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The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H2). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O2 sensitivity, binding specificity, or H2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS]4 linker group (CbHydA1-PsaE). We demonstrate that the CbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H2 at a rate of 84.9 ± 3.1 µmol H2 mgchl-1 h-1. Further, when prepared and illuminated in the presence of O2, the nanoconstruct retains the ability to generate H2, though at a diminished rate of 2.2 ± 0.5 µmol H2 mgchl-1 h-1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O2-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H2 generating system that can function in the presence of O2.

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Hyoscyamine 6ß-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would─analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N-acetylnorloline synthase (LolO)─coordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6ß-hydroxyhyoscyamine suggest that a modest (∼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2H2O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.

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An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

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[FeFe] hydrogenase from Clostridium beijerinkii (CbHydA1) is an unusual hydrogenase in that it can withstand prolonged exposure to O2 by reversibly converting into an O2-protected, inactive state (Hinact). It has been indicated in the past that an atypical conformation of the "SC367CP" loop near the [2Fe]H portion of the six-iron active site (H-cluster) allows the Cys367 residue to adopt an "off-H+-pathway" orientation, promoting a facile transition of the cofactor to Hinact. Here, we investigated the electronic structure of the H-cluster in the oxidized state (Hox) that directly converts to Hinact under oxidizing conditions and the related CO-inhibited state (Hox-CO). We demonstrate that both states exhibit two distinct forms in electron paramagnetic resonance (EPR) spectroscopy. The ratio between the two forms is pH-dependent but also sensitive to the buffer choice. Our IR and EPR analyses illustrate that the spectral heterogeneity is due to a perturbation of the coordination environment of the H-cluster's [4Fe4S]H subcluster without affecting the [2Fe]H subcluster. Overall, we conclude that the observation of two spectral components per state is evidence of heterogeneity of the environment of the H-cluster likely associated with conformational mobility of the SCCP loop. Such flexibility may allow Cys367 to switch rapidly between off- and on-H+-pathway rotamers. Consequently, we believe such structural mobility may be the key to maintaining high enzymatic activity while allowing a facile transition to the O2-protected state.

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An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl - or Br - ), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three non-gaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O 2 . Halide, 2OG, and (lastly) O 2 all coordinate directly to the cofactor to initiate its conversion to a cis -halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l -lysine 4-chlorinase, BesD. After 2OG adds, subsequent coordination of the halide to the cofactor and binding of cationic l -Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon addition of O 2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l -Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate• l -Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l -Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply that (i) BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l -Lys binding and chloride coordination following loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

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[FeFe] hydrogenases comprise an important class of H2 evolving enzymes; however, these proteins are often oxygen sensitive and require anaerobic environments for characterization. Understanding the electrochemical relationships between various active and inactive states of these enzymes is instrumental in uncovering the reaction mechanisms of the complex six-iron active center of [FeFe] hydrogenases called H-cluster. Since states of the H-cluster exhibit distinct fingerprint-like spectra in the mid-IR range, IR spectroelectrochemical experiments provide a powerful methodological framework for this goal. This chapter describes protocols for performing Fourier-transform infrared (FTIR) spectroelectrochemical experiments on [FeFe] hydrogenases under anaerobic conditions. Topics included experimental design, data acquisition, and data analysis.

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Bacteria that infect the human gut must compete for essential nutrients, including iron, under a variety of different metabolic conditions. Several enteric pathogens, including Vibrio cholerae and Escherichia coli O157:H7, have evolved mechanisms to obtain iron from heme in an anaerobic environment. Our laboratory has demonstrated that a radical S-adenosylmethionine (SAM) methyltransferase is responsible for the opening of the heme porphyrin ring and release of iron under anaerobic conditions. Furthermore, the enzyme in V. cholerae, HutW, has recently been shown to accept electrons from NADPH directly when SAM is utilized to initiate the reaction. However, how NADPH, a hydride donor, catalyzes the single electron reduction of a [4Fe-4S] cluster, and/or subsequent electron/proton transfer reactions, was not addressed. In this work, we provide evidence that the substrate, in this case, heme, facilitates electron transfer from NADPH to the [4Fe-4S] cluster. This study uncovers a new electron transfer pathway adopted by radical SAM enzymes and further expands our understanding of these enzymes in bacterial pathogens.

