful strategy in n-DAMO application but also provide insights into the stimulating mechanism of magnetite in n-DAMO process.

Diffusional and Biochemical Limitations to Photosynthesis Under Water Deficit for Field-Grown Cotton.

Water deficit stress limits net photosynthetic rate (AN), but the relative sensitivities of underlying processes such as thylakoid reactions, ATP production, carbon fixation reactions, and carbon loss processes to water deficit stress in field-grown upland cotton require further exploration. Therefore, the objective of the present study was to assess (1) the diffusional and biochemical mechanisms associated with water deficit-induced declines in AN and (2) associations between water deficit-induced variation in oxidative stress and energy dissipation for field-grown cotton. Water deficit stress was imposed for three weeks during the peak bloom stage of cotton development, causing significant reductions in leaf water potential and AN. Among diffusional limitations, mesophyll conductance was the major contributor to the AN decline. Several biochemical processes were adversely impacted by water deficit. Among these, electron transport rate and RuBP regeneration were most sensitive to AN-limiting water deficit. Carbon loss processes (photorespiration and dark respiration) were less sensitive than carbon assimilation, contributing to the water deficit-induced declines in AN. Increased energy dissipation via non-photochemical quenching or maintenance of electron flux to photorespiration prevented oxidative stress. Declines in AN were not associated with water deficit-induced variation in ATP production. It was concluded that diffusional limitations followed by biochemical limitations (ETR and RuBP regeneration) contributed to declines in AN, carbon loss processes partially contributed to the decline in AN, and increased energy dissipation prevented oxidative stress under water deficit in field-grown cotton.

Progress and Perspective in harnessing MXene-carbon-based composites (0-3D): Synthesis, performance, and applications.

MXene is recognized as a promising catalyst for versatile applications due to its abundant metal sites, physicochemical properties, and structural formation. This comprehensive review offers an in-depth analysis of the incorporation of carbon into MXene, resulting in the formation of MXene-carbon-based composites (MCCs). Pristine MXene exhibits numerous outstanding characteristics, such as its atomically thin 2D structure, hydrophilic surface nature, metallic electrical conductivity, and substantial specific surface area. The introduction of carbon guides the assembly of MCCs through electrostatic self-assembly, pairing positively charged carbon with negatively charged MXene. These interactions result in increased interlayer spacing, reduced ion/electron transport distances, and enhanced surface hydrophilicity. Subsequent sections delve into the synthesis methods for MCCs, focusing on MXene integrated with various carbon structures, including 0D, 1D, 2D, and 3D carbon. Comprehensive discussions explore the distinctive properties of MCCs and the unique advantages they offer in each application domain, emphasizing the contributions and advancements they bring to specific fields. Furthermore, this comprehensive review addresses the challenges encountered by MCCs across different applications. Through these analyses, the review promotes a deeper understanding of exceptional characteristics and potential applications of MCCs. Insights derived from this review can serve as guidance for future research and development efforts, promoting the widespread utilization of MCCs across a broad spectrum of disciplines and spurring future innovations.

External Electrons Directly Stimulate Escherichia coli for Enhancing Biological Hydrogen Production.

External electric field has the potential to influence metabolic processes such as biological hydrogen production in microorganisms. Based on this concept, we designed and constructed an electroactive hybrid system for microbial biohydrogen production under an electric field comprised of polydopamine (PDA)-modified Escherichia coli (E. coli) and Ni foam (NF). In this system, electrons generated from NF directly migrate into E. coli cells to promote highly efficient biocatalytic hydrogen production. Compared to that generated in the absence of electric field stimulation, biohydrogen production by the PDA-modified E. coli-based system is significantly enhanced. This investigation has demonstrated the mechanism for electron transfer in a biohybrid system and gives insight into precise basis for the enhancement of hydrogen production by using the multifield coupling technology.

Species-resolved, single-cell respiration rates reveal dominance of sulfate reduction in a deep continental subsurface ecosystem.

Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~103 cells mL-1) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers including Candidatus Desulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell-1 h-1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L-1 d-1, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable to Ca. Desulforudis audaxviator.

