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Flavin monooxygenases (FMOs) have been widely used in the biosynthesis of natural compounds due to their excellent stereoselectivity, regioselectivity and chemoselectivity. Stenotrophomonas maltophilia flavin monooxygenase (SmFMO) has been reported to catalyze the oxidation of various thiols to corresponding sulfoxides, but its activity is relatively low. Herein, we obtained a mutant SmFMO F52G which showed 4.35-fold increase in k cat /K m (4.96 mM -1 s -1 ) and 6.84-fold increase in enzyme activity (81.76 U/g) compared to the SmFMO WT (1.14 mM -1 s -1 and 11.95 U/g) through semi-rational design guided by structural analysis and catalytic mechanism combined with high-throughput screening. By forming hydrogen bond with O4 atom of FAD isoalloxazine ring and reducing steric hindrance, the conformation of FAD isoalloxazine ring in SmFMO F52G is more stable, and NADPH and substrate are closer to FAD isoalloxazine ring, shortening the distances of hydrogen transfer and substrate oxygenation, thereby increasing the rate of reduction and oxidation reactions and enhancing enzyme activity. Additionally, the overall structural stability and substrate binding capacity of the SmFMO F52G have significant improved than that of SmFMO WT . The strategy used in this study to improve the enzyme activity of FMOs may have generality, providing important references for the rational and semi-rational engineering of FMOs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Misfolding and amyloid fibrillogenesis of proteins have close relationships with several neurodegenerative diseases. The present work investigates the inhibitive activities of ankaflavin (AK) and monascin (MS), two yellow pigments separated from Monascus -fermented rice, on hen egg white lysozyme (HEWL) fibrillation. The results demonstrated that AK/MS suppressed HEWL fibrillation through interfering with the nucleation period and AK was more potent. Fluorescence quenching and in silico docking studies revealed that AK/MS bond to HEWL by the formation of noncovalent forces with some critical amino acid residues that tend to form fibrils. Compared to those of AK, hydrogen bonding interactions between MS and Asn46, Trp62, and Trp63 residues in HEWL were slightly weaker. Besides, the covalent interaction between MS and HEWL with the binding site of Arg68 was found. These observations offered reasonable explanations for the difference in the mechanisms of AK and MS inhibiting HEWL fibrillogenesis. In a word, all data acquired herein indicated AK/MS as potent candidates for the improvement and treatment of neurological disorders.

Bioluminescence of bacteria is widely applied in biological imaging, environmental toxicant detection, and many other situations. Understanding the spectral tuning mechanism not only helps explain the diversity of colors observed in nature but also provides principles for bioengineering new color variants for practical applications. In this study, time-dependent density functional theory (TD-DFT) and quantum mechanics and molecular mechanics (QM/MM) calculations have been employed to understand the fluorescence spectral tuning mechanism of bacterial luciferase with a focus on the electrostatic effect. The spectrum can be tuned by both a homogeneous dielectric environment and oriented external electric fields (OEEFs). Increasing the solvent polarity leads to a redshift of the fluorescence emission maximum, λ F , accompanied by a substantial increase in density. In contrast, applying an OEEF along the long axis of the isoalloxazine ring ( X -axis) leads to a significant red- or blue-shift in λ F , depending on the direction of the OEEF, yet with much smaller changes in intensity. The effect of polar solvents is directionless, and the red-shifts can be attributed to the larger dipole moment of the S 1 state compared with that of the S 0 state. However, the effect of OEEFs directly correlates with the difference dipole moment between the S 1 and S 0 states, which is directional and is determined by the charge redistribution upon deexcitation. Moreover, the electrostatic effect of bacterial luciferase is in line with the presence of an internal electric field (IEF) pointing in the negative X direction. Finally, the key residues that contribute to this IEF and strategies for modulating the spectrum through site-directed point mutations are discussed.

Significance: Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications., Aim: We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells., Approach: We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids., Results: The use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins., Conclusions: The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids., (© 2024 The Authors.)

