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The hydroxylase component (S5HH) of salicylate-5-hydroxylase catalyzes C5 ring hydroxylation of salicylate but switches to methyl hydroxylation when a C5 methyl substituent is present. The use of

Human histidine triad nucleotide binding (hHINT) proteins catalyze nucleotide phosphoramidase and acyl-phosphatase reactions that are essential for the activation of antiviral proTides, such as Sofosbuvir and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic surface residues (Q62, E100) which gate a conserved water channel leading to the active site 13 Å away. hHINT2 crystal structures identify analogous residues (R99, D137) and water channel. hHINT1 Q62 variants significantly alter the steady-state k(cat) and K(m) for turnover of the fluorescent substrate (TpAd), while stopped-flow kinetics indicate the K(D) also changes. hHINT2, like hHINT1, exhibits a burst-phase of adenylation, monitored by fluorescent tryptamine release, prior to rate-limiting hydrolysis and nucleotide release. hHINT2 exhibits a much smaller burst-phase amplitude than hHINT1, which is further diminished in hHINT2 R99Q. Kinetic simulations suggest that amplitude variations can be accounted for by a variable fluorescent yield of the E•S complex from changes in the environment of bound TpAd. Isothermal titration calorimetry measurements of inhibitor binding shows that these hHINT variants also alter the thermodynamic binding profile. We propose that these altered surface residues engender long-range dynamic changes that affect the orientation of bound ligands, altering the thermodynamic and kinetic characteristics of hHINT active site function. Thus, studies of the cellular roles and proTide activation potential by hHINTs should consider the importance of long-range interactions and possible protein binding surfaces far from the active site.

Methanotrophic bacteria utilize the nonheme diiron enzyme soluble methane monooxygenase (sMMO) to convert methane to methanol in the first step of their metabolic cycle under copper-limiting conditions. The structure of the sMMO Fe(IV)2 intermediate Q responsible for activating the inert C-H bond of methane (BDE = 104 kcal/mol) remains controversial, with recent studies suggesting both "open" and "closed" core geometries for its active site. In this study, we employ nuclear resonance vibrational spectroscopy (NRVS) to probe the geometric and electronic structure of intermediate Q at cryogenic temperatures. These data demonstrate that Q decays rapidly during the NRVS experiment. Combining data from several years of measurements, we derive the NRVS vibrational features of intermediate Q as well as its cryoreduced decay product. A library of 90 open and closed core models of intermediate Q is generated using density functional theory to analyze the NRVS data of Q and its cryoreduced product as well as prior spectroscopic data on Q. Our analysis reveals that a subset of closed core models reproduce these newly acquired NRVS data as well as prior data. The reaction coordinate with methane is also evaluated using both closed and open core models of Q. These studies show that the potent reactivity of Q toward methane resides in the "spectator oxo" of its Fe(IV)2O2 core, in contrast to nonheme mononuclear Fe(IV)═O enzyme intermediates that H atoms abstract from weaker C-H bonds.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 μM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.

Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme FeIV2 intermediate MMOHQ, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent FeIV sites in catalysis; however, MMOHQ is unique as Nature’s only identified FeIV2 intermediate. Previous 57Fe Mössbauer spectroscopic studies have shown that MMOHQ employs antiferromagnetic coupling of the two FeIV sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (Sloc) of each of the irons by most spectroscopic techniques. Here, we use Fe Kβ X-ray emission spectroscopy (XES) to characterize the local spin states of the key intermediates of the sMMO catalytic cycle, including MMOHQ trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOHQ is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOHQ spectrum with those of known Sloc = 1 and Sloc = 2 FeIV sites in chemical and biological models reveal that MMOHQ possesses Sloc = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis. Graphical abstract

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high-resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O2 access routes enhanced when the complex forms. Previously reported, lower-resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O2 conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O2 conduits in the M. trichosporium OB3b sMMOH:MMOB complex revealed a narrow molecular tunnel, termed the W308-tunnel. This tunnel is sized appropriately for O2 and traverses the sMMOH-MMOB interface before accessing the active site. The kinetics of reaction of O2 with the diferrous sMMOH:MMOB complex in solution show that use of the MMOB V41R variant decreases the rate constant for O2 binding >25000-fold without altering the component affinity. The location of Val41 near the entrance to the W308-tunnel is consistent with the tunnel serving as the primary route for the transfer of O2 into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-tunnel.