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Nosiheptide is a ribosomally produced and post-translationally modified thiopeptide antibiotic that displays potent antibacterial activity in vitro, especially against Gram-positive pathogens. It comprises a core peptide macrocycle that contains multiple thiazole rings, dehydrated serine and threonine residues, a tri-substituted 3-hydroxypyridine ring and several other modifications. Among these additional modifications includes a 3,4-dimethyl-2-indolic acid (DMIA) moiety that bridges Glu6 and Cys8 of the core peptide to form a second smaller ring system. This side-ring system is formed by the action of NosN, a radical S-adenosylmethionine (SAM) enzyme that falls within the class C radical SAM methylase (RSMT) family. However, the true function of NosN is to transfer a methylene group from the methyl moiety of SAM to C4 of 3-methylindolic acid (MIA) attached in a thioester linkage to Cys8 of the core peptide to set up a highly electrophilic species. This species is then trapped by the side chain of Glu6, resulting in formation of a lactone and the side-ring system. The NosN reaction requires two simultaneously bound molecules of SAM. The first, SAMI, is cleaved to generate a 5'-deoxyadenosyl 5'-radical, which abstracts a hydrogen atom from the methyl group of the second molecule of SAM, SAMII. The resulting SAMII radical is believed to add to C4 of MIA, affording a radical intermediate on the MIA substrate. Herein we describe synthetic approaches that allow detection of this radical by electron paramagnetic resonance (EPR) spectroscopy.

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Important bioactive natural products, including prostaglandin H2 and artemisinin, contain reactive endoperoxides. Known enzymatic pathways for endoperoxide installation require multiple hydrogen-atom transfers (HATs). For example, iron(II)- and 2-oxoglutarate-dependent verruculogen synthase (FtmOx1; EC 1.14.11.38) mediates HAT from aliphatic C21 of fumitremorgin B, capture of O2 by the C21 radical (C21•), addition of the peroxyl radical (C21-O-O•) to olefinic C27, and HAT to the resultant C26•. Recent studies proposed conflicting roles for FtmOx1 tyrosine residues, Tyr224 and Tyr68, in the HATs from C21 and to C26•. Here, analysis of variant proteins bearing a ring-halogenated tyrosine or (amino)phenylalanine in place of either residue establishes that Tyr68 is the hydrogen donor to C26•, while Tyr224 has no essential role. The radicals that accumulate rapidly in FtmOx1 variants bearing a HAT-competent tyrosine analog at position 68 exhibit hypsochromically shifted absorption and, in cases of fluorine substitution, 19F-coupled electron-paramagnetic-resonance (EPR) spectra. By contrast, functional Tyr224-substituted variants generate radicals with unaltered light-absorption and EPR signatures as they produce verruculogen. The alternative major product of the Tyr68Phe variant, which forms competitively with verruculogen also in wild-type FtmOx1 in 2H2O and in the variant with the less readily oxidized 2,3-F2Tyr at position 68, is identified by mass spectrometry and isotopic labeling as the 26-hydroxy-21,27-endoperoxide compound formed after capture of another equivalent of O2 by the longer lived C26•. The results highlight the considerable chemical challenges the enzyme must navigate in averting both oxygen rebound and a second O2 coupling to obtain verruculogen selectively over other possible products.

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The diversity of the reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM enzymes, the radical quenching step is thought to involve an electron transfer to or from an auxiliary 4Fe-4S cluster in or adjacent to the active site. However, experimental demonstration of such functions remains limited. As a representative member of this family, MoaA has one radical SAM cluster ([4Fe-4S]RS) and one auxiliary cluster ([4Fe-4S]AUX), and catalyzes a unique 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) in the molybdenum cofactor (Moco) biosynthesis. Here, we report a mechanistic investigation of the radical quenching step in MoaA, a chemically challenging reduction of 3',8-cyclo-GTP-N7 aminyl radical. We first determined the reduction potentials of [4Fe-4S]RS and [4Fe-4S]AUX as -510 mV and -455 mV, respectively, using a combination of protein film voltammogram (PFV) and electron paramagnetic resonance (EPR) spectroscopy. Subsequent Q-band EPR characterization of 5'-deoxyadenosine C4' radical (5'-dA-C4'•) trapped in the active site revealed isotropic exchange interaction (∼260 MHz) between 5'-dA-C4'• and [4Fe-4S]AUX1+, suggesting that [4Fe-4S]AUX is in the reduced (1+) state during the catalysis. Together with density functional theory (DFT) calculation, we propose that the aminyl radical reduction proceeds through a proton-coupled electron transfer (PCET), where [4Fe-4S]AUX serves as an electron donor and R17 residue acts as a proton donor. These results provide detailed mechanistic insights into the radical quenching step of radical SAM enzyme catalysis.

