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Radical enzymes, including the evolutionarily ancient glycyl radical enzyme (GRE) family, catalyze chemically challenging reactions that are involved in a myriad of important biological processes. All GREs possess an essential, conserved backbone glycine that forms a stable, catalytically essential α-carbon radical. Through close examination of the GRE family, we unexpectedly identified hundreds of noncanonical GRE homologs that encode either an alanine, serine, or threonine in place of the catalytic glycine residue. Contrary to a long-standing belief, we experimentally demonstrate that these aminoacyl radical enzymes (AAREs) form stable α-carbon radicals on the three cognate residues when activated by partner activating enzymes. The previously unrecognized AAREs are widespread in microbial genomes, highlighting their biological importance and potential for exhibiting new reactivity. Collectively, these studies expand the known radical chemistry of living systems while raising questions about the evolutionary emergence of the AAREs.

Oxygen-independent alkyl radicals (R • ) have demonstrated great promise in combating tumor hypoxia. Currently, Azo compounds have been the primary source of R • , suffering from external stimuli and decomposition during circulation. Herein, we developed a self-catalytic ATRP nanoinitiator that could generate R • via glutathione (GSH) reduction and thus selectively induce apoptosis of tumor cells. Specifically, a conjugation of laccase (possessing a copper(II) complex) and polymeric alkyl bromide, poly(iBBr), was fabricated to yield an ATRP nanoinitiator (Lac-P(iBBr)). After internalization by cells featured with overexpressed GSH, copper(II) in Lac-P(iBBr) was reduced to copper(I) by GSH, which abstracted the Br atom in poly(iBBr) to yield toxic R • . Moreover, GSH-depletion intensified the oxidative damage caused by R • . Efficient generation of R • by Lac-P(iBBr) could happen in lab flasks, living cells, and tumor-bearing mice without any external stimuli, as demonstrated by the radical product, as well as the consumption of GSH. Moreover, the self-catalytic ATRP nanoinitiator significantly induced cell apoptosis and suppressed tumor growth. Our study expands the chemical toolbox to manipulate cell fates.

Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C-O cleavage. This "anhydroglycolysis" pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu 2+ center must undergo formal reduction to Cu 1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast ( Hansenula polymorpha ) amine oxidase (HPAO) that has undergone site-specific insertion of a para -aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and p K a , we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu 2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.

Hydroxyprolines are abundant in nature and widely utilized by many living organisms. Isomerization of trans-4-hydroxy-d-proline (t4D-HP) to generate 2-amino-4-ketopentanoate has been found to need a glycyl radical enzyme HplG, which catalyzes the cleavage of the C-N bond, while dehydration of trans-4-hydroxy-l-proline involves a homologous enzyme of HplG. Herein, molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations are employed to understand the reaction mechanism of HplG. Two possible reaction pathways of HplG have been explored to decipher the origin of its chemoselectivity. The QM/MM calculations reveal that the isomerization proceeds via an initial hydrogen shift from the Cγ site of t4D-HP to a catalytic cysteine radical, followed by cleavage of the Cδ-N bond in t4D-HP to form a radical intermediate that captures a hydrogen atom from the cysteine. Activation of the Cδ-H bond in t4D-HP to bring about dehydration of t4D-HP possesses an extremely high energy barrier, thus rendering the dehydration pathway implausible in HplG. On the basis of the current calculations, conserved residue Glu429 plays a pivotal role in the isomerization pathway: the hydrogen bonding between it and t4D-HP weakens the hydroxyalkyl Cγ-Hγ bond, and it acts as a proton acceptor to trigger the cleavage of the C-N bond in t4D-HP. Our current QM/MM calculations rationalize the origin of the experimentally observed chemoselectivity of HplG and propose an H-bond-assisted bond activation strategy in radical-containing enzymes. These findings have general implications on radical-mediated enzymatic catalysis and expand our understanding of how nature wisely and selectively activates the C-H bond to modulate catalytic selectivity.

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.

Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.

Pyruvate Formate Lyase (PFL) catalyzes acetyl transfer from pyruvate to coenzyme a by a mechanism involving multiple amino acid radicals. A post-translationally installed glycyl radical (G 734 · in Escherichia coli ) is essential for enzyme activity and two cysteines (C 418 and C 419 ) are proposed to form thiyl radicals during turnover, yet their unique roles in catalysis have not been directly demonstrated with both structural and electronic resolution. Methacrylate is an isostructural analog of pyruvate and an informative irreversible inhibitor of pfl. Here we demonstrate the mechanism of inhibition of pfl by methacrylate. Treatment of activated pfl with methacrylate results in the conversion of the G 734 · to a new radical species, concomitant with enzyme inhibition, centered at g = 2.0033. Spectral simulations, reactions with methacrylate isotopologues, and Density Functional Theory (DFT) calculations support our assignment of the radical to a C2 tertiary methacryl radical. The reaction is specific for C 418 , as evidenced by mass spectrometry. The methacryl radical decays over time, reforming G 734 ·, and the decay exhibits a H/D solvent isotope effect of 3.4, consistent with H-atom transfer from an ionizable donor, presumably the C 419 sulfhydryl group. Acrylate also inhibits PFL irreversibly, and alkylates C 418 , but we did not observe an acryl secondary radical in H 2 O or in D 2 O within 10 s, consistent with our DFT calculations and the expected reactivity of a secondary versus tertiary carbon-centered radical. Together, the results support unique roles of the two active site cysteines of PFL and a C 419 S-H bond dissociation energy between that of a secondary and tertiary C-H bond.