Flavin cofactors offer a wide range of chemical mechanisms to support a great diversity in catalytic function. As a corollary, such diversity necessitates careful control within each flavoprotein to limit its function to an appropriate subset of possible reactions and substrates. This task falls to the protein environment surrounding the flavin in most enzymes. For iodotyrosine deiodinase that catalyzes a reductive dehalogenation of halotyrosines, substrates can dictate the chemistry available to the flavin. Their ability to stabilize the necessary one-electron reduced semiquinone form of flavin strictly depends on a direct coordination between the flavin and α-ammonium and carboxylate groups of its substrates. While perturbations to the carboxylate group do not significantly affect binding to the resting oxidized form of the deiodinase, dehalogenation ( k cat / K m ) is suppressed by over 2000-fold. Lack of the α-ammonium group abolishes detectable binding and dehalogenation. Substitution of the ammonium group with a hydroxyl group does not restore measurable binding but does support dehalogenation with an efficiency greater than those of the carboxylate derivatives. Consistent with these observations, the flavin semiquinone does not accumulate during redox titration in the presence of inert substrate analogues lacking either the α-ammonium or carboxylate groups. As a complement, a nitroreductase activity based on hydride transfer is revealed for the appropriate substrates with perturbations to their zwitterion.

The prenylated-flavin mononucleotide-dependent decarboxylases (also known as UbiD-like enzymes) are the most recently discovered family of decarboxylases. The modified flavin facilitates the decarboxylation of unsaturated carboxylic acids through a novel mechanism involving 1,3-dipolar cyclo-addition chemistry. UbiD-like enzymes have attracted considerable interest for biocatalysis applications due to their ability to catalyse (de)carboxylation reactions on a broad range of aromatic substrates at otherwise unreactive carbon centres. There are now ∼35 000 protein sequences annotated as hypothetical UbiD-like enzymes. Sequence similarity network analyses of the UbiD protein family suggests that there are likely dozens of distinct decarboxylase enzymes represented within this family. Furthermore, many of the enzymes so far characterized can decarboxylate a broad range of substrates. Here we describe a strategy to identify potential substrates of UbiD-like enzymes based on detecting enzyme-catalysed solvent deuterium exchange into potential substrates. Using ferulic acid decarboxylase (FDC) as a model system, we tested a diverse range of aromatic and heterocyclic molecules for their ability to undergo enzyme-catalysed H/D exchange in deuterated buffer. We found that FDC catalyses H/D exchange, albeit at generally very low levels, into a wide range of small, aromatic molecules that have little resemblance to its physiological substrate. In contrast, the sub-set of aromatic carboxylic acids that are substrates for FDC-catalysed decarboxylation is much smaller. We discuss the implications of these findings for screening uncharacterized UbiD-like enzymes for novel (de)carboxylase activity.

The question about acceptor molecules of optical radiation that determine the effects of photobiomodulation in relation to various types of cells still remains the focus of attention of researchers. This issue is most relevant for cancer cells, since, depending on the parameters of optical radiation, light can either stimulate their growth or inhibit them and lead to death. This study shows that endogenous porphyrins, which have sensitizing properties, may play an important role in the implementation of the effects of photobiomodulation, along with flavins. For the first time, using steady-state and kinetic spectrofluorimetry, free-base porphyrins and their zinc complexes were discovered and identified in living human cervical epithelial carcinoma (HeLa) cells, as well as in their extracts. It has been shown that reliable detection of porphyrin fluorescence in cells is hampered by the intense fluorescence of flavins due to their high concentration (micromolar range) and higher (compared to tetrapyrroles) fluorescence quantum yield. Optimization of the spectral range of excitation and the use of extractants that provide multiple quenching of the flavin component while increasing the emission efficiency of tetrapyrroles makes it possible to weaken the contribution of the flavin component to the recorded fluorescence spectra., (© 2024 American Society for Photobiology.)

