Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para-aminobenzoate in Chlamydia trachomatis.

Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of para-aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen Chlamydia trachomatis. The pathway used by Chlamydiae for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from l-tyrosine. As a member of the emerging superfamily of heme oxygenase-like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is ∼14 Šfrom the dimetal site. We propose that this self-sacrificial reaction occurs through O2 activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the "substrate" Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in C. trachomatis, which will inform the design of novel therapeutics.

Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

NMR and Mössbauer Studies Reveal a Temperature-Dependent Switch from S = 1 to 2 in a Nonheme Oxoiron(IV) Complex with Faster C-H Bond Cleavage Rates.

S = 2 FeIV═O centers generated in the active sites of nonheme iron oxygenases cleave substrate C-H bonds at rates significantly faster than most known synthetic FeIV═O complexes. Unlike the majority of the latter, which are S = 1 complexes, [FeIV(O)(tris(2-quinolylmethyl)amine)(MeCN)]2+ (3) is a rare example of a synthetic S = 2 FeIV═O complex that cleaves C-H bonds 1000-fold faster than the related [FeIV(O)(tris(pyridyl-2-methyl)amine)(MeCN)]2+ complex (0). To rationalize this significant difference, a systematic comparison of properties has been carried out on 0 and 3 as well as related complexes 1 and 2 with mixed pyridine (Py)/quinoline (Q) ligation. Interestingly, 2 with a 2-Q-1-Py donor combination cleaves C-H bonds at 233 K with rates approaching those of 3, even though Mössbauer analysis reveals 2 to be S = 1 at 4 K. At 233 K however, 2 becomes S = 2, as shown by its 1H NMR spectrum. These results demonstrate a unique temperature-dependent spin-state transition from triplet to quintet in oxoiron(IV) chemistry that gives rise to the high C-H bond cleaving reactivity observed for 2.

New Frontiers in Nonheme Enzymatic Oxyferryl Species.

Non-heme mononuclear iron dependent (NHM-Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2-4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high-valent reactive iron intermediate, the S = 2 oxoferryl (Fe(IV)-oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM-Fe enzymes containing a 2-His-Fe or 2-His-1-carboxylate-Fe center. However, for 3-His/4-His-Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S = 1 Fe(IV) intermediate in a 3-His-Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon-sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S = 2 Fe(IV)-oxo intermediates, the discovery of the first S = 1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S = 2 and the S = 1 Fe(IV)-oxo species. Finally, we postulate the possible reasons to utilize an S = 1 Fe(IV) species in OvoA and their implications to other 3-His/4-His-Fe enzymes.

Reconstitution of the Final Steps in the Biosynthesis of Valanimycin Reveals the Origin of Its Characteristic Azoxy Moiety.

Valanimycin is an azoxy-containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456-hF10. While the biosynthesis of valanimycin has been partially characterized, how the azoxy group is constructed remains obscure. Herein, the membrane protein VlmO and the putative hydrazine synthetase ForJ from the formycin biosynthetic pathway are demonstrated to catalyze N-N bond formation converting O-(l-seryl)-isobutyl hydroxylamine into N-(isobutylamino)-l-serine. Subsequent installation of the azoxy group is shown to be catalyzed by the non-heme diiron enzyme VlmB in a reaction in which the N-N single bond in the VlmO/ForJ product is oxidized by four electrons to yield the azoxy group. The catalytic cycle of VlmB appears to begin with a resting µ-oxo diferric complex in VlmB, as supported by Mössbauer spectroscopy. This study also identifies N-(isobutylamino)-d-serine as an alternative substrate for VlmB leading to two azoxy regioisomers. The reactions catalyzed by the kinase VlmJ and the lyase VlmK during the final steps of valanimycin biosynthesis are established as well. The biosynthesis of valanimycin was thus fully reconstituted in vitro using the enzymes VlmO/ForJ, VlmB, VlmJ and VlmK. Importantly, the VlmB-catalyzed reaction represents the first example of enzyme-catalyzed azoxy formation and is expected to proceed by an atypical mechanism.

Formation of a Reactive [Mn(III)-O-Ce(IV)] Species and its Facile Equilibrium with Related Mn(IV)(OX) (X = Sc or H) Complexes.