Acetaminophen overdoses cause cell injury in the liver. It is widely accepted that liver toxicity is initiated by the reactive N -acetyl- para -aminophenol (APAP) metabolite N -acetyl- p -benzoquinone imine (NAPQI), which first depletes glutathione and then irreversibly binds to mitochondrial proteins and nuclear DNA. As a consequence, mitochondrial respiration is inhibited, and DNA strands break. NAPQI also promotes the oxidative stress since glutathione is one of the main free-radical scavengers in the cell. However, so far it is unknown where exactly free radicals are generated. In this study, we used relaxometry, a novel technique that allows nanoscale magnetic resonance imaging detection of free radicals. The method is based on fluorescent nanodiamonds, which change their optical properties based on their magnetic surrounding. To achieve subcellular resolution, these nanodiamonds were targeted to cellular locations, that is, the cytoplasm, mitochondria, and the nucleus. Since relaxometry is sensitive to spin noise from radicals, we were able to measure the radical load in these different organelles. For the first time, we measured APAP-induced free-radical production in an organelle-specific manner, which helps predict and better understand cellular toxicity.

Metastasis is the predominant cause of cancer deaths due to solid organ malignancies; however, anticancer drugs are not effective in treating metastatic cancer. Here we report a nanotherapeutic approach that combines magnetic nanocluster-based hyperthermia and free radical generation with an immune checkpoint blockade (ICB) for effective suppression of both primary and secondary tumors. We attached 2,2'-azobis(2-midinopropane) dihydrochloride (AAPH) molecules to magnetic iron oxide nanoclusters (IONCs) to form an IONC-AAPH nanoplatform. The IONC can generate a high level of localized heat under an alternating magnetic field (AMF), which decomposes the AAPH on the cluster surface and produces a large number of carbon-centered free radicals. A combination of localized heating and free radicals can effectively kill tumor cells under both normoxic and hypoxic conditions. The tumor cell death caused by the combination of magnetic heating and free radicals led to the release or exposure of various damage-associated molecule patterns, which promoted the maturation of dendritic cells. Treating the tumor-bearing mice with IONC-AAPH under AMF not only eradicated the tumors but also generated systemic antitumor immune responses. The combination of IONC-AAPH under AMF with anti-PD-1 ICB dramatically suppressed the growth of untreated distant tumors and induced long-term immune memory. This IONC-AAPH based magneto-immunotherapy has the potential to effectively combat metastasis and control cancer recurrence.

Free radicals play a major role in sperm development, including maturation and fertilization, but they are also linked to infertility. Since they are short-lived and reactive, they are challenging to detect with state of the art methodologies. Thus, many details surrounding their role remain unknown. One unknown factor is the source of radicals that plays a role in the sperm maturation process. Two alternative sources have been postulated: First, the NADPH-oxidase system embedded in the plasma membrane (NOX5) and second, the NADH-dependent oxidoreductase of mitochondria. Due to a lack of localized measurements, the relative contribution of each source for capacitation remains unknown. To answer this question, we use a technique called diamond magnetometry, which allows nanoscale MRI to perform localized free radical detection. With this tool, we were able to quantify radical formation in the acrosome of sperm heads. This allowed us to quantify radical formation locally in real time during capacitation. We further investigated how different inhibitors or triggers alter the radical generation. We were able to identify NOX5 as the prominent source of radical generation in capacitation while the NADH-dependent oxidoreductase of mitochondria seems to play a smaller role.

The radical S -adenosyl-l-methionine (SAM) enzyme HydG cleaves tyrosine to generate CO and CN - ligands of the [FeFe] hydrogenase H-cluster, accompanied by the formation of a 4-oxidobenzyl radical (4-OB • ), which is the precursor to the HydG p -cresol byproduct. Native HydG only generates a small amount of 4-OB • , limiting detailed electron paramagnetic resonance (EPR) spectral characterization beyond our initial EPR lineshape study employing various tyrosine isotopologues. Here, we show that the concentration of trapped 4-OB • is significantly increased in reactions using HydG variants, in which the "dangler Fe" to which CO and CN - bind is missing or substituted by a redox-inert Zn 2+ ion. This allows for the detailed characterization of 4-OB • using high-field EPR and electron nuclear double resonance spectroscopy to extract its g -values and 1 H/ 13 C hyperfine couplings. These results are compared to density functional theory-predicted values of several 4-OB • models with different sizes and protonation states, with a best fit to the deprotonated radical anion configuration of 4-OB • . Overall, our results depict a clearer electronic structure of the transient 4-OB • radical and provide new insights into the radical SAM chemistry of HydG.

Two alkylated verdazyl radicals (AlkVZs) were investigated as active compounds for photoinitiated controlled MCF-7 cell death. Observed results unambiguously showed that AlkVZ could be a potential structural moiety for the design of a novel family of photodynamic therapy agents. The main advantage of the proposed substances is an oxygen-independent generation of active radicals, which play a pivotal role in the treatment of oxygen-deficient tumors.

Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR β 2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122· of E. coli RNR β 2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the β 2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity ( K d ∼ 6 nM) of 3AP-Fe(II) for β 2 . Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.

