Threonine Aldolase-Catalyzed Enantioselective α-Alkylation of Amino Acids through Unconventional Photoinduced Radical Initiation.

Visible light-driven pyridoxal radical biocatalysis has emerged as a promising strategy for the stereoselective synthesis of valuable noncanonical amino acids (ncAAs). Previously, the use of well-tailored photoredox catalysts represented the key to enable efficient pyridoxal phosphate (PLP) enzyme-catalyzed radical reactions. Here, we report a PLP-dependent threonine aldolase-catalyzed asymmetric α-C-H alkylation of abundant amino acids using Katritzky pyridinium salts as alkylating agents. The use of engineered threonine aldolases allowed for this redox-neutral radical alkylation to proceed efficiently, giving rise to challenging α-trisubstituted and -tetrasubstituted ncAA products in a protecting-group-free fashion with excellent enantiocontrol. Mechanistically, this enantioselective α-alkylation capitalizes on the unique reactivity of the persistent enzymatic quinonoid intermediate derived from the PLP cofactor and the amino acid substrate to allow for novel radical C-C coupling. Surprisingly, this photobiocatalytic process does not require the use of well-established photoredox catalysts and operates through an unconventional photoinduced radical generation involving a PLP-derived aldimine. The ability to develop photobiocatalytic reactions without relying on classic photocatalysts or photoenzymes opens up new avenues for advancing stereoselective intermolecular radical reactions that are not known in either organic chemistry or enzymology.

Analysis of heaping-induced decline in olive quality: insights from integrated analysis between phenotypic traits and gene expression profiles.

Heaping is an unavoidable process before olive milling, and its duration significantly affects the olive quality. However, there is limited research on the quality changes of olive fruits on a short-time scale. To gain a better understanding of the molecular mechanisms underlying postharvest deterioration of olives, this study piled olives at room temperature and extracted oil at 0 h, 8 h, 24 h, 48 h and 72 h to analyze oil quality parameters. GC/LC-MS techniques were employed to investigate variations in metabolite contents. Concurrently, the transcriptional profiles of olives during heaping were examined. As piling time progressed, quality indicators declined, and stored fruit were categorized into three groups based on their quality characters: '0 h' belongs to the first category, '8 h' and '24 h' to the second category, and '48 h' and '72 h' to the third category. Metabolite changes were consistent with the expression patterns of genes related to their synthesis pathways. Additionally, ethylene was identified as a crucial factor influencing fruit senescence. These findings establish a foundation for further research on olive deterioration after harvesting and offer insights for optimizing olive oil production.

Panoply of P: An Array of Rhenium-Phosphorus Complexes Generated from a Transition Metal Anion.

We expand upon the synthetic utility of anionic rhenium complex Na[(BDI)ReCp] (1, BDI = N,N'-bis(2,6-diisopropylphenyl)-3,5-dimethyl-ß-diketiminate) to generate several rhenium-phosphorus complexes. Complex 1 reacts in a metathetical manner with chlorophosphines Ph2PCl, MeNHP-Cl, and OHP-Cl to generate XL-type phosphido complexes 2, 3, and 4, respectively (MeNHP-Cl = 2-chloro-1,3-dimethyl-1,3,2-diazaphospholidine; OHP-Cl = 2-chloro-1,3,2-dioxaphospholane). Crystallographic and computational investigations of phosphido triad 2, 3, and 4 reveal that increasing the electronegativity of the phosphorus substituent (C < N < O) results in a shortening and strengthening of the rhenium-phosphorus bond. Complex 1 reacts with iminophosphane Mes*NPCl (Mes* = 2,4,6-tritert-butylphenyl) to generate linear iminophosphanyl complex 5. In the presence of a suitable halide abstraction reagent, 1 reacts with the dichlorophosphine iPr2NPCl2 to afford cationic phosphinidene complex 6+. Complex 6+ may be reduced by one electron to form 6•, a rare example of a stable, paramagnetic phosphinidene complex. Spectroscopic and structural investigations, as well as computational analyses, are employed to elucidate the influence of the phosphorus substituent on the nature of the rhenium-phosphorus bond in 2 through 6. Furthermore, we examine several common analogies employed to understand metal phosphido, phosphinidene, and iminophosphanyl complexes.

The H-cluster of [FeFe] Hydrogenases: Its Enzymatic Synthesis and Parallel Inorganic Semisynthesis.