Lewis acid-bound high valent Mn-oxo species are of great importance due to their relevance to photosystem II. Here, we report the synthesis of a unique [(BnTPEN)Mn(III)-O-Ce(IV)(NO3 )4 ]+ adduct (2) by the reaction of (BnTPEN)Mn(II) (1) with 4 eq. ceric ammonium nitrate. 2 has been characterized using UV/Vis, NMR, resonance Raman spectroscopy, as well as by mass spectrometry. Treatment of 2 with Sc(III)(OTf)3 results in the formation of (BnTPEN)Mn(IV)-O-Sc(III) (3), while HClO4 addition to 2 forms (BnTPEN)Mn(IV)-OH (4), reverting to 2 upon Ce(III)(NO3 )3 addition. 2 can also be prepared by the oxidation of 1 eq. Ce(III)(NO3 )3 with [(BnTPEN)Mn(IV)=O]2+ (5). In addition, the EPR spectroscopy revealed the elegant temperature-dependent equilibria between 2 and Mn(IV) species. The binding of redox-active Ce(IV) boosts electron transfer efficiency of 2 towards ferrocenes. Remarkably, the newly characterized Mn(III)-O-Ce(IV) species can carry out O-atom and H-atom transfer reactions.

Direct Activation of the C(sp3)-NH2 Bond of Primary Aliphatic Alkylamines by a High-Valent CoIII,IV2(µ-O)2 Diamond Core Complex.

Aliphatic alkylamines are abundant feedstock and versatile building blocks for many organic transformations. While remarkable progress has been made to construct C-N bonds on aliphatic and aromatic carbon centers, the activation and functionalization of C(sp3)-NH2 bonds in primary alkylamines remain a challenging process. In the present work, we discovered an unprecedented method to directly activate the C(sp3)-NH2 bond of primary alkylamines by a high-valent dinuclear CoIII,IV2(µ-O)2 diamond core complex. This reaction results in the installation of other functional groups such as halides and alkenes onto the α-carbon center concomitant with the 2-e- oxidation of the nitrogen atom on the amino group to form NH2OH. These results shed light on future development enabling versatile functionalization of primary alkylamines based on the dinuclear cobalt system. Moreover, our work suggests that a related high-valent copper-oxo intermediate is likely generated in the ammonia monooxygenase catalytic cycle to affect the oxidation of NH3 to NH2OH.

Mechanistic Studies of Aziridine Formation Catalyzed by Mononuclear Non-Heme Iron Enzymes.

Aziridines are compounds with a nitrogen-containing three-membered ring. When it is incorporated into natural products, the reactivity of the strained ring often drives the biological activities of aziridines. Despite its importance, the enzymes and biosynthetic strategies deployed to install this reactive moiety remain understudied. Herein, we report the use of in silico methods to identify enzymes with potential aziridine-installing (aziridinase) functionality. To validate candidates, we reconstitute enzymatic activity in vitro and demonstrate that an iron(IV)-oxo species initiates aziridine ring closure by the C-H bond cleavage. Furthermore, we divert the reaction pathway from aziridination to hydroxylation using mechanistic probes. This observation, isotope tracing experiments using H218O and 18O2, and quantitative product analysis, provide evidence for the polar capture of a carbocation species by the amine in the pathway to aziridine installation.

Seeing the cis-Dihydroxylating Intermediate: A Mononuclear Nonheme Iron-Peroxo Complex in cis-Dihydroxylation Reactions Modeling Rieske Dioxygenases.

The nature of reactive intermediates and the mechanism of the cis-dihydroxylation of arenes and olefins by Rieske dioxygenases and synthetic nonheme iron catalysts have been the topic of intense research over the past several decades. In this study, we report that a spectroscopically well characterized mononuclear nonheme iron(III)-peroxo complex reacts with olefins and naphthalene derivatives, yielding iron(III) cycloadducts that are isolated and characterized structurally and spectroscopically. Kinetics and product analysis reveal that the nonheme iron(III)-peroxo complex is a nucleophile that reacts with olefins and naphthalenes to yield cis-diol products. The present study reports the first example of the cis-dihydroxylation of substrates by a nonheme iron(III)-peroxo complex that yields cis-diol products.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

Mechanistic Analysis of Stereodivergent Nitroalkane Cyclopropanation Catalyzed by Nonheme Iron Enzymes.