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Electronic cigarettes (ECs) are categorized into generations which differ in terms of design, aerosol production, and customizability. Current and former smokers prefer third-generation devices that satisfy tobacco cravings more effectively than older generations. Recent studies indicate that EC aerosols from first- and second-generation devices contain reactive carbonyls and free radicals and can cause in vitro cytotoxicity. Third-generation ECs have not been adequately studied. Further, previous studies have focused on cells from the respiratory tract, whereas those of the oral cavity, which is exposed to high levels of EC aerosols, have been understudied. We quantified the production of reactive carbonyls and free radicals by a third-generation EC and investigated the induction of cytotoxicity and oxidative stress in normal and cancerous human oral cell lines using a panel of eight commercial EC liquids. We found that EC aerosols produced using a new atomizer contained formaldehyde, acetaldehyde, and acrolein, but did not contain detectable levels of free radicals. We found that EC aerosols generated from only one of the eight liquids tested using a new atomizer induced cytotoxicity against two human oral cells in vitro. Treatment of oral cells with the cytotoxic EC aerosol caused a concomitant increase in intracellular oxidative stress. As atomizer age increased with repeated use of the same atomizer, carbonyl production, radical emissions, and cytotoxicity increased. Overall, our results suggest that third-generation ECs may cause adverse effects in the oral cavity and normal EC use, which involves repeated use of the same atomizer to generate aerosol, may enhance the potential toxic effects of third-generation ECs.

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Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical S-adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen Helicobacter pylori (Hp), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of Hp growth that exclusively targets MqnE. These data provide the foundation for designing effective Hp therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.

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[FeFe] hydrogenases are enzymes capable of producing and oxidizing H2 at staggering submillisecond time scales. A major limitation in applying these enzymes for industrial hydrogen production is their irreversible inactivation by oxygen. Recently, an [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) was reported to regain its catalytic activity after exposure to oxygen. In this report, we have determined that artificially matured CbHydA1 is indeed oxygen tolerant in the absence of reducing agents and sulfides by means of reaching an O2-protected state (Hinact). We were also able to generate the Hinact state anaerobically via both chemical and electrochemical oxidation. We use a combination of spectroscopy, electrochemistry, and density functional theory (DFT) to uncover intrinsic properties of the active center of CbHydA1, leading to its unprecedented oxygen tolerance. We have observed that reversible, low-potential oxidation of the active center leads to the protection against O2-induced degradation. The transition between the active oxidized state (Hox) and the Hinact state appears to proceed without any detectable intermediates. We found that the Hinact state is stable for more than 40 h in air, highlighting the remarkable resilience of CbHydA1 to oxygen. Using a combination of DFT and FTIR, we also provide a hypothesis for the chemical identity of the Hinact state. These results demonstrate that CbHydA1 has remarkable stability in the presence of oxygen, which will drive future efforts to engineer more robust catalysts for biofuel production.

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The enzyme MiaB catalyzes the attachment of a methylthio (-SCH3) group at the C2 position of N6-(isopentenyl)adenosine (i6A) in the final step of the biosynthesis of the hypermodified tRNA nucleotide 2-methythio-N6-(isopentenyl)adenosine (ms2i6A). MiaB belongs to the expanding subgroup of enzymes of the radical S-adenosylmethionine (SAM) superfamily that harbor one or more auxiliary [4Fe-4S] clusters in addition to the [4Fe-4S] cluster that all family members require for the reductive cleavage of SAM to afford the common 5'-deoxyadenosyl 5'-radical (5'-dA•) intermediate. While the role of the radical SAM cluster in generating the 5'-dA• is well understood, the detailed role of the auxiliary cluster, which is essential for MiaB catalysis, remains unclear. It has been proposed that the auxiliary cluster may serve as a coordination site for exogenously derived sulfur destined for attachment to the substrate or that the cluster itself provides the sulfur atom and is sacrificed during turnover. In this work, we report spectroscopic and biochemical evidence that the auxiliary [4Fe-4S]2+ cluster in Bacteroides thetaiotaomicron (Bt) MiaB is converted to a [3Fe-4S]0-like cluster during the methylation step of catalysis. Mössbauer characterization of the MiaB [3Fe-4S]0-like cluster revealed unusual spectroscopic properties compared to those of other well-characterized cuboidal [3Fe-4S]0 clusters. Specifically, the Fe sites of the mixed-valent moiety do not have identical Mössbauer parameters. Our results support a mechanism where the auxiliary [4Fe-4S] cluster is the direct sulfur source during catalysis.

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Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.

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Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.

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MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA• and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.

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All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.

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Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (2H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors 1H or 2H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors 2H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.

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A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.

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