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Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD •− isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine–aspartate salt bridge allows proton transfer from arginine to FAD •− . Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis. A reduction reaction is usually equated with an electron transfer reaction. Now, ultrafast time-resolved serial femtosecond X-ray crystallography has enabled the visualization of the stepwise structural changes that occur after electron transfers have been observed in the light-triggered reduction of flavin adenine dinucleotide catalysed by DNA photolyase.

Extracellular redox-active compounds, flavins and other quinones, have been hypothesized to play a major role in the delivery of electrons from cellular metabolic systems to extracellular insoluble substrates by a diffusion-based shuttling two-electron-transfer mechanism. Here we show that flavin molecules secreted by Shewanella oneidensis MR-1 enhance the ability of its outer-membrane c -type cytochromes (OM c- Cyts) to transport electrons as redox cofactors, but not free-form flavins. Whole-cell differential pulse voltammetry revealed that the redox potential of flavin was reversibly shifted more than 100 mV in a positive direction, in good agreement with increasing microbial current generation. Importantly, this flavin/OM c- Cyts interaction was found to facilitate a one-electron redox reaction via a semiquinone, resulting in a 10 ³- to 10 ⁵-fold faster reaction rate than that of free flavin. These results are not consistent with previously proposed redox-shuttling mechanisms but suggest that the flavin/OM c- Cyts interaction regulates the extent of extracellular electron transport coupled with intracellular metabolic activity.

Structural analysis and spectroscopy elucidate how pairs of electrons are bifurcated in a flavoenzyme by generating an unstable flavin semiquinone, thus coupling exergonic and endergonic oxidation–reduction reactions. 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.

The introduction of fluoroalkyl groups into pharmaceutical compounds has the potential to enhance their therapeutic properties. Nevertheless, the synthesis of enantiomerically pure C( sp³ )–CF₃ compounds poses a significant challenge. Biocatalysis offers precise stereochemical control, however, the scarcity of fluorine-containing natural products makes it difficult to find enzymes capable of incorporating fluoroalkyl groups. Herein, we develop a ground-state flavin-dependent enzyme-catalyzed strategy for the radical-mediated enantioselective trifluoromethylation. Two engineered flavin-dependent enzymes are successfully developed to catalyze stereoselective hydrotrifluoromethylation and trifluoromethyl-alkyl cross-electrophile coupling reactions using trifluoromethyl thianthrenium triflate as a radical donor. Experimental investigations and computational simulations demonstrate that the reaction is initiated through single-electron transfer from the ground state flavin hydroquinone (FMN hq ) and quenched through hydrogen atom transfer by flavin semiquinone (FMN sq ). This strategy provides an opportunity to bridge the gap between biocatalysis and organic fluorides but also introduces an alternative approach to address challenging stereoselective fluoroalkylation reactions in organic synthesis. The introduction of fluoroalkyl groups into pharmaceutical compounds has the potential to enhance their therapeutic properties, but synthesis of enantiomerically pure C(sp³)–CF₃ compounds is challenging. Here, the authors develop a ground-state flavin-dependent enzyme catalyzed strategy for the radical-mediated enantioselective trifluoromethylation.

Controlled halogenation of chemically versatile substrates is difficult to achieve. Here we describe a unique flavin-dependent halogenase, PltM, which is capable of utilizing a wide range of halides for installation on a diverse array of phenolic compounds, including FDA-approved drugs and natural products, such as terbutaline, fenoterol, resveratrol, and catechin. Crystal structures of PltM in complex with phloroglucinol and FAD in different states yield insight into substrate recognition and the FAD recycling mechanism of this halogenase. Halogenase enzymes are of interest as halogenating tools for organic synthesis. Here the authors show that the bacterial FAD-dependent phenolic halogenase PltM chlorinates, brominates and iodinates a variety of substrates and reveal the structural basis for its substrate versatility and provide insights into the FAD recycling mechanism of PltM.

The RNA polymerase NS5B of HCV is capable of catalyzing the addition of flavin adenine dinucleotide (FAD) to its RNA as a 5′ cap structure, aiding the virus in evading host immune responses. However, the exact mechanism underlying the 5′-FAD capping process of HCV RNA remains to be elucidated. Here, we determine crystal structures of the HCV NS5B de novo initiation, primed initiation and elongation complexes in presence of FAD. Structural analysis and comparisons show that residues M447 and Y448 of the β loop in the priming element (PE) of NS5B are the determinants for specific recognition of FAD. The adenine group of FAD is exclusively paired with the uracil base at the 3′ end of the template RNA strand. At the initial elongation stage, the C-terminal linker (residues 530−570) of NS5B is involved in stabilizing the 5′ FAD, which in turn induces sequential conformational changes of the bases in the product strand and creates a unique intermediate state of the RNA duplex, facilitating the translocation of the product strand. Our study offers novel insights for developments of new anti-HCV therapies. Using high-resolution crystal structures, the authors discover that flavin adenine dinucleotide (FAD) facilitates the initiation of HCV RNA synthesis by interacting with key amino acids M447 and Y448 in NS5B. They also observe an elongation complex with an FAD-capped primer/product strand, explaining how the product strand moves for efficient RNA synthesis.