BelL and HrmJ are α-ketoglutarate-dependent nonheme iron enzymes that catalyze the oxidative cyclization of 6-nitronorleucine, resulting in the formation of two diastereomeric 3-(2-nitrocyclopropyl)alanine (Ncpa) products containing trans-cyclopropane rings with (1'R,2'R) and (1'S,2'S) configurations, respectively. Herein, we investigate the catalytic mechanism and stereodivergency of the cyclopropanases. The results suggest that the nitroalkane moiety of the substrate is first deprotonated to produce the nitronate form. Spectroscopic analyses and biochemical assays with substrates and analogues indicate that an iron(IV)-oxo species abstracts proS-H from C4 to initiate intramolecular C-C bond formation. A hydroxylation intermediate is unlikely to be involved in the cyclopropanation reaction. Additionally, a genome mining approach is employed to discover new homologues that perform the cyclopropanation of 6-nitronorleucine to generate cis-configured Ncpa products with (1'R,2'S) or (1'S,2'R) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.

Enabling Valence Delocalization in Iron(III) Macrocyclic Complexes through Ring Unsaturation.

The complexes [FeIII(HMC)(C2DMA)2]CF3SO3 ([2]OTf) and [FeIII(HMTI)(C2Y)2]CF3SO3 ([3a-c]OTf) have been prepared and thoroughly characterized (HMC = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane; HMTI = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-1,3,8,10-tetraene; Y = Fc (ferrocenyl, [3a]OTf), 4-(N,N-dimethyl)anilino (DMA, [3b]OTf), or 4-(N,N-bis(4-methoxyphenyl)anilino (TPA, [3c]OTf); OTf- = CF3SO3-)). Vibrational and electronic absorption spectroelectrochemical analyses following one-electron oxidation of the ethynyl substituent Y revealed evidence of strong coupling in the resultant mixed valent species for all HMTI-based complexes. However, the analogous mixed valent ion based on [2]OTf appeared to be more localized. Thus, the tetra-imino macrocycle HMTI has enabled significant valence delocalization along the -C2-FeIII-C2- bridge. Electron paramagnetic resonance and Mössbauer spectroscopic studies of [3b]OTf reveal that the π-acidity of HMTI lowers the energy of the FeIII dπ orbitals compared to the purely σ-donating HMC. This observation provides a basis for the interpretation of the macrocycle-dependent valence (de)localization.

Synthesis, Spectroscopy, and Structure of [FeRu(µ-dithiolate)(CN)2(CO)4]2.

The salt [K(18-crown-6)]2[Ru(CN)2(CO)3] ([K(18-crown-6)]2[1]) was generated by the reaction of Ru(C2H4)(CO)4 with [K(18-crown-6)]CN. An initial thermal reaction gives [Ru(CN)(CO)4]-, which, upon ultraviolet (UV) irradiation, reacts with a second equiv of CN-. Protonation of [1]2- gave [HRu(CN)2(CO)3]- ([H1]-), which was isolated as a single isomer with mutually trans cyanide ligands. The complex cis,cis,cis-[Ru(pdt)(CN)2(CO)2]2- ([2]2-) was prepared by the UV-induced reaction of [1]2- with propanedithiol (pdtH2). The corresponding iron complex cis,cis,cis-[Fe(pdt)(CN)2(CO)2]2- ([3]2-) was prepared similarly. The pdt complexes [2]2- and [3]2- were treated with Fe(benzylideneacetone)(CO)3 to give, respectively, [RuFe (µ-pdt)(CN)2(CO)4]2- ([5]2-) and [Fe2(µ-pdt)(CN)2(CO)4]2- ([4]2-). The pathway from [3]2- to Fe2 complex [4]2- implicates intermetallic migration of CN-. In contrast, the formation of [5]2- leaves the Ru(CN)2(CO) center intact, as confirmed by X-ray crystallography. The structure of [5]2- features a "rotated" square-pyramidal Fe(CO)2(µ-CO) site. NMR measurements indicate that the octahedral Ru site is stereochemically rigid, whereas the Fe site dynamically undergoes turnstile rotation. 57Fe Mössbauer spectral parameters are very similar for rotated [5]2- and unrotated Fe2 complex [4]2-, indicating the insensitivity of that technique to both the geometry and the oxidation state of the Fe site. According to cyclic voltammetry, [5]2- oxidizes at E1/2 ∼ -0.8 V vs Fc+/0. Electron paramagnetic resonance (EPR) measurements show that 1e- oxidation of [5]2- gives an S = 1/2 rhombic species, consistent with the formulation Ru(II)Fe(I), related to the Hox state of the [FeFe] hydrogenases. Density functional theory (DFT) studies reproduce the structure, 1H NMR shifts, and infrared (IR) spectra observed for [5]2-. Related homometallic complexes with both cyanides on a single metal are predicted to not adopt rotated structures. These data suggest that [5]2- is best described as Ru(II)Fe(0). This conclusion raises the possibility that for some reduced states of the [FeFe]-hydrogenases, the [2Fe]H site may be better described as Fe(II)Fe(0) than Fe(I)Fe(I).