Modulating molecular structure and function at the nanoscale drives innovation across wide-ranging technologies. Electrical control of the bonding of individual DNA base pairs endows DNA with precise nanoscale structural reconfigurability, benefiting efforts in DNA origami and actuation. Here, alloxazine DNA base surrogates were synthesized and incorporated into DNA duplexes to function as a redox-active switch of hydrogen bonding. Circular dichroism (CD) revealed that 24-mer DNA duplexes containing one or two alloxazines exhibited CD spectra and melting transitions similar to DNA with only canonical bases, indicating that the constructs adopt a B-form conformation. However, duplexes were not formed when four or more alloxazines were incorporated into a 24-mer strand. Thiolated duplexes incorporating alloxazines were self-assembled onto multiplexed gold electrodes and probed electrochemically. Square-wave voltammetry (SWV) revealed a substantial reduction peak centered at -0.272 V vs Ag/AgCl reference. Alternating between alloxazine oxidizing and reducing conditions modulated the SWV peak in a manner consistent with the formation and loss of hydrogen bonding, which disrupts the base pair stacking and redox efficiency of the DNA construct. These alternating signals support the assertion that alloxazine can function as a redox-active switch of hydrogen bonding, useful in controlling DNA and bioinspired assemblies.

Herein, we report a novel flavin analogue as singular chemical component for lysosome bioimaging, and inherited photosensitizer capability of the flavin core was demonstrated as a promising candidate for photodynamic therapy (PDT) application. Fine-tuning the flavin core with the incorporation of methoxy naphthyl appendage provides an appropriate chemical design, thereby offering photostability, selectivity, and lysosomal colocalization, along with the aggregation-induced emissive nature, making it suitable for lysosomal bioimaging applications. Additionally, photosensitization capability of the flavin core with photostable nature of the synthesized analogue has shown remarkable capacity for generating reactive oxygen species (ROS) within cells, making it a promising candidate for photodynamic therapy (PDT) application., (© 2024 Wiley-VCH GmbH.)

ConspectusOrganohalogen compounds are extensively used as building blocks, intermediates, pharmaceuticals, and agrochemicals due to their unique chemical and biological properties. Installing halogen substituents, however, frequently requires functionalized starting materials and multistep functional group interconversion. Several classes of halogenases evolved in nature to enable halogenation of a different classes of substrates; for example, site-selective halogenation of electron rich aromatic compounds is catalyzed by flavin-dependent halogenases (FDHs). Mechanistic studies have shown that these enzymes use FADH 2 to reduce O 2 to water with concomitant oxidation of X - to HOX (X = Cl, Br, I). This species travels through a tunnel within the enzyme to access the FDH active site. Here, it is believed to interact with an active site lysine proximal to bound substrate, enabling electrophilic halogenation with selectivity imparted via molecular recognition, rather than directing groups or strong electronic activation.The unique selectivity of FDHs led to several early biocatalysis efforts, preparative halogenation was rare, and the hallmark catalyst-controlled selectivity of FDHs did not translate to non-native substrates. FDH engineering was limited to site-directed mutagenesis, which resulted in modest changes in site-selectivity or substrate preference. To address these limitations, we optimized expression conditions for the FDH RebH and its cognate flavin reductase (FRed), RebF. We then showed that RebH could be used for preparative halogenation of non-native substrates with catalyst-controlled selectivity. We reported the first examples in which the stability, substrate scope, and site selectivity of a FDH were improved to synthetically useful levels via directed evolution. X-ray crystal structures of evolved FDHs and reversion mutations showed that random mutations throughout the RebH structure were critical to achieving high levels of activity and selectivity on diverse aromatic substrates, and these data were used in combination with molecular dynamics simulations to develop predictive model for FDH selectivity. Finally, we used family wide genome mining to identify a diverse set of FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds.The diversity of our evolved and mined FDHs allowed us to pursue synthetic applications beyond simple aromatic halogenation. For example, we established that FDHs catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. These results highlight the ability of FDH active sites to tolerate different substrate topologies. This utility was further expanded by our recent studies on the single component FDH/FRed, AetF. While we were initially drawn to AetF because it does not require a separate FRed, we found that it halogenates substrates that are not halogenated efficiently or at all by other FDHs and provides high enantioselectivity for reactions that could only be achieved using RebH variants after extensive mutagenesis. Perhaps most notably, AetF catalyzes site-selective aromatic iodination and enantioselective iodoetherification. Together, these studies highlight the origins of FDH engineering, the utility and limitations of the enzymes developed to date, and the promise of FDHs for an ever-expanding range of biocatalytic halogenation reactions.