Radicals in biology, once thought to all be bad actors, are now known to play a central role in many enzymatic reactions. Of the known radical-based enzymes, ribonucleotide reductases (RNRs) are pre-eminent as they are essential in the biology of all organisms by providing the building blocks and controlling the fidelity of DNA replication and repair. Intense examination of RNRs has led to the development of new tools and a guiding framework for the study of radicals in biology, pointing the way to future frontiers in radical enzymology.

The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate 17 O high-frequency electron-nuclear double resonance (ENDOR) in conjunction with H 2 17 O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α 2 β 2 E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O··· 17 O distances in the range 2.8-3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.

Atom-transfer radical polymerization (ATRP) is a well-known technique for the controlled polymerization of vinyl monomers under mild conditions. However, as with any other radical polymerization, ATRP typically requires rigorous oxygen exclusion, making it time-consuming and challenging to use by nonexperts. In this Account, we discuss various approaches to achieving oxygen tolerance in ATRP, presenting the overall progress in the field.Copper-mediated ATRP, which we first discovered in the late 1990s, uses a Cu I /L activator that reversibly reacts with the dormant C(sp 3 )-X polymer chain end, forming a X-Cu II /L deactivator and a propagating radical. Oxygen interferes with activation and chain propagation by quenching the radicals and oxidizing the activator. At ATRP equilibrium, the activator is present at a much higher concentration than the propagating radicals. Thus, oxidation of the activator is the dominant inhibition pathway. In conventional ATRP, this reaction is irreversible, so oxygen must be strictly excluded to achieve good results.Over the last two decades, our group has developed several ATRP techniques based on the concept of regenerating the activator. When the oxidized activator is continuously converted back to its active reduced form, then the catalytic system itself can act as an oxygen scavenger. Regeneration can be accomplished by reducing agents and photo-, electro-, and mechanochemical stimuli. This family of methods offers a degree of oxygen tolerance, but most of them can tolerate only a limited amount of oxygen and do not allow polymerization in an open vessel.More recently, we discovered that enzymes can be used in auxiliary catalytic systems that directly deoxygenate the reaction medium and protect the polymerization process. We developed a method that uses glucose oxidase (GOx), glucose, and sodium pyruvate to very effectively scavenge oxygen and enable open-vessel ATRP. By adding a second enzyme, horseradish peroxidase (HPR), we managed to extend the role of the auxiliary enzymatic system to generating carbon-based radicals and changed ATRP from an oxygen-sensitive to an oxygen-fueled reaction.While performing control experiments for the enzymatic methods, we noticed that using sodium pyruvate under UV irradiation triggers polymerization without the presence of GOx. This serendipitous discovery allowed us to develop the first oxygen-proof, small-molecule-based, photoinduced ATRP system. It has oxygen tolerance similar to that of the enzymatic methods, exhibits superior compatibility with both aqueous media and organic solvents, and avoids problems associated with purifying polymers from enzymes. The system was able to rapidly polymerize N -isopropylacrylamide, a challenging monomer, with a high degree of control.These contributions have substantially simplified the use of ATRP, making it more practical and accessible to everyone.

Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron-transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP + oxidoreductase from Bacillus subtilis ( Bs FNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50) is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants, with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that, upon the excitation of WT Bs FNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ∼260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan-bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effects of protein environment and relaxations on the spectral properties of those radical intermediates and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counterions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins.

Organelle-targeted activatable photosensitizers are attractive to improve the specificity and controllability of photodynamic therapy (PDT), however, they suffer from a big problem in the photoactivity under both normoxia and hypoxia due to the limited diversity of phototoxic species (mainly reactive oxygen species). Herein, by effectively photocaging a π-conjugated donor-acceptor (D-A) structure with an N-nitrosamine substituent, we established a unimolecular glutathione and light coactivatable photosensitizer, which achieved its high performance PDT effect by targeting mitochondria through both type I and type II (dual type) reactions as well as secondary radicals-participating reactions. Of peculiar interest, hydrogen radical (H • ) was detected by electron spin resonance technique. The generation pathway of H • via reduction of proton and its role in type I reaction were discussed. We demonstrated that the synergistic effect of multiple reactive species originated from tandem cascade reactions comprising reduction of O 2 by H • to form O 2 •- /HO 2 • and downstream reaction of O 2 •- with • NO to yield ONOO - . With a relatively large two-photon absorption cross section for photoexcitation in the near-infrared region (166 ± 22 GM at 800 nm) and fluorogenic property, the new photosensitizing system is very promising for broad biomedical applications, particularly low-light dose PDT, in both normoxic and hypoxic environments.

Microbes employ a remarkably intricate electron transport system to extract energy from the environment. The respiratory cascade of bacteria culminates in the terminal transfer of electrons onto higher redox potential acceptors in the extracellular space. This general and inducible mechanism of electron efflux during normal bacterial proliferation leads to a characteristic fall in bulk redox potential ( E h ), the degree of which is dependent on growth phase, the microbial taxa, and their physiology. Here, we show that the general reducing power of bacteria can be subverted to induce the abiotic production of a carbon-centered radical species for targeted bioorthogonal molecular synthesis. Using two species, Escherichia coli and Salmonella enterica serovar Typhimurium as model microbes, a common redox active aryldiazonium salt is employed to intervene in the terminal respiratory electron flow, affording radical production that is mediated by native redox-active molecular shuttles and active bacterial metabolism. The aryl radicals are harnessed to initiate and sustain a bioorthogonal controlled radical polymerization via reversible addition-fragmentation chain transfer (BacRAFT), yielding a synthetic extracellular matrix of "living" vinyl polymers with predetermined molecular weight and low dispersity. The ability to interface the ubiquitous reducing power of bacteria into synthetic materials design offers a new means for creating engineered living materials with promising adaptive and self-regenerative capabilities.