ConspectusNature's prototypical hydrogen-forming catalysts─hydrogenases─have attracted much attention because they catalyze hydrogen evolution at near zero overpotential and ambient conditions. Beyond any possible applications in the energy sphere, the hydrogenases feature complicated active sites, which implies novel biosynthetic pathways. In terms of the variety of cofactors, the [FeFe]-hydrogenase is among the most complex.For more than a decade, we have worked on the biosynthesis of the active site of [FeFe] hydrogenases. This site, the H-cluster, is a six-iron ensemble consisting of a [4Fe-4S]H cluster linked to a [2Fe]H cluster that is coordinated to CO, cyanide, and a unique organic azadithiolate ligand. Many years ago, three enzymes, namely, HydG, HydE, and HydF, were shown to be required for the biosynthesis and the in vitro maturation of [FeFe] hydrogenases. The structures of the maturases were determined crystallographically, but still little progress was made on the biosynthetic pathway. As described in this Account, the elucidation of the biosynthetic pathway began in earnest with the identification of a molecular iron-cysteinate complex produced within HydG.In this Account, we present our most recent progress toward the molecular mechanism of [2Fe]H biosynthesis using a collaborative approach involving cell-free biosynthesis, isotope and element-sensitive spectroscopies, as well as inorganic synthesis of purported biosynthetic intermediates. Our study starts from the radical SAM enzyme HydG that lyses tyrosine into CO and cyanide and forms an Fe(CO)2(CN)-containing species. Crystallographic identification of a unique auxiliary 5Fe-4S cluster in HydG leads to a proposed catalytic cycle in which a free cysteine-chelated "dangler" Fe serves as the platform for the stepwise formation of a [4Fe-4S][Fe(CO)(CN)(cysteinate)] intermediate, which releases the [Fe(CO)2(CN)(cysteinate)] product, Complex B. Since Complex B is unstable, we applied synthetic organometallic chemistry to make an analogue, syn-B, and showed that it fully replaces HydG in the in vitro maturation of the H-cluster. Syn-B serves as the substrate for the next radical SAM enzyme HydE, where the low-spin Fe(II) center is activated by 5'-dAdo• to form an adenosylated Fe(I) intermediate. We propose that this Fe(I) species strips the carbon backbone and dimerizes in HydE to form a [Fe2(SH)2(CO)4(CN)2]2- product. This mechanistic scenario is supported by the use of a synthetic version of this dimer complex, syn-dimer, which allows for the formation of active hydrogenase with only the HydF maturase. Further application of this semisynthesis strategy shows that an [Fe2(SCH2NH2)2(CO)4(CN)2]2- complex can activate the apo hydrogenase, marking it as the last biosynthetic intermediate en route to the H-cluster. This combined enzymatic and semisynthetic approach greatly accelerates our understanding of H-cluster biosynthesis. We anticipate additional mechanistic details regarding H-cluster biosynthesis to be gleaned, and this methodology may be further applied in the study of other complex metallocofactors.

Quantification of Free Radicals from Vaping Electronic Cigarettes Containing Nicotine Salt Solutions with Different Organic Acid Types and Concentrations.

Electronic (e-) cigarette formulations containing nicotine salts from a range of organic acid conjugates and pH values have dominated the commercial market. The acids in the nicotine salt formulations may alter the redox environment in e-cigarettes, impacting free radical formation in e-cigarette aerosol. Here, the generation of aerosol mass and free radicals from a fourth-generation e-cigarette device was evaluated at 2 wt % nicotine salts (pH 7, 30:70 mixture propylene glycol to vegetable glycerin) across eight organic acids used in e-liquids: benzoic acid (BA), salicylic acid (SLA), lactic acid (LA), levulinic acid (LVA), succinic acid (SA), malic acid (MA), tartaric acid (TA), and citric acid (CA). Furthermore, 2 wt % BA nicotine salts were studied at the following nicotine to acid ratios: 1:2 (pH 4), 1:1 (pH 7), and 2:1 (pH 8), in comparison with freebase nicotine (pH 10). Radical yields were quantified by spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. The EPR spectra of free radicals in the nicotine salt aerosol matched those generated from the Fenton reaction, which are primarily hydroxyl (OH) radicals and other reactive oxygen species (ROS). Although the aerosol mass formation was not significantly different for most of the tested nicotine salts and acid concentrations, notable ROS yields were observed only from BA, CA, and TA under the study conditions. The e-liquids with SLA, LA, LVA, SA, and MA produced less ROS than the 2 wt % freebase nicotine e-liquid, suggesting that organic acids may play dual roles in the production and scavenging of ROS. For BA nicotine salts, it was found that the ROS yield increased with a higher acid concentration (or a lower nicotine to acid ratio). The observation that BA nicotine salts produce the highest ROS yield in aerosol generated from a fourth-generation vape device, which increases with acid concentration, has important implications for ROS-mediated health outcomes that may be relevant to consumers, manufacturers, and regulatory agencies.

