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.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

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.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-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.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

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 ß-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.

Reengineering of a flavin-binding fluorescent protein using ProteinMPNN.

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.

Redox Properties of Flavin in BLUF and LOV Photoreceptor Proteins from Hybrid QM/MM Molecular Dynamics Simulation.

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.

Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain.

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.

Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.

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.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

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.

Selective C-H Halogenation of Alkenes and Alkynes Using Flavin-Dependent Halogenases.

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.

Origin of the multi-phasic quenching dynamics in the BLUF domains across the species.

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 Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin 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 WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

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.

Deciphering Photoreceptors Through Atomistic Modeling from Light Absorption to Conformational Response.

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.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State.

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.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor.

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.

Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91out and W91in, 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 W91out 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 W91in conformation. This leads to a lower activation energy for the charge separation in the W91out conformation, resulting in a faster charge separation compared to that in the W91in conformation. In the W91out 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 W91in 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 W91out conformation.

A novel eco-friendly polymeric photosensitizer based on chitosan and flavin mononucleotide.

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 × 103 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 1O2 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.

Further Characterization of Fungal Halogenase RadH and Its Homologs.

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.

Crystal structure of MAB_4123, a putative flavin-dependent monooxygenase from Mycobacterium abscessus.

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.

Spin Dynamics of Flavoproteins.

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.