Our understanding of lipid peroxidation in biology and medicine is rapidly evolving, as it is increasingly implicated in various diseases but also recognized as a key part of normal cell function, signaling, and death (ferroptosis). Not surprisingly, the root and consequences of lipid peroxidation have garnered increasing attention from multiple disciplines in recent years. Here we "connect the dots" between the fundamental chemistry underpinning the cascade reactions of lipid peroxidation (enzymatic or free radical), the reactive nature of the products formed (lipid-derived electrophiles), and the biological targets and mechanisms associated with these products that culminate in cellular responses. We additionally bring light to the use of highly sensitive, fluorescence-based methodologies. Stemming from the foundational concepts in chemistry and biology, these methodologies enable visualizing and quantifying each reaction in the cascade in a cellular and ultimately tissue context, toward deciphering the connections between the chemistry and physiology of lipid peroxidation. The review offers a platform in which the chemistry and biomedical research communities can access a comprehensive summary of fundamental concepts regarding lipid peroxidation, experimental tools for the study of such processes, as well as the recent discoveries by leading investigators with an emphasis on significant open questions.

Flavin-dependent "ene"-reductases can generate stabilized alkyl radicals when irradiated with visible light; however, they are not known to form unstabilized radicals. Here, we report an enantioselective radical cyclization using alkyl iodides as precursors to unstabilized nucleophilic radicals. Evidence suggests this species is accessed by photoexcitation of a charge-transfer complex that forms between flavin and substrate within the protein active site. Stereoselective delivery of a hydrogen atom from the flavin semiquinone to the prochiral radical formed after cyclization provides high levels of enantioselectivity across a variety of substrates. Overall, this transformation demonstrates that photoenzymatic catalysis can address long-standing selectivity challenges in the radical literature.

Current chemodynamic therapy (CDT) primarily relies on the delivery of transition metal ions with Fenton activity to trigger hydroxyl radical production from hydrogen peroxide. However, administration of an excess amount of exogenous Fenton-type heavy metals may cause potential adverse effects to human health, including acute and chronic damages. Here, we present a new CDT strategy that uses intracellular labile iron pool (LIP) as the endogenous source of Fenton-reactive metals for eliciting free radical generation, and the discovery of hydroperoxides (R'OOH) as an optimal LIP-mediated chemodynamic agent against cancer. By simulating the metabolic fates of peroxo compounds within cells, R'OOH was found to have excellent free radical-producing ability in the presence of labile iron(II) and to suffer only moderate elimination by glutathione/glutathione peroxidase, which contributes to its superior chemodynamic efficacy. The LIP-initiated nontoxic-to-toxic transition of R'OOH, together with increased LIP levels in tumor cells, enabled efficient and specific CDT of cancer. Moreover, pH/labile iron(II) cascade-responsive nanomedicines comprising encapsulated methyl linoleate hydroperoxide and LIP-increasing agent in pH-sensitive polymer particles were fabricated to realize enhanced CDT. This work not only paves the way to using endogenous Fenton-type metals for cancer therapy but also offers a paradigm for the exploration of high-performance chemodynamic agents activated by intracellular LIP.

All kingdoms of life use the transient 5'-deoxyadenosyl radical (5'-dAdo•) to initiate a wide range of difficult chemical reactions. Because of its high reactivity, the 5'-dAdo• must be generated in a controlled manner to abstract a specific H atom and avoid unproductive reactions. In radical S -adenosylmethionine (SAM) enzymes, the 5'-dAdo• is formed upon reduction of SAM by an [Fe 4 S 4 ] cluster. An organometallic precursor featuring an Fe-C bond between the [Fe 4 S 4 ] cluster and the 5'-dAdo group was recently characterized and shown to be competent for substrate radical generation, presumably via Fe-C bond homolysis. Such reactivity is without precedent for Fe-S clusters. Here, we show that synthetic [Fe 4 S 4 ]-alkyl clusters undergo Fe-C bond homolysis when the alkylated Fe site has a suitable coordination number, thereby providing support for the intermediacy of organometallic species in radical SAM enzymes. Addition of pyridine donors to [(IMes) 3 Fe 4 S 4 -R] + clusters (R = alkyl or benzyl; IMes = 1,3-dimesitylimidazol-2-ylidene) generates R•, ultimately forming R-R coupled hydrocarbons. This process is facile at room temperature and allows for the generation of highly reactive radicals including primary carbon radicals. Mechanistic studies, including use of the 5-hexenyl radical clock, demonstrate that Fe-C bond homolysis occurs reversibly. Using these experimental insights and kinetic simulations, we evaluate the circumstances in which an organometallic intermediate can direct the 5'-dAdo• toward productive H-atom abstraction. Our findings demonstrate that reversible homolysis of even weak M-C bonds is a feasible protective mechanism for the 5'-dAdo• that can allow selective X-H bond activation in both radical SAM and adenosylcobalamin enzymes.