Functional differentiation of olive PLP_deC genes: insights into metabolite biosynthesis and genetic improvement at the whole-genome level.

KEY MESSAGE: This study identified 16 pyridoxal phosphate-dependent decarboxylases in olive at the whole-genome level, conducted analyses on their physicochemical properties, evolutionary relationships and characterized their activity. Group II pyridoxal phosphate-dependent decarboxylases (PLP_deC II) mediate the biosynthesis of characteristic olive metabolites, such as oleuropein and hydroxytyrosol. However, there have been no report on the functional differentiation of this gene family at the whole-genome level. This study conducted an exploration of the family members of PLP_deC II at the whole-genome level, identified 16 PLP_deC II genes, and analyzed their gene structure, physicochemical properties, cis-acting elements, phylogenetic evolution, and gene expression patterns. Prokaryotic expression and enzyme activity assays revealed that OeAAD2 and OeAAD4 could catalyze the decarboxylation reaction of tyrosine and dopa, resulting in the formation of their respective amine compounds, but it did not catalyze phenylalanine and tryptophan. Which is an important step in the synthetic pathway of hydroxytyrosol and oleuropein. This finding established the foundational data at the molecular level for studying the functional aspects of the olive PLP_deC II gene family and provided essential gene information for genetic improvement of olive.

Final Stages in the Biosynthesis of the [FeFe]-Hydrogenase Active Site.

The paper aims to elucidate the final stages in the biosynthesis of the [2Fe]H active site of the [FeFe]-hydrogenases. The recently hypothesized intermediate [Fe2(SCH2NH2)2(CN)2(CO)4]2- ([1]2-) was prepared by a multistep route from [Fe2(S2)(CN)(CO)5]-. The following synthetic intermediates were characterized in order: [Fe2(SCH2NHFmoc)2(CNBEt3)(CO)5]-, [Fe2(SCH2NHFmoc)2(CN)-(CO)5]-, and [Fe2(SCH2NHFmoc)2(CN)2(CO)4]2-, where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18-crown-6)]+, PPh4 + and PPN+ salts as well as the 13CD2-isotopologues. These Fe2 species exist as a mixture of two isomers attributed to diequatorial (ee) and axial-equatorial (ae) stereochemistry at sulfur. In vitro experiments demonstrate that [1]2- maturates HydA1 in the presence of HydF and a cocktail of reagents. HydA1 can also be maturated using a highly simplified cocktail, omitting HydF and other proteins. This result is consistent with HydA1 participating in the maturation process and refines the roles of HydF.

Bioinformatic Discovery of a Cambialistic Monooxygenase.

Dinuclear monooxygenases mediate challenging C-H bond oxidation reactions throughout nature. Many of these enzymes are presumed to exclusively utilize diiron cofactors. Herein we report the bioinformatic discovery of an orphan dinuclear monooxygenase that preferentially utilizes a heterobimetallic manganese-iron (Mn/Fe) cofactor to mediate an O2-dependent C-H bond hydroxylation reaction. Unlike the structurally similar Mn/Fe-dependent monooxygenase AibH2, the diiron form of this enzyme (SfbO) exhibits a nascent enzymatic activity. This behavior raises the possibility that many other dinuclear monooxygenases may be endowed with the capacity to harness cofactors with a variable metal content.

Characterizing the Biosynthesis of the [Fe(II)(CN)(CO)2(cysteinate)]- Organometallic Product of the Radical-SAM Enzyme HydG by EPR and Mössbauer Spectroscopy.