Methanotrophic bacteria utilize the non-heme diiron enzyme soluble methane monooxygenase (sMMO) to convert methane to methanol in the first step of their metabolic cycle under copper-limiting conditions. The structure of the sMMO Fe(IV)(2) intermediate Q responsible for activating the inert C-H bond of methane (BDE = 104 kcal/mol) remains controversial, with recent studies suggesting both “open” and “closed” core geometries for its active site. In this study, we employ nuclear resonance vibrational spectroscopy (NRVS) to probe the geometric and electronic structure of intermediate Q at cryogenic temperatures. These data demonstrate that Q decays rapidly during the NRVS experiment. Combining data from several years of measurements, we derive the NRVS vibrational features of intermediate Q as well as its cryoreduced decay product. A library of 90 open and closed core models of intermediate Q is generated using density functional theory (DFT) to analyze the NRVS data of Q and its cryoreduced product, as well as prior spectroscopic data on Q. Our analysis reveals that a subset of closed core models reproduce these newly acquired NRVS data as well as prior data. The reaction coordinate with methane is also evaluated using both closed and open core models of Q. These studies show that the potent reactivity of Q towards methane resides in the “spectator oxo” of its Fe(IV)(2)O(2) core, in contrast to non-heme mononuclear Fe(IV)=O enzyme intermediates that H-atom abstract from weaker C-H bonds.

Aerobic life is possible because the molecular structure of oxygen (O2) makes direct reaction with most organic materials at ambient temperatures an exceptionally slow process. Of course, these reactions are inherently very favorable, and they occur rapidly with the release of a great deal of energy at high temperature. Nature has been able to tap this sequestered reservoir of energy with great spatial and temporal selectivity at ambient temperatures through the evolution of oxidase and oxygenase enzymes. One mechanism used by these enzymes for O2activation has been studied in detail for the soluble form of the enzyme methane monooxygenase. These studies have revealed the step-by-step process of O2activation and insertion into the ultimately stable C–H bond of methane. Additionally, an elegant regulatory mechanism has been defined that enlists size selection and quantum tunneling to allow methane oxidation to occur specifically in the presence of more easily oxidized substrates.

Rigorous substrate selectivity is a hallmark of enzyme catalysis. This selectivity is generally ascribed to a thermodynamically favorable process of substrate binding to the enzyme active site based upon complementary physiochemical characteristics, which allows both acquisition and orientation. However, this chemical selectivity is more difficult to rationalize for diminutive molecules that possess too narrow a range of physical characteristics to allow either precise positioning or discrimination between a substrate and an inhibitor. Foremost among these small molecules are the dissolved gases such as H(2), N(2), O(2), CO, CO(2), NO, N(2)O, NH(3) and CH(4) so often encountered in metalloenzyme catalysis. Nevertheless, metalloenzymes have evolved to metabolize these small molecule substrates with high selectivity and efficiency. The soluble methane monooxygenase enzyme (sMMO) acts upon two of these small molecules, O(2) and CH(4) to generate methanol as part of the C1 metabolic pathway of methanotrophic organisms. sMMO is capable of oxidizing many alternative hydrocarbon substrates. Yet remarkably, it will preferentially oxidize methane, the substrate with the fewest discriminating physical characteristics and the strongest C-H bond. Early studies led us to broadly attribute this specificity to formation of a ‘molecular sieve’ in which a methane- and oxygen-sized tunnel provides a size-selective route from bulk solvent to the completely buried sMMO active site. Indeed, recent cryogenic and serial femtosecond ambient temperature crystallographic studies have revealed such a route in sMMO. A detailed study of the sMMO tunnel considered here in the context of small molecule tunnels identified in other metalloenzymes, reveals three discrete characteristics that contribute to substrate selectivity and positioning beyond that which can be provided by the active site itself. Moreover, the dynamic nature of many tunnels allows exquisite coordination of substrate binding and reaction phases of the catalytic cycle. Here we differentiate between the highly selective molecular tunnel, which only allows one-dimensional transit of small molecules, and the larger, less selective, channels found in typical enzymes. Methods are described to identify and characterize tunnels as well as to differentiate them from channels. In metalloenzymes which metabolize dissolved gases, we posit that the contribution of tunnels is so great that they should be considered as extensions of the active site itself. A full understanding of catalysis by these enzymes requires an appreciation for the roles played by tunnels. Such understanding will also facilitate the use of the enzymes or their synthetic mimics in industrial or pharmaceutical applications.

Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O(6)-POB-G) and O(6)-methylguanine (O(6)-Me-G) adducts in DNA. These adducts can be directly repaired by O(6)-alkylguanine-DNA alkyltransferase (AGT). Repair begins by flipping the damaged base out of the DNA helix. AGT binding and base-flipping have been previously studied using pyrrolocytosine as a fluorescent probe paired to the O(6)-alkyl-guanine lesion, but low fluorescence yield limited the resolution of steps in the repair process. Here, we utilize the highly fluorescent 6-phenylpyrrolo-2’-deoxycytidine (6-phenylpyrrolo-C) to investigate AGT-DNA interactions. Synthetic oligodeoxynucleotide duplexes containing O(6)-POB-G and O(6)-Me-G adducts were placed within the CpG sites of codons 158, 245, and 248 of the p53 tumor suppressor gene and base-paired to 6-phenylpyrrolo-C in the opposite strand. Neighboring cytosine was