Linalool is one of the most commonly used fragrance terpenes in consumer products. While pure linalool is considered as non-allergenic because it has a very low skin sensitization potential, its autoxidation on air leads to allylic hydroperoxides that have been shown to be major skin sensitizers. These hydroperoxides have the potential to form antigens via radical mechanisms. In order to obtain in-depth insights of such reactivity, we first investigated the formation of free radicals derived from linalool hydroperoxides in situ in a model of human reconstructed epidermis by electron paramagnetic resonance combined with spin trapping. The formation of carbon- and oxygen-centered radical species derived from the hydroperoxides was especially evidenced in an epidermis model, mimicking human skin and thus closer to what may happen in vivo . To further investigate these results, we synthesized linalool hydroperoxides containing a 13 C-substitution at positions precursor of carbon radicals to elucidate if one of these positions could react with cysteine, its thiol chemical function being one of the most labile groups prone to react through radical mechanisms. Reactions were followed by mono- and bidimensional 13 C NMR. We validated that carbon radicals derived from allylic hydrogen abstraction by the initially formed alkoxyl radical and/or from its β-scission can alter directly the lateral chain of cysteine forming adducts via radical processes. Such results provide an original vision on the mechanisms likely involved in the reaction with thiol groups that might be present in the skin environment. Consequently, the present findings are a step ahead toward the understanding of protein binding processes to allergenic allylic hydroperoxides of linalool through the involvement of free radical species and thus of their sensitizing potential.

While the number of characterized radical S -adenosyl-l-methionine (SAM) enzymes is increasing, the roles of these enzymes in radical catalysis remain largely ambiguous. In radical SAM enzymes, the slow radical initiation step kinetically masks the subsequent steps, making it impossible to study the kinetics of radical chemistry. Due to this kinetic masking, it is unknown whether the subsequent radical reactions require rate acceleration by the enzyme active site. Here, we report the first evidence that a radical SAM enzyme MoaA accelerates the radical-mediated C-C bond formation. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH 2 GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterizations, we found that MoaA catalyzes a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from (4' R )-5'-deoxyadenosine (5'-dA) to transiently generate 5'-deoxyadenos-4'-yl radical (5'-dA-C4' • ) that is subsequently reduced stereospecifically to yield (4' S )-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics and determined the rate of the on-pathway 3',8-cyclization step as 2.7 ± 0.7 s -1 . Together with DFT calculations, this observation suggested that the 3',8-cyclization by MoaA is accelerated by 6-9 orders of magnitude. Further experimental and theoretical characterizations suggested that the rate acceleration is achieved mainly by constraining the triphosphate and guanine base positions while leaving the ribose flexible, and a transition state stabilization through H-bond and electrostatic interactions with the positively charged R17 residue. This is the first evidence for rate acceleration of radical reactions by a radical SAM enzyme and provides insights into the mechanism by which radical SAM enzymes accelerate radical chemistry.

G-Quadruplexes are formed by guanine rich DNA/RNA sequences in the presence of metal ions, which occupy the central cavity of these four-stranded structures. We show that these metal ions have a significant effect on the photogeneration and the reactivity of guanine radicals. Transient absorption experiments on G-quadruplexes formed by association of four TGGGGT strands in the presence of K + reveal that the quantum yield of one-photon ionization at 266 nm (8.1 × 10 -3 ) is twice as high as that determined in the presence of Na + . Replacement of Na + with K + also suppresses one reaction path involving deprotonated radicals, (G-H2) • → (G-H1) • tautomerization. Such behavior shows that the underlying mechanisms are governed by dynamical processes, controlled by the mobility of metal ions, which is higher for Na + than for K + . These findings may contribute to our understanding of the ultraviolet-induced DNA damage and optimize optoelectronic devices based on four-stranded structures, beyond DNA.

( S )-2-Hydroxypropylphosphonate [( S )-2-HPP, 1 ] epoxidase (HppE) reduces H 2 O 2 at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis -epoxide of the drug [(1 R ,2 S )-epoxypropylphosphonic acid ( cis -Fos, 2 )]. Here we show that HppE achieves ∼95% selectivity for C1 inversion and cis -epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE·Fe II · 1 and HppE·Zn II · 2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues-Leu120, Leu144, Phe182, and Leu193-close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans ∼99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (∼82:18). Retention of C1 configuration and trans -epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (∼13:87), trifluorination of C3 (∼23:77), or both (∼1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (∼56:44) and Leu144Phe/Leu193Phe (∼90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical-as proposed in a recent computational study-to direct formation of the more potently antibacterial cis -epoxide by radicaloid C-O coupling.

Breaking the threshold of intracellular reactive oxygen species (ROS) levels can cause nonspecific oxidative damage to proteins and lead to the Fenton reaction-mediated exogenous ROS production to be a new promising anticancer strategy. However, the problems, including the inefficient transport of metal catalysts and insufficient endogenous hydrogen peroxide (H 2 O 2 ) content in cells, still need to be improved. In this study, a functional nanosystem encapsulated with benzothiazole complexes (FeTB 2 ) and the photosensitizer indocyanine green (ICG) was designed for highly effective antitumor therapy. The surface of the nanocarriers was modified with dihydroartemisinin (DHA)-grafted polyglutamic acid. The induced hyperthermia enables the lipid-polymer shell to depolymerize, releasing FeTB 2 . The released FeTB 2 could kill tumor cells in two different ways by inhibiting DNA replication and catalyzing H 2 O 2 to produce active •OH. Moreover, the conjugated DHA could increase the amount of peroxides in tumor cells and significantly enhance the ROS yield. This work has provided solid evidence that the present nanosystem enables a significant effect on tumor killing through the combined inhibition of DNA replication and ROS-mediated oxidative damage by regulation of the tumor microenvironment, providing a ROS-mediated high-efficiency antitumor strategy.