Recent mechanistic developments for cytochrome c nitrite reductase, the key enzyme in the dissimilatory nitrate reduction to ammonium pathway.

Cytochrome c nitrite reductase, NrfA, is a soluble, periplasmic pentaheme cytochrome responsible for the reduction of nitrite to ammonium in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway, a vital reaction in the global nitrogen cycle. NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we summarize the latest progress in elucidating the reaction mechanism of ammonia production, including new findings about the active site architecture of NrfA, as well as recent results that elucidate electron transfer and storage in the pentaheme scaffold of this enzyme.

Cu/N co-doped biochar activating PMS for selective degrading paracetamol via a non-radical pathway dominated by singlet oxygen and electron transfer.

The non-free radical oxidation pathway (PMS-NOPs) of peroxymonosulfate (PMS) holds significant promise for practical wastewater treatment applications, owing to its low oxidation potential, high PMS utilization rate, and robust anti-interference capability in the degradation of pollutants. A novel activator copper nitrogen co-doped porous biochar (Cu-N-BC) with rich defect edges and functional groups was obtained by adding Cu and N to the biochar matrix generated by sodium alginate through pyrolysis in this study. Under the condition of 1 mM PMS, 30 mg/L activator was used to activate PMS and achieve efficient degradation of 10 mg/L paracetamol (PCT) within 15 min, with a high reaction rate constants (kobs) of 0.391 min-1. The activation mechanism of the Cu-N-BC/PMS/PCT system was a non-radical activation pathway with the dominance of singlet oxygen (1O2) and the presence of catalyst-mediated electron transfer. The graphite nitrogen, pyridine nitrogen, and Cu-N coordination introduced by Cu/N co-doping, as well as the carbon skeleton and CO functional group of biochar, were considered active sites that promote the 1O2 generation. The Cu-N-BC/PMS system exhibits strong stability, eco-friendliness, effective mineralization, and interference resistance across diverse pH levels (3-11) and interfering ions, including Cl-, H2PO4-, NO3-, SO42-, and humic acid. Remarkably, it efficiently degrades PCT in tap and lake water, achieving a notable 63.73% TOC mineralization rate, with leached copper ions below 0.02 mg/L. This research introduces a novel method for obtaining metal nitrogen carbon activators and enhances understanding of PMS non-radical activation pathways and active sites.

Modulation of Electron Transfer Branches by Atrazine and Triazine Herbicides in Photosynthetic Reaction Centers.

Quinone analogue molecules, functioning as herbicides, bind to the secondary quinone site, QB, in type-II photosynthetic reaction centers, including those from purple bacteria (PbRC). Here, we investigated the impact of herbicide binding on electron transfer branches, using herbicide-bound PbRC crystal structures and employing the linear Poisson-Boltzmann equation. In contrast to urea and phenolic herbicides [Fufezan, C. Biochemistry 2005, 44, 12780-12789], binding of atrazine and triazine did not cause significant changes in the redox-potential (Em) values of the primary quinone (QA) in these crystal structures. However, a slight Em difference at the bacteriopheophytin in the electron transfer inactive branch (HM) was observed between the S(-)- and R(+)-triazine-bound PbRC structures. This discrepancy is linked to variations in the protonation pattern of the tightly coupled Glu-L212 and Glu-H177 pairs, crucial components of the proton uptake pathway in native PbRC. These findings suggest the existence of a QB-mediated link between the electron transfer inactive HM and the proton uptake pathway in PbRCs.

Surface wettability control and electron transport regulation in zerovalent iron for enhanced removal of emerging polystyrene microplastics-heavy metal contaminants.