The inherent structural properties of enzymes are critical in defining catalytic function. Often, studies to evaluate the relationship between structure and function are limited to only one defined structural element. The two-component flavin-dependent desulfonase family of enzymes involved in bacterial sulfur acquisition utilize a comprehensive range of structural features to carry out the desulfonation of organosulfur compounds. These metabolically essential two-component FMN-dependent desulfonase systems have been proposed to utilize oligomeric changes, protein-protein interactions for flavin transfer, and common mechanistic steps for carbon-sulfur bond cleavage. This review is focused on our current functional and structural understanding of two-component FMN-dependent desulfonase systems from multiple bacterial sources. Mechanistic and structural comparisons from recent independent studies provide fresh insights into the overall functional properties of these systems and note areas in need of further investigation. The review acknowledges current studies focused on evaluating the structural properties of these enzymes in relationship to their distinct catalytic function. The role of these enzymes in maintaining adequate sulfur levels, coupled with the conserved nature of these enzymes in diverse bacteria, underscore the importance in understanding the functional and structural nuances of these systems., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Flavin-containing monooxygenase from Methylophaga sp. ( m FMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored m FMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis ( Ni FMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of m FMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

Hydrophobic peptide models derived from the α-helical transmembrane segment of the epidermal growth factor receptor were synthetically modified with a flavin amino acid as a photo-inducible charge donor and decorated with tryptophans along the helix as charge acceptors. The helical conformation of the peptides was conserved despite the modifications, notably also in lipid vesicles and multibilayers. Their ability to facilitate photo-induced transmembrane charge transport was examined by means of steady-state and time-resolved optical spectroscopy. The first tryptophan next to the flavin donor plays a major role in initiating the charge transport near the N-terminus, while the other tryptophans might promote charge transport along the transmembrane helix. These artificially modified, but still naturally derived helical peptides are important models for studying transmembrane electron transfer and the principles of photosynthesis.

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (Fl C4a-OOH ), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H 2 O 2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that Fl C4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from Fl C4a-OOH cannot halogenate their substrates. Remarkably, in situ H 2 O 2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming Fl C4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

Flavoenzymes catalyze numerous redox reactions including the transfer of an O 2 -derived oxygen atom to organic substrates, while the other one is reduced to water. Investigation of some of these monooxygenases led to a detailed understanding of their catalytic cycle, which involves the flavin-C 4α -(hydro)peroxide as hallmark oxygenating species, and newly discovered flavoprotein monooxygenases were generally assumed to operate similarly. However, discoveries in recent years revealed a broader mechanistic versatility, including enzymes that utilize flavin-N 5 oxygen adducts for catalysis in the form of the flavin-N 5 -(hydro)peroxide and the flavin-N 5 -oxide species. In this review, I will highlight recent developments in that area, including noncanonical flavoenzymes from natural product biosynthesis and sulfur metabolism that provide first insights into the chemical properties of these species. Remarkably, some enzymes may even combine the flavin-N 5 -peroxide and the flavin-N 5 -oxide species for consecutive oxygen-transfers to the same substrate and thereby in essence operate as dioxygenases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Enzymes 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 (FMN hq ) 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.

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering., (© 2024 Wiley-VCH GmbH.)