Efficient organic photosensitizers (PSs) have attracted much attention because of their promising applications in photodynamic therapy (PDT). However, guidelines on their molecular design are rarely reported. In this work, a series of PSs are designed and synthesized based on a triphenylamine-azafluorenone core. Their structure-property-application relationships are systematically studied. Cationization is an effective strategy to enhance the PDT efficiency of PSs by targeting mitochondria. From the molecularly dispersed state to the aggregate state, the fluorescence and the reactive oxygen species generation efficiency of PSs with aggregation-induced emission (AIE) increase due to the restriction of the intramolecular motions and enhancement of intersystem crossing. Cationized mitochondrion-targeting PSs show higher PDT efficiency than that of nonionized ones targeting lipid droplets. The ability of AIE PSs to kill cancer cells can be further enhanced by combination of PDT with radiotherapy. Such results should trigger research enthusiasm for designing and synthesizing AIE PSs with better PDT efficiency and properties.

The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S -adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5'-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S] + cluster to the coordinated S -adenosylmethionine (SAM) to induce homolytic S-C5' bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5' (2pπ) radical (∼70% spin occupancy); the C5'(H) 2 plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5'-C4'-(C4'-H) plane, placing a C5'-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4'-H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.

S -Adenosyl-l-methionine (SAM) is the central cofactor in the radical SAM enzyme superfamily, responsible for a vast number of transformations in primary and secondary metabolism. In nearly all of these reactions, the reductive cleavage of SAM is proposed to produce a reactive species, 5'-deoxyadenosyl radical, which initiates catalysis. While the mechanistic details in many cases are well-understood, the reductive cleavage of SAM remains elusive. In this manuscript, we have measured the solution peak potential of SAM to be ∼-1.4 V (v SHE) and show that under controlled potential conditions, it undergoes irreversible fragmentation to the 5'-deoxyadenosyl radical. While the radical intermediate is not directly observed, its presence as an initial intermediate is inferred by the formation of 8,5'-cycloadenosine and by H atom incorporation into 5'-deoxyadenosine from solvent exchangeable site. Similarly, 2-aminobutyrate is also observed under electrolysis conditions. The implications of these results in the context of the reductive cleavage of SAM by radical SAM enzymes are discussed.

The aggregation of α-synuclein (α-Syn) has been implicated strongly in Parkinson's disease (PD). The intrinsically disordered nature of α-Syn makes this protein prone to self-association or heteroassociation with another protein or lipid. While conformational fluctuation and free radical chemistry have been shown to play important roles in its ability toward self- and heteroassociation, any systematic understanding of their contributions is missing. Here, we report an in vitro investigation of the interaction between α-Syn and cytochrome c in the oxidized (cyt c III) and reduced forms (cyt c II), in which cyt c III was found to induce a large compaction of α-Syn and inhibit the aggregation by favoring a hetero-dityrosine bond formation. In contrast, the presence of cyt c II did not result in any compaction and its presence was found to facilitate α-Syn aggregation. The variation in the charge distribution of the surface residues of cyt c III and cyt c II is expected to play a decisive role in their interaction with α-Syn.

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the β 2 subunit (Y 122 • ) necessary for nucleotide reductase activity. Upon binding the cognate α 2 subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active α 2 β 2 complex from the Y 122 • site in β 2 to the substrate activating cysteine residue (C 439 ) in α 2 via a pathway of redox active amino acids (Y 122 [β] ↔ W 48 [β]? ↔ Y 356 [β] ↔ Y 731 [α] ↔ Y 730 [α] ↔ C 439 [α]) spanning >35 Å. Ionizable residues at the α 2 β 2 interface are essential in mediating RT, and therefore control activity. One of these mutations, E 350 X (where X = A, D, Q) in β 2 , obviates all RT, though the mechanism of control by which E 350 mediates RT remains unclear. Herein, we utilize an E 350 Q-photoβ 2 construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y 122 • → Y 356 ) is replaced by direct photochemical radical generation of Y 356 • . These data present compelling evidence that E 350 conveys allosteric information between the α 2 and β 2 subunits facilitating conformational gating of RT that specifically targets Y 122 • reduction, while the fidelity of the remainder of the RT pathway is retained.

Free radicals have emerged as new-type and promising candidates for hypoxic tumor treatment, and further study of their therapeutic mechanism by real-time imaging is of great importance to explore their biomedical applications. Herein, we present a smart free-radical generator AuNC-V057-TPP for hypoxic tumor therapy; the AuNC-V057-TPP not only exhibits good therapeutic effect under both hypoxic and normoxic conditions but also can monitor the release of free radicals in real-time both in vitro and in vivo. What is more, with the mitochondria-targeting ability, the AuNC-V057-TPP is demonstrated with improved antitumor efficacy through enhanced free radical level in mitochondria, which leads to mitochondrial membrane damage and ATP production reduction and finally induces cancer cell apoptosis.