[FeFe]-hydrogenases employ a catalytic H-cluster, consisting of a [4Fe-4S]H cluster linked to a [2Fe]H subcluster with CO, CN- ligands, and an azadithiolate bridge, which mediates the rapid redox interconversion of H+ and H2. In the biosynthesis of this H-cluster active site, the radical S-adenosyl-l-methionine (radical SAM, RS) enzyme HydG plays the crucial role of generating an organometallic [Fe(II)(CN)(CO)2(cysteinate)]- product that is en route to forming the H-cluster. Here, we report direct observation of this diamagnetic organometallic Fe(II) complex through Mössbauer spectroscopy, revealing an isomer shift of δ = 0.10 mm s-1 and quadrupole splitting of ΔEQ = 0.66 mm s-1. These Mössbauer values are a change from the starting values of δ = 1.15 mm s-1 and ΔEQ = 3.23 mm s-1 for the ferrous "dangler" Fe in HydG. These values of the observed product complex B are in good agreement with Mössbauer parameters for the low-spin Fe2+ ions in synthetic analogues, such as 57Fe Syn-B, which we report here. These results highlight the essential role that HydG plays in converting a resting-state high-spin Fe(II) to a low-spin organometallic Fe(II) product that can be transferred to the downstream maturase enzymes.

Cross-coupling by a noncanonical mechanism involving the addition of aryl halide to Cu(II).

Copper complexes are widely used in the synthesis of fine chemicals and materials to catalyze couplings of heteroatom nucleophiles with aryl halides. We show that cross-couplings catalyzed by some of the most active catalysts occur by a mechanism not previously considered. Copper(II) [Cu(II)] complexes of oxalamide ligands catalyze Ullmann coupling to form the C-O bond in aryl ethers by concerted oxidative addition of an aryl halide to Cu(II) to form a high-valent species that is stabilized by radical character on the oxalamide ligand. This mechanism diverges from those involving Cu(I) and Cu(III) intermediates that have been posited for other Ullmann-type couplings. The stability of the Cu(II) state leads to high turnover numbers, >1000 for the coupling of phenoxide with aryl chloride electrophiles, as well as an ability to run the reactions in air.

Genome-Wide Analysis of the Polyphenol Oxidase Gene Family in Olea europaea Provides Insights into the Mechanism of Enzymatic Browning in Olive Fruit.

Browning of olive (Olea europaea L.) fruit reduces the sensory and nutritional qualities of olive oil, thereby increasing production costs. Polyphenol oxidases (PPOs) are the key enzymes that catalyze phenolic substance oxidation and mediate enzymatic browning in olive fruit, but the exact regulatory mechanism remains unclear. The main challenge is the lack of comprehensive information on OePPOs at the genome-wide level. In this study, 18 OePPO genes were identified. Subsequently, we performed a bioinformatic analysis on them. We also analyzed the expression patterns and determined the relationship among browning degree, PPO activity, and expression of OePPOs in the fruits of three olive varieties. Based on our analysis, we identified the four most conserved motifs. OePPOs were classified into two groups, with OePPOs from Group 1 showing only diphenolase activity and OePPOs from Group 2 exhibiting both mono-/diphenolase activities. Seven pairs of gene duplication events were identified, and purifying selection was found to have played a critical role in the evolution of the OePPO gene family. A positive correlation was observed between the browning degree of olive fruit and PPO activity across different olive varieties. Moreover, two important genes were found: OePPO-5 the main effector gene responsible for fruit browning, and OePPO-8, a key gene associated with specialized metabolite synthesis in the olive fruit. In short, our discoveries provide a basis for additional functional studies on OePPO genes and can help elucidate the mechanism of enzymatic browning in olive fruit in the future.

Fully Refined Semisynthesis of the [FeFe] Hydrogenase H-Cluster.

[FeFe] hydrogenases contain a 6-Fe cofactor that serves as the active site for efficient redox interconversion between H2 and protons. The biosynthesis of the so-called H-cluster involves unusual enzymatic reactions that synthesize organometallic Fe complexes containing azadithiolate, CO, and CN- ligands. We have previously demonstrated that specific synthetic [Fe(CO)x(CN)y] complexes can be used to functionally replace proposed Fe intermediates in the maturation reaction. Here, we report the results from performing such cluster semisynthesis in the context of a recent fully defined cluster maturation procedure, which eliminates unknown components previously employed from Escherichia coli cell lysate and demonstrate this provides a concise route to H-cluster synthesis. We show that formaldehyde can be used as a simple reagent as the carbon source of the bridging adt ligand of H-cluster in lieu of serine/serine hydroxymethyltransferase. In addition to the actual H-cluster, we observe the formation of several H-cluster-like species, the identities of which are probed by cryogenic photolysis combined with EPR/ENDOR spectroscopy.