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae , can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation., Competing Interests: IP, AV, MT, AH, FD, JD, VC, JM, SS, FF No competing interests declared, (© 2024, Petit-Hartlein et al.)

Flavins play an important role in many oxidation and reduction processes in biological systems. For example, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are common cofactors found in enzymatic proteins that use the special redox properties of these flavin molecules for their catalytic or photoactive functions. The redox potential of the flavin is strongly affected by its (protein) environment; however, the underlying molecular interactions of this effect are still unknown. Using hybrid quantum mechanics/molecular mechanics (QM/MM) simulation techniques, we have studied the redox properties of flavin in the gas phase, aqueous solution, and two different protein environments, in particular, a BLUF and a LOV photoreceptor domain. By mapping the changes in electrostatic potential and solvent structure, we gain insight into how specific polarization of the flavin by its environment tunes the reduction potential. We find also that accurate calculation of the reduction potentials of these systems by using the hybrid QM/MM approach is hampered by a too limited sampling of the counterion configurations and by artifacts at the QM/MM boundary. We make suggestions for how these issues can be overcome.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH • and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide- and protein-binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non-proteinaceous ligand-binding protein, flavin-based fluorescent protein CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN) while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids so that the resulting sequences are 55%-66% identical to the original one. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability, and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities., (© 2024 The Protein Society.)

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and β-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Single component flavin-dependent halogenases (FDHs) possess both flavin reductase and FDH activity in a single enzyme. We recently reported that the single component FDH AetF catalyzes site-selective bromination and iodination of a variety of aromatic substrates and enantioselective bromolactonization and iodoetherification of styrenes bearing pendant carboxylic acid or alcohol substituents. Given this inherent reactivity and selectivity, we explored the utility of AetF as catalyst for alkene and alkyne C-H halogenation. We find that AetF catalyzes halogenation of a range of 1,1-disubstituted styrenes, often with high stereoselectivity. Despite the utility of haloalkenes for cross-coupling and other applications, accessing these compounds in a stereoselective manner typically requires functional group interconversion processes, and selective halogenation of 1,1'-disubstituted olefins remains rare. We also establish that AetF and homologues of this enzyme can halogenate terminal alkynes. Mutagenesis studies and deuterium kinetic isotope effects are used to support a mechanistic proposal involving covalent catalysis for halogenation of unactivated alkynes by AetF homologues. These findings expand the scope of FDH catalysis and continue to show the unique utility of single component FDHs for biocatalysis., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The blue light using the flavin (BLUF) domain is one of the smallest photoreceptors in nature, which consists of a unique bidirectional electron-coupled proton relay process in its photoactivation reaction cycle. This perspective summarizes our recent efforts in dissecting the photocycle into three elementary processes, including proton-coupled electron transfer (PCET), proton rocking, and proton relay. Using ultrafast spectroscopy, we have determined the temporal sequence, rates, kinetic isotope effects (KIEs), and concertedness of these elementary steps. Our findings provide important implications for illuminating the photoactivation mechanism of the BLUF domain and suggest an engineering platform to characterize intricate reactions involving proton motions that are ubiquitous in nonphotosensitive protein machines.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this review, we discuss the successes and challenges of the atomistic modeling of photoreceptors. Throughout our presentation, we integrate explanations of the primary methodological approaches, ranging from quantum mechanical descriptions to classical enhanced sampling methods, all while providing illustrative examples of their practical application to specific systems. To enhance the effectiveness of our analysis, our primary focus has been directed towards the examination of applications across three distinct photoreceptors. These include an example of Blue Light-Using Flavin (BLUF) domains, a bacteriophytochrome, and the orange carotenoid protein (OCP) employed by cyanobacteria for photoprotection. Particular emphasis will be placed on the pivotal role played by the protein matrix in fine-tuning the initial photochemical event within the embedded chromophore. Furthermore, we will investigate how this localized perturbation initiates a cascade of events propagating from the binding pocket throughout the entire protein structure, thanks to the intricate network of interactions between the chromophore and the protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the W out , W in NH in and W in NH out conformations using 19 F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key W in NH in conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of W in NH in , which encodes a Tyr-Gln configuration that is not conducive to proton transfer., (© 2024. The Author(s).)