Indoxyl sulfate (IS) is a uremic toxin that has been associated with inflammation and oxidative stress as well as with the progression of chronic kidney disease (CKD). IS is a protein metabolite that is concentrated in the serum of CKD patients. IS is a well-known uremic toxin, but there are very few reports on the effect of IS on cells including mononuclear cells (MNCs). We hypothesized that a high concentration of IS in CKD patients may induce changes in redox balance in the in vitro cells exposed. In the present study, we investigated the effect of IS on free radical production, antioxidant capacity, and protein damage in the mononuclear blood cells. As already determined, the concentrations (0.2 or 1 mM) of IS used in this study do not affect the survival rate of MNCs. For both the concentrations of IS, there was an increase in superoxide and nitric oxide and a release of other reactive oxygen species (ROS) inside the cells, as measured using fluorescent probes. However, an increase in other ROS as indicated by H 2 DCF-DA was found only for 1 mM of IS. Moreover, a decrease in the non-enzymatic antioxidant capacity and an increase in the superoxide dismutase activity after incubation of the cells with IS were observed. Furthermore, we found an increase in the levels of carbonyl compounds and peroxides in the cells treated with both the concentrations of IS. The obtained results show that IS induces oxidative stress and a decrease in antioxidant defense in cells leading to lipid and protein damage.

It is a remarkable fact that ∼50 μT magnetic fields can alter the rates and yields of certain free-radical reactions and that such effects might be the basis of the light-dependent ability of migratory birds to sense the direction of the Earth's magnetic field. The most likely sensory molecule at the heart of this chemical compass is cryptochrome, a flavin-containing protein that undergoes intramolecular, blue-light-induced electron transfer to produce magnetically sensitive radical pairs. To learn more about the factors that control the magnetic sensitivity of cryptochromes, we have used a set of de novo designed protein maquettes that self-assemble as four-α-helical proteins incorporating a single tryptophan residue as an electron donor placed approximately 0.6, 1.1, or 1.7 nm away from a covalently attached riboflavin as chromophore and electron acceptor. Using a specifically developed form of cavity ring-down spectroscopy, we have characterized the photochemistry of these designed flavoprotein maquettes to determine the identities and kinetics of the transient radicals responsible for the magnetic field effects. Given the gross structural and dynamic differences from the natural proteins, it is remarkable that the maquettes show magnetic field effects that are so similar to those observed for cryptochromes.

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of ( S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B 12 -dependent propanediol dehydratase (B 12 -PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B 12 -PD. Here we report 18 O labeling experiments that suggest PD and B 12 -PD employ distinct mechanisms. Unlike B 12 -PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1- gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.

Multiple pathogenic factors [e.g., amyloid-β (Aβ), metal ions, metal-bound Aβ (metal-Aβ), reactive oxygen species (ROS)] are found in the brain of patients with Alzheimer's disease (AD). In order to elucidate the roles of pathological elements in AD, chemical tools able to regulate their activities would be valuable. Due to the complicated link among multiple pathological factors, however, it has been challenging to invent such chemical tools. Herein, we report novel small molecules as chemical tools toward modulation of single or multiple target(s), designed via a rational structure-property-directed strategy. The chemical properties (e.g., oxidation potentials) of our molecules and their coverage of reactivities toward the pathological targets were successfully differentiated through a minor structural variation [i.e., replacement of one nitrogen (N) or sulfur (S) donor atom in the framework]. Among our compounds (1-3), 1 with the lowest oxidation potential is able to noticeably modify the aggregation of both metal-free Aβ and metal-Aβ, as well as scavenge free radicals. Compound 2 with the moderate oxidation potential significantly alters the aggregation of Cu(II)-Aβ 42 . The hardly oxidizable compound, 3, relative to 1 and 2, indicates no noticeable interactions with all pathogenic factors, including metal-free Aβ, metal-Aβ, and free radicals. Overall, our studies demonstrate that the design of small molecules as chemical tools able to control distinct pathological components could be achieved via fine-tuning of structures and properties.

Sulfur mustard (SM) is a chemical alkylating compound that primary targets lung tissue. It causes a wide variety of pathological effects in respiratory system such as chronic bronchitis, bronchiolitis obliterans, necrosis of the mucosa and inflammation, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. However, molecular and cellular mechanisms for these pathologies are still unclear. Oxidative stress (OS) induced by reactive oxygen species (ROS) is likely a significant mechanism by which SM leads to cell death and tissues injury. SM can trigger various molecular and cellular pathways that are linked to ROS generation, OS, and inflammation. Hypoxia-induced oxidative stress, reduced activity of enzymatic antioxidants, depletion of intercellular glutathione (GSH), decreased productivity of GSH-dependent antioxidants, mitochondrial dysfunction, accumulation of leukocytes and proinflammatory cytokines, and increased expression of ROS producing-related enzymes and inflammatory mediators are the major events in which SM leads to massive production of ROS and OS in pulmonary system. Therefore, understanding of these molecules and signaling pathways gives us valuable information about toxicological effects of SM on injured tissues and the way for developing a suitable clinical treatment. In this review, we aim to discuss the possible mechanisms by which SM induces excessive production of ROS, OS, and antioxidants depletion in lung tissue of exposed patients.