Emerging microplastics-heavy metal (MPs-HM) contaminants in wastewaters pose an emerging health and environmental risk due to their persistence and increasing ecological risks (e.g., "Trojan horse" effect). Hence, removing MPs in solution and preventing secondary releases of HM has become a key challenge when tackling with MPs pollution. Leveraging the hydrophobic nature of MPs and the electron transfer efficiency from Fe0 to HM, we demonstrate an alkylated and sulfidated nanoscale zerovalent iron (AS-nZVI) featuring a delicate "core-shell-hydrophobic film" nanostructure. Exemplified by polystyrene (PS) MPs-Pb(II) removal, the three nanocomponents offer synergistic functions for the rapid separation of MPs, as well as the reduction and stabilization of Pb(II). The outmost hydrophobic film of AS-nZVI greatly improves the anticorrosion performance of nanoscale zerovalent iron and the enrichment abilities of hydrophobic MPs, achieving a maximum removal capacity of MPs to 2725.87 mgMPs·gFe-1. This MPs enrichment promotes the subsequent reductive removal of Pb(II) through the electron transfer from the iron core of AS-nZVI to Pb(II), a process further strengthened by the introduced sulfur. When considering the inevitable aging of MPs in wastewaters due to mechanical wear or microbial degradation, our study concurrently examines the efficiencies and behaviors of AS-nZVI in removing virgin-MPs-Pb(II) and aged-MPs-Pb(II). The batch results reveal that AS-nZVI has an exceptional ability to remove above 99.6 % Pb(II) for all reaction systems. Overall, this work marks a pioneering effort in highlighting the importance of MPs-toxin combinations in dealing with MPs contamination and in demonstrating the utility of nZVI techniques for MPs-contaminated water purification.

From Ground-State to Excited-State Activation Modes: Flavin-Dependent "Ene"-Reductases Catalyzed Non-natural Radical Reactions.

ConspectusEnzymes are desired catalysts for chemical synthesis, because they can be engineered to provide unparalleled levels of efficiency and selectivity. Yet, despite the astonishing array of reactions catalyzed by natural enzymes, many reactivity patterns found in small molecule catalysts have no counterpart in the living world. With a detailed understanding of the mechanisms utilized by small molecule catalysts, we can identify existing enzymes with the potential to catalyze reactions that are currently unknown in nature. Over the past eight years, our group has demonstrated that flavin-dependent "ene"-reductases (EREDs) can catalyze various radical-mediated reactions with unparalleled levels of selectivity, solving long-standing challenges in asymmetric synthesis.This Account presents our development of EREDs as general catalysts for asymmetric radical reactions. While we have developed multiple mechanisms for generating radicals within protein active sites, this account will focus on examples where flavin mononucleotide hydroquinone (FMNhq) serves as an electron transfer radical initiator. While our initial mechanistic hypotheses were rooted in electron-transfer-based radical initiation mechanisms commonly used by synthetic organic chemists, we ultimately uncovered emergent mechanisms of radical initiation that are unique to the protein active site. We will begin by covering intramolecular reactions and discussing how the protein activates the substrate for reduction by altering the redox-potential of alkyl halides and templating the charge transfer complex between the substrate and flavin-cofactor. Protein engineering has been used to modify the fundamental photophysics of these reactions, highlighting the opportunity to tune these systems further by using directed evolution. This section highlights the range of coupling partners and radical termination mechanisms available to intramolecular reactions.The next section will focus on intermolecular reactions and the role of enzyme-templated ternary charge transfer complexes among the cofactor, alkyl halide, and coupling partner in gating electron transfer to ensure that it only occurs when both substrates are bound within the protein active site. We will highlight the synthetic applications available to this activation mode, including olefin hydroalkylation, carbohydroxylation, arene functionalization, and nitronate alkylation. This section also discusses how the protein can favor mechanistic steps that are elusive in solution for the asymmetric reductive coupling of alkyl halides and nitroalkanes. We are aware of several recent EREDs-catalyzed photoenzymatic transformations from other groups. We will discuss results from these papers in the context of understanding the nuances of radical initiation with various substrates.These biocatalytic asymmetric radical reactions often complement the state-of-the-art small-molecule-catalyzed reactions, making EREDs a valuable addition to a chemist's synthetic toolbox. Moreover, the underlying principles studied with these systems are potentially operative with other cofactor-dependent proteins, opening the door to different types of enzyme-catalyzed radical reactions. We anticipate that this Account will serve as a guide and inspire broad interest in repurposing existing enzymes to access new transformations.