A Porphyrin Iron(III) π-Dication Species and its Relevance in Catalyst Design for the Umpolung of Nucleophiles.

Isoporphyrins have recently been identified as remarkable species capable of turning the nucleophile attached to the porphyrin ring into an electrophile, thereby providing umpolung of reactivity (Inorg. Chem. 2022, 61, 8105-8111). They are generated by nucleophilic attack on an iron(III) π-dication, a class of species that has received scant attention. Here, we explore the effect of the porphyrin meso-substituent and report a iron(III) π-dication bearing the meso-tetraphenylporphyrin (TPP) ligand. We provide an extensive study of the species by UV/Vis absorption, 2 H NMR, EPR, applied field Mössbauer, and resonance Raman spectroscopy. We further explore the system's highly dynamic and tunable properties and address the nature of the axial ligands as well as the conformation of the porphyrin ring. The insights presented are essential for the rational design of catalysts for the umpolung of nucleophiles. Such catalytic avenues could for example provide a novel method for electrophilic chlorinations. We further examine the importance of electronic tuning of the porphyrin by nature of the meso-substituent as a factor in catalyst design.

Repurposing Iron- and 2-Oxoglutarate-Dependent Oxygenases to Catalyze Olefin Hydration.

Mononuclear nonheme iron(II) and 2-oxoglutarate (Fe/2OG)-dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C-H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration-a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron-oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG-independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.

An S=1 Iron(IV) Intermediate Revealed in a Non-Heme Iron Enzyme-Catalyzed Oxidative C-S Bond Formation.

Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol-histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2 -dependent C-S bond formation catalyzed by non-heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four-histidine ligand environment (three from the protein residues and one from the substrate) in enabling C-S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non-heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C-S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure-function relationship of high-valent iron intermediates supported by a histidine rich ligand environment.

Mechanism of Methyldehydrofosmidomycin Maturation: Use Olefination to Enable Chain Elongation.

Utilization of mononuclear iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes to enable C-H bond functionalization is a widely used strategy to diversify the structural complexity of natural products. Besides those well-studied reactions including hydroxylation, epoxidation, and halogenation, in the biosynthetic pathway of dehydrofosmidomycin, an Fe/2OG enzyme is reported to catalyze desaturation, alkyl chain elongation, along with demethylation in which trimethyl-2-aminoethylphosphonate is converted into methyldehydrofosmidomycin. How this transformation takes place is largely unknown. Herein, we characterized the reactive species, revealed the structure of the reaction intermediate, and used mechanistic probes to investigate the reaction pathway and mechanism. These results led to the elucidation of a two-step process in which the first reaction employs a long-lived Fe(IV)-oxo species to trigger C═C bond installation. During the second reaction, the olefin installed in situ enables C-C bond formation that is accompanied with a C-N bond cleavage and hydroxylation to furnish the alkyl chain elongation and demethylation. This work expands the reaction repertoire of Fe/2OG enzymes by introducing a new pathway to the known C-C bond formation mechanisms utilized by metalloenzymes.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae.

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.

BesC Initiates C-C Cleavage through a Substrate-Triggered and Reactive Diferric-Peroxo Intermediate.

BesC catalyzes the iron- and O2-dependent cleavage of 4-chloro-l-lysine to form 4-chloro-l-allylglycine, formaldehyde, and ammonia. This process is a critical step for a biosynthetic pathway that generates a terminal alkyne amino acid which can be leveraged as a useful bio-orthogonal handle for protein labeling. As a member of an emerging family of diiron enzymes that are typified by their heme oxygenase-like fold and a very similar set of coordinating ligands, recently termed HDOs, BesC performs an unusual type of carbon-carbon cleavage reaction that is a significant departure from reactions catalyzed by canonical dinuclear-iron enzymes. Here, we show that BesC activates O2 in a substrate-gated manner to generate a diferric-peroxo intermediate. Examination of the reactivity of the peroxo intermediate with a series of lysine derivatives demonstrates that BesC initiates this unique reaction trajectory via cleavage of the C4-H bond; this process represents the rate-limiting step in a single turnover reaction. The observed reactivity of BesC represents the first example of a dinuclear-iron enzyme that utilizes a diferric-peroxo intermediate to capably cleave a C-H bond as part of its native function, thus circumventing the formation of a high-valent intermediate more commonly associated with substrate monooxygenations.