Function/location of menaquinone (MQ) was studied in the photosynthetic reaction center of Heliobacterium (Hbt.) modesticaldum (hRC), which is one of the most primitive homodimeric type I RCs. The spin-polarized electron paramagnetic resonance signals of light-induced radical pair species, which are made of oxidized electron donor bacteriochlorophyll g (P800 + ) and reduced menaquinone (MQ - ) or iron-sulfur cluster (F X - ), were measured in the oriented membranes of Hbt. modesticaldum at cryogenic temperature. The spectral shape of transient electron spin-polarized signal of P800 + F X - radical pair state varied little with respect to the direction of the external magnetic field. It suggested a dominant contribution of the spin evolution on the precursor primary radical pair P800 + A 0 - state with the larger isotropic magnetic exchange interaction J than the anisotropic dipole interaction D. The pure P800 + MQ - signal was simulated by subtracting the effects of spin evolution during the electron-transfer process. It was concluded that the J value of the P800 + MQ - radical pair is negative with an amplitude almost comparable to | D|. It is in contrast to a positive and small J value of the P700 + PhyQ - state in photosystem I (PS I). The results indicate similar but somewhat different locations/binding sites of quinones between hRC and PS I.

Regiospecific dehydration of vicinal diols by enzymes is a difficult reaction that usually requires activation by dedicated organic cofactors. The enzymatic use of radical-based chemistry is an effective but challenging alternative as radical intermediates are difficult to control. Here we report the X-ray structure of the radical S-adenosyl-l-methionine (SAM) dehydratase AprD4 involved in the biosynthesis of the aminoglycoside (AG) antibiotic apramycin. Using in vitro characterizations and theoretical calculations based on our crystal structure, we have been able to propose a detailed mechanism of AprD4 catalysis, which involves a complex partially substrate-induced proton relay network in the enzyme active site and highlights the key role of the protein matrix in driving high-energy intermediates.

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C 439 • ) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α 2 :β 2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y 122 β ↔ [W 48 β?] ↔ Y 356 β ↔ Y 731 α ↔ Y 730 α ↔ C 439 α). The mutant R 411 A(α) disrupts the H-bonding environment and conformation of Y 731 , ostensibly breaking the RT pathway in α 2 . However, the R 411 A protein retains significant enzymatic activity, suggesting Y 731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH 2 Y) by amber codon suppression at positions Y 731 or Y 730 and investigation of the NH 2 Y • trapped state in the active α 2 :β 2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y 731 in R 411 A-α 2 is dynamic, reforming the H-bond between Y 731 and Y 730 to allow RT to propagate to Y 730 . Kinetic studies facilitated by photochemical radical generation reveal that Y 731 changes conformation on the ns-μs time scale, significantly faster than the enzymatic k cat . Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 10 4 s -1 when comparing wild type to R 411 A-α 2 , respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.

Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically 13 C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary Q A and secondary Q B ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides 13 C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected 13 C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and p π -orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.

A purple color is formed during the fibrillation of lysozyme, a well-studied protein lacking a prosthetic group. The application of Raman spectroscopy, electron paramagnetic resonance and UV-vis absorption spectroscopy indicates the formation of a sulfur∴π-bonded radical cation due to the methionine-phenylalanine interaction, which is consistent with a small molecule model reported in the literature. A purple chromophore with characteristic 550 nm absorption is formed due to a specific orientation of the sulfur-centered radical cation and a phenyl ring stabilized by the fibril framework. A specific fibril conformation and the resulting formation of the chromophore are controlled reversibly by varying the pH. This is the first known example of a side chain self-assembled chromophore formed due to protein aggregation.

S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C 5',adenosine -S bond of SAM to generate a 5'-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the C γ,Met -S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the C methyl -S bond, forming 5'-deoxy-5'-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S] + cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters.

Thiomuracin is a thiopeptide antibiotic with potent activity toward Gram-positive drug-resistant bacteria. Thiomuracin is biosynthesized from a precursor peptide, TbtA, by a complex array of posttranslational modifications. One of several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp 2 carbon. Herein, we report the in vitro reconstitution of TbtI, the responsible radical S-adenosyl-methionine (rSAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies demonstrate that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. In determining the minimal substrate for TbtI, we found that the enzyme is functional when most of the leader peptide has been removed. The in vitro reconstitution of TbtI, a class C rSAM methyltransferase, further adds to the chemical versatility of rSAM enzymes, and informs on the complexity of thiomuracin biosynthesis.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y 122 • ) cofactor in β2. The Y 122 • -mediated oxidation of C 439 in α2 occurs by a pathway (Y 122 ⇆ [W 48 ] ⇆ Y 356 in β2 to Y 731 ⇆ Y 730 ⇆ C 439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y 356 and Y 731 at the subunit interface. The proximity in the primary sequence of the conserved E 350 to Y 356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F 2 Y) replaces residue 356, 731, or both are reported in the presence of E 350 or E 350 X (X = A, D, or Q) mutants. With E 350 , activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F 2 Y 356 and F 2 Y 731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E 350 X mutants, all RNRs were inactive, suggesting that E 350 could be a proton acceptor during oxidation of the interface Ys. To determine if E 350 plays a role in conformational gating, the strong oxidants, NO 2 Y 122 • -β2 and 2,3,5-F 3 Y 122 • -β2, were reacted with α2, CDP, and ATP in E 350 and E 350 X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E 350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical S-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on p-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also sec-butyl and t-butyl groups. To our knowledge, this is the first in vitro reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t-butyl group.