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o -hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o -position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o -hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o -hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Oxidoreductases facilitating electron transfer between molecules are pivotal in metabolic pathways. Flavin-based electron bifurcation (FBEB), a recently discovered energy coupling mechanism in oxidoreductases, enables the reversible division of electron pairs into two acceptors, bridging exergonic and otherwise unfeasible endergonic reactions. This chapter explores the four distinct FBEB complex families and highlights a decade of structural insights into FBEB complexes. In this chapter, we discuss the architecture, electron transfer routes, and conformational changes across all FBEB families, revealing the structural foundation that facilitate these remarkable functions., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91 out and W91 in , with Trp91 either proximal or distal to flavin (FMN). Using a quantum mechanical/molecular mechanical/polarizable continuum model approach, the energetics of charge-separated and biradical states in the two conformations were investigated. In the W91 out conformation, the charge-separated state (FMN •- ) is more stable than the photoexcited state (FMN*), whereas it is less stable due to an electrostatic repulsive interaction with the Ser28 side chain in the W91 in conformation. This leads to a lower activation energy for the charge separation in the W91 out conformation, resulting in a faster charge separation compared to that in the W91 in conformation. In the W91 out conformation, the radical state (FMNH • ) is more stable than FMN •- and forms from FMN •- , leading to reorientation of the Gln50 side chain adjacent to FMN and formation of a hydrogen bond between Gln50 and FMN. Subsequently, a signaling state forms through charge recombination. In contrast, in the W91 in conformation, FMN •- cannot proceed further, returning to the dark-adapted state, as FMNH • is less stable. Thus, formation of the signaling state exclusively occurs in the W91 out conformation.

Flavin mononucleotide (FMN) is a dye belonging to the flavin family. These dyes produce photosensitized degradation of organic compounds via reaction with the excited states of the dye or with reactive oxygen species photogenerated from the triplet of the dye. This article presents a new polymeric dye (FMN-CS) composed of the photosensitizer FMN covalently bonded to chitosan polysaccharide (CS). FMN-CS obtained has a molecular weight of 230 × 10 3  g mol -1 and a deacetylation degree of 74.8%. The polymeric dye is an environmentally friendly polymer with spectroscopic and physicochemical properties similar to those of FMN and CS, respectively. Moreover, under sunlight, it is capable of generating 1 O 2 with a quantum yield of 0.31. FMN-CS, like CS, is insoluble in basic media. This allows easy recovery of the polymeric dye once the photosensitized process has been carried out and makes FMN-CS a suitable photosensitizer for the degradation of pollutants in contaminated waters. To evaluate whether FMN-CS may be used for pollutant degradation, the photosensitized degradation of two trihydroxybenzenes by FMN-CS was studied., (© 2023. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

In nature, flavin-dependent halogenases (FDHs) catalyze site-selective chlorination and bromination of aromatic natural products. This ability has led to extensive efforts to engineer FDHs for selective chlorination, bromination, and iodination of electron rich aromatic compounds. On the other hand, FDHs are unique among halogenases and haloperoxidases that exhibit catalyst-controlled site selectivity in that no examples of enantioselective FDH catalysis in natural product biosynthesis have been characterized. Over the past several years, our group has established that FDHs can catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. Achieving high activity and selectivity for these reactions has required extensive mutagenesis and mitigation of problems resulting from hypohalous acid generated during FDH catalysis. The single-component flavin reductase/FDH AetF is unique among the wild type enzyme we have studied in that it provides high activity and selectivity toward several asymmetric transformations. These results highlight the ability of FDH active sites to tolerate different substrate topologies and suggest that they could be useful for a broad range of oxidative halogenations., (© 2023 The Authors. Chirality published by Wiley Periodicals LLC.)