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor.

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

A Bifunctional Ionic Liquid for Capture and Electrochemical Conversion of CO2 to CO over Silver.

Electrochemical conversion of CO2 requires selective catalysts and high solubility of CO2 in the electrolyte to reduce the energy requirement and increase the current efficiency. In this study, the CO2 reduction reaction (CO2RR) over Ag electrodes in acetonitrile-based electrolytes containing 0.1 M [EMIM][2-CNpyr] (1-ethyl-3-methylimidazolium 2-cyanopyrolide), a reactive ionic liquid (IL), is shown to selectively (>94%) convert CO2 to CO with a stable current density (6 mA·cm-2) for at least 12 h. The linear sweep voltammetry experiments show the onset potential of CO2 reduction in acetonitrile shifts positively by 240 mV when [EMIM][2-CNpyr] is added. This is attributed to the pre-activation of CO2 through the carboxylate formation via the carbene intermediate of the [EMIM]+ cation and the carbamate formation via binding to the nucleophilic [2-CNpyr]- anion. The analysis of the electrode-electrolyte interface by surface-enhanced Raman spectroscopy (SERS) confirms the catalytic role of the functionalized IL where the accumulation of the IL-CO2 adduct between -1.7 and -2.3 V vs Ag/Ag+ and the simultaneous CO formation are captured. This study reveals the electrode surface species and the role of the functionalized ions in lowering the energy requirement of CO2RR for the design of multifunctional electrolytes for the integrated capture and conversion.

An Artifact of Perfluoroalkyl Acid (PFAA) Removal Attributed to Sorption Processes in a Laccase Mediator System.

Fungi and laccase mediator systems (LMSs) have a proven track record of oxidizing recalcitrant organic compounds. There has been considerable interest in applying LMSs to the treatment of perfluoroalkyl acids (PFAAs), a class of ubiquitous and persistent environmental contaminants. Some laboratory experiments have indicated modest losses of PFAAs over extended periods, but there have been no clear demonstrations of a transformation mechanism or the kinetics that would be needed for remediation applications. We set out to determine if this was a question of identifying and optimizing a rate-limiting step but discovered that observed losses of PFAAs were experimental artifacts. While unable to replicate the oxidation of PFAAs, we show that interactions of the PFAA compounds with laccase and laccase mediator mixtures could cause an artifact that mimics transformation (≲60%) of PFAAs. Furthermore, we employed a surrogate compound, carbamazepine (CBZ), and electron paramagnetic resonance spectroscopy to probe the formation of the radical species that had been proposed to be responsible for contaminant oxidation. We confirmed that under conditions where sufficient radical concentrations were produced to oxidize CBZ, no PFAA removal took place.

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor.

Manganese cofactors activate strong chemical bonds in many essential enzymes. Yet very few manganese-dependent enzymes are known to functionalize ubiquitous carbon-hydrogen (C-H) bonds, and those that catalyze this important reaction display limited intrinsic reactivity. Herein, we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis requires manganese to functionalize a C-H bond possessing a bond dissociation enthalpy (BDE) exceeding 100 kcal/mol. Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site that utilizes a manganese ion at the locus for O 2 activation and substrate coordination. Accordingly, this enzyme represents the first documented Mn-dependent monooxygenase in biology. Related proteins are widespread in microorganisms suggesting that many uncharacterized monooxygenases may utilize manganese-containing cofactors to accomplish diverse biological tasks.

Tryptophan Can Promote Oxygen Reduction to Water in a Biosynthetic Model of Heme Copper Oxidases.