Chlorobaculum tepidum ferredoxin: NADP+ oxidoreductase (CtFNR) is a dimeric thioredoxin reductase (TrxR)-type FNR, whose mechanism and redox properties are poorly characterized. In this work, we focused on the reoxidation mechanisms of its flavin adenine dinucleotide (FAD) cofactor using quinones (Q), nitroaromatics (ArNO2), and other nonphysiological oxidants with different single-electron reduction midpoint potentials (E71) and electrostatic charge. Like in other FNRs, the rate-limiting step of the reaction is the reoxidation of FAD semiquinone (FADH•). However, only one FAD per dimer functions in CtFNR due to some nonequivalence of the NADP(H) binding domains in separate subunits. The reactivity of Q increases with increasing E71, while ArNO2 form another analogous series of lower reactivity. The compounds are reduced in a dominant single-electron way. These data are consistent with an “outer sphere” electron transfer mechanism. On the basis of reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of FAD at pH 7.0 is −0.282 V. In CtFNR, 11% FADH• was stabilized at equilibrium. Calculated electron transfer distances in reactions with Q and ArNO2 were in the range of 2.6–3.4 Å. Taken together with previous studies of Rhodopseudomonas palustris and Bacillus subtilis FNRs, this work allows us to generalize the information on the catalytic ant thermodynamic properties of TrxR-type FNRs. In addition, our data may be valuable from an applied perspective, e.g., the use of redox mediators in photobioelectrochemical systems or microbial cells based on anoxygenic phototrophic bacteria.

Chromatin modifiers often work in concert with transcription factors (TFs) and other complex members, where they can serve both enzymatic and scaffolding functions. Due to this, active site inhibitors targeting chromatin modifiers may perturb both enzymatic and nonenzymatic functions. For instance, the antiproliferative effects of active-site inhibitors targeting lysine-specific histone demethylase 1A (LSD1) are driven by disruption of a protein-protein interaction with growth factor independence 1B (GFI1B) rather than inhibition of demethylase activity. Recently, next-generation precision LSD1 covalent inhibitors have been developed, which selectively block LSD1 enzyme activity by forming a compact N -formyl flavin adenine dinucleotide (FAD) adduct that spares the GFI1B interaction. However, the mechanism accounting for N -formyl-FAD formation remains unclear. Here we clarify the mechanism of these demethylase-specific inhibitors of LSD1, demonstrating that the covalent inhibitor-FAD adduct undergoes a Grob fragmentation. Using inhibitor analogs and structural biology, we identify structure-activity relationships that promote this transformation. Furthermore, we unveil an unusual drug resistance mechanism whereby distal active-site mutations can promote inhibitor-adduct Grob fragmentation even for previous generation compounds. Our study uncovers the unique Grob fragmentation underlying the mechanism of action of precision LSD1 enzyme inhibitors, offering insight into their reactivity with broader implications for drug resistance. Next generation precision lysine-specific histone demethylase 1A (LSD1) covalent inhibitors which selectively block LSD1 enzyme activity by forming a compact N -formyl-FAD adduct have been developed, but the mechanism of adduct formation was unclear. Here, the authors show that the covalent inhibitor-FAD adduct undergoes a Grob fragmentation and elucidate the structure-activity relationships that promote this transformation.

The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus species require suitable redox mediators to transfer electrons from the enzyme to the electrode surface for the application of bioelectrical devices. Although several mediators for FAD-GDH are already in use, they are still far from optimum in view of potential, kinetics, sustainability, and cost-effectiveness. Herein, we investigated the efficiency of various phenothiazines and quinones in the electrochemical oxidation of FAD-GDH from Aspergillus terreus. At pH 7.0, the logarithm of the bimolecular oxidation rate constants appeared to depend on the redox potentials of all the mediators tested. Notably, the rate constant of each molecule for FAD-GDH was approximately 2.5 orders of magnitude higher than that for glucose oxidase from Aspergillus sp. The results suggest that the electron transfer kinetics is mainly determined by the formal potential of the mediator, the driving force of electron transfer, and the electron transfer distance between the redox active site of the mediator and the FAD, affected by the steric or chemical interactions. Higher k2 values were found for ortho-quinones than for para-quinones in the reactions with FAD-GDH and glucose oxidase, which was likely due to less steric hindrance in the active site in the case of the ortho-quinones.

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.