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.

This short review reports the surprising phenomenon of nuclear hyperpolarization occurring in chemical reactions, which is called CIDNP (chemically induced dynamic nuclear polarization) or photo-CIDNP if the chemical reaction is light-driven. The phenomenon occurs in both liquid and solid-state, and electron transfer systems, often carrying flavins as electron acceptors, are involved. Here, we explain the physical and chemical properties of flavins, their occurrence in spin-correlated radical pairs (SCRP) and the possible involvement of flavin-carrying SCRPs in animal magneto-reception at earth's magnetic field.

Numerous bacteria from different phylae can perform desulfurization reactions of organosulfur compounds. In these degradation or detoxification pathways, two-component flavin-dependent monooxygenases that use flavins (FMN or FAD) as a cofactor play important roles as they catalyse the first steps of these metabolic routes. The TdsC or DszC and MsuC proteins belong to this class of enzymes as they process dibenzothiophene (DBT) and methanesulfinate. Elucidation of their X-ray structures in apo, ligand-bound and cofactor-bound forms has provided important molecular insights into their catalytic reaction. Mycobacterial species have also been shown to possess a DBT degradation pathway, but no structural information is available on these two-component flavin-dependent monooxygenases. In this study, the crystal structure of the uncharacterized MAB_4123 protein from the human pathogen Mycobacterium abscessus is presented. The structure solved at high resolution displays high similarity to homologs from Rhodococcus, Paenibacillus and Pseudomonas species. In silico docking approaches suggest that MAB_4123 binds FMN and may use it as a cofactor. Structural analysis strongly suggests that MAB_4123 is a two-component flavin-dependent monooxygenase that could act as a detoxifying enzyme of organosulfur compounds in mycobacteria.

A bioinspired mimic for the stabilization of hydroperoxyflavin intermediate formation was designed and investigated for monooxygenase like catalytic properties. A suitable peptide appendage was covalently linked to the C7-position of the neutral isoalloxazine core to synthesize Fl-G, Fl-F, Fl-P, and Fl-βA analogues. While the presence and identity of the peptide appendage were found to be crucial for catalytic efficiency, corroborative observations were made from theoretical studies as well, supporting the precise conformational and accessibility requirements for the stabilization of the key hydroperoxyflavin intermediate. A simple yet elegant flavopeptide model (Fl-G) was found to achieve almost quantitative catalytic efficiency compared to the control flavin analogue without a peptide appendage.

Flavins are a class of organic compounds with the basic structure of 7,8-dimethy-10-alkyl isoalloxazine. They are ubiquitous in nature and participate in many biochemical reactions. Due to various existing forms, there is a lack of systematic research on the absorption and fluorescence spectra of flavins. In this study, employing the density functional theory (DFT) and time-dependent (TD) DFT, we calculated the pH-dependent absorption and fluorescence spectra of flavin of three redox states (quinone, semiquinone, and hydroquinone) in solvents. The chemical equilibrium of three redox states of flavins and the pH effect on the absorption spectra and fluorescence spectra of flavins were carefully discussed. The conclusion helps with identifying the existing forms of flavins in solvent with different pH values.

Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H 4 to FMN, generating a NAD(P)H 4 cation radical and anionic FMN semiquinone. This electron transfer occurs in ∼1 ps and appears to kinetically outcompete reductive quenching from aromatic residues in the active site. Time-resolved infrared measurements show that relaxation processes appear to be largely localized on the FMN and the charge-separated state is short-lived, with relaxation, presumably via back electron transfer, occurring over ∼3-30 ps. While this demonstrates the potential for non-natural photoactivity, useful photocatalysis will likely require longer-lived excited states, which may be accessible by enzyme engineering and/or a judicious choice of substrate.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC 50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC 50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses., Competing Interests: Declaration of Competing Interest SE is owner and ACP is an employee of Collaborations Pharmaceuticals, Inc. All other co-authors have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re -side and si -side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