Heme-copper oxidases (HCOs) utilize tyrosine (Tyr) to donate one of the four electrons required for the reduction of O2 to water in biological respiration, while tryptophan (Trp) is speculated to fulfill the same role in cyt bd oxidases. We previously engineered myoglobin into a biosynthetic model of HCOs and demonstrated the critical role that Tyr serves in the oxygen reduction reaction (ORR). To address the roles of Tyr and Trp in these oxidases, we herein report the preparation of the same biosynthetic model with the Tyr replaced by Trp and further demonstrate that Trp can also promote the ORR, albeit with lower activity. An X-ray crystal structure of the Trp variant shows a hydrogen-bonding network involving two water molecules that are organized by Trp, similar to that in the Tyr variant, which is absent in the crystal structure with the native Phe residue. Additional electron paramagnetic resonance measurements are consistent with the formation of a Trp radical species upon reacting with H2O2. We attribute the lower activity of the Trp variant to Trp's higher reduction potential relative to Tyr. Together, these findings demonstrate, for the first time, that Trp can indeed promote the ORR and provides a structural basis for the observation of varying activities. The results support a redox role for the conserved Trp in bd oxidase while suggesting that HCOs use Tyr instead of Trp to achieve higher reactivity.

Proposed Mechanism for the Biosynthesis of the [FeFe] Hydrogenase H-Cluster: Central Roles for the Radical SAM Enzymes HydG and HydE.

Radical S-adenosylmethionine (radical SAM or rSAM) enzymes use their S-adenosylmethionine cofactor bound to a unique Fe of a [4Fe-4S] cluster to generate the "hot" 5'-deoxyadenosyl radical, which drives highly selective radical reactions via specific interactions with a given rSAM enzyme's substrate. This Perspective focuses on the two rSAM enzymes involved in the biosynthesis of the organometallic H-cluster of [FeFe] hydrogenases. We present here a detailed sequential model initiated by HydG, which lyses a tyrosine substrate via a 5'-deoxyadenosyl H atom abstraction from those amino acid's amino group, initially producing dehydroglycine and an oxidobenzyl radical. In this model, two successive radical cascade reactions lead ultimately to the formation of HydG's product, a mononuclear Fe organometallic complex: [Fe(II)(CN)(CO)2(cysteinate)]-, with the iron originating from a unique "dangler" Fe coordinated by a cysteine ligand providing a sulfur bridge to another [4Fe-4S] auxiliary cluster in the enzyme. In turn, in this model, [Fe(II)(CN)(CO)2(cysteinate)]- is the substrate for HydE, the second rSAM enzyme in the biosynthetic pathway, which activates this mononuclear organometallic unit for dimerization, forming a [Fe2S2(CO)4(CN)2] precursor to the [2Fe] H component of the H-cluster, requiring only the completion of the bridging azadithiolate (SCH2NHCH2S) ligand. This model is built upon a foundation of data that incorporates cell-free synthesis, isotope sensitive spectroscopies, and the selective use of synthetic complexes substituting for intermediates in the enzymatic "assembly line". We discuss controversies pertaining to this model and some remaining open issues to be addressed by future work.

Accumulation and Pulse Electron Paramagnetic Resonance Spectroscopic Investigation of the 4-Oxidobenzyl Radical Generated in the Radical S-Adenosyl-l-methionine Enzyme HydG.

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 Zn2+ 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 1H/13C 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.

Quantum Chemical Study of a Radical Relay Mechanism for the HydG-Catalyzed Synthesis of a Fe(II)(CO)2(CN)cysteine Precursor to the H-Cluster of [FeFe] Hydrogenase.

The [FeFe] hydrogenase catalyzes the redox interconversion of protons and H2 with a Fe-S "H-cluster" employing CO, CN, and azadithiolate ligands to two Fe centers. The biosynthesis of the H-cluster is a highly interesting reaction carried out by a set of Fe-S maturase enzymes called HydE, HydF, and HydG. HydG, a member of the radical S-adenosylmethionine (rSAM) family, converts tyrosine, cysteine, and Fe(II) into an organometallic Fe(II)(CO)2(CN)cysteine "synthon", which serves as the substrate for HydE. Although key aspects of the HydG mechanism have been experimentally determined via isotope-sensitive spectroscopic methods, other important mechanistic questions have eluded experimental determination. Here, we use computational quantum chemistry to refine the mechanism of the HydG catalytic reaction. We utilize quantum mechanics/molecular mechanics simulations to investigate the reactions at the canonical Fe-S cluster, where a radical cleavage of the tyrosine substrate takes place and proceeds through a relay of radical intermediates to form HCN and a COO•- radical anion. We then carry out a broken-symmetry density functional theory study of the reactions at the unusual five-iron auxiliary Fe-S cluster, where two equivalents of CN- and COOH• coordinate to the fifth "dangler iron" in a series of substitution and redox reactions that yield the synthon as the final product for further processing by HydE.