Structure-photosensitizing activity relationships for a series of flavin analogues were investigated with the final goal of identifying the most potent photosensitizer in these series. The main structural modifications involved the introduction of various halogen atoms in C 7 - and/or C 8 -positions on the isoalloxazine ring. These compounds were synthesized by reacting judiciously-functionalized anilines with alloxan. The SAR trends showed that the photosensitizing activity increased with the size of the halogen atoms, confirming the importance of the heavy-atom effect on the photosensitizer's activity. The halogens in C 8 were more active than the di-substituted halogens, which in turn were more active than the C 7 -substituted equivalents. However, even if the photosensitizing activity is slightly less important for the 7- compared to the 8-substituted derivatives, the 7-haloisoalloxazines are promising photosensitizers, as they present a better cellular toxicity profile than the 8-substituted analoges. The photosensitizing activity perfectly correlated with the determined fluorescence for the same compounds. Except for the dihalogeno derivatives, all the compounds were not toxic up to a 50 μM range., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin's C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin's N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4-8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pK a1 = 6.6 and pK a2 = 9.2 at 2 μM FMN while it contained only a descending limb pK a of 9.7 at 50 μM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS -hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin.

The excited-state dynamics of molecules embedded in complex (bio)matrices is still a challenging goal for quantum chemical models. Hybrid QM/MM models have proven to be an effective strategy, but an optimal combination of accuracy and computational cost still has to be found. Here, we present a method which combines the accuracy of a polarizable embedding QM/MM approach with the computational efficiency of an excited-state self-consistent field method. The newly implemented method is applied to the photoactivation of the blue-light-using flavin (BLUF) domain of the AppA protein. We show that the proton-coupled electron transfer (PCET) process suggested for other BLUF proteins is still valid also for AppA.

Fatty acid photodecarboxylase is a newly discovered flavin photoenzyme that converts a carboxylic acid into a hydrocarbon and a carbon dioxide molecule through decarboxylation. The enzymatic reactions are poorly understood. In this study, we carefully characterized its dynamic evolution with femtosecond spectroscopy. We observed initial electron transfer from the substrate to the flavin cofactor in 347 ps with a stretched dynamic behavior and subsequently captured the critical carbonyloxy radical. The dominant process following this step was decarboxylation in 5.8 ns to form an alkyl radical and a carbon dioxide molecule. We further identified the absorption bands of two carbonyloxy and alkyl radical intermediates. The overall enzymatic quantum efficiency determined by our obtained timescales is 0.81, consistent with the steady-state value. The results are essential to the elucidation of the enzyme mechanism and catalytic photocycle, providing a molecular basis for potential design of flavin-based artificial photoenzymes., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Photoenzymes use light to initiate biochemical reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biological photocatalysis., Competing Interests: Declaration of competing interest The authors confirm there are no conflicts of interest with this submission., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lumichrome (LC) is the major photodegradation product of biologically important flavin cofactors. Since LC serves as a structural comparison with the flavins; understanding excited states of LC is fundamentally important to establish a connection with photophysics of different flavins, such as lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Herein, we deduce the initial excited state structural dynamics of LC using UV resonance Raman (UVRR) intensity analysis. The UVRR spectra at wavelengths across the 260 nm absorption band of LC were measured and resulting Raman excitation profiles and absorption spectrum were self-consistently simulated using a time-dependent wave packet formalism to extract the initial excited state structural and solvent broadening parameters. These results are compared with those obtained for other flavins following UV excitations. We find that LC undergoes a very distinct instantaneous charge redistribution than flavins, which is attributed to the extended π-conjugation present in flavins but missing in LC. The homogeneous broadening linewidth of LC appears to be lower than that of LF, while the inhomogeneous broadening values are comparable, indicating greater solvent interaction with excited flavin on ultrafast timescale compared with LC, whereas on longer timescale these interactions are almost similar., (© 2022 American Society for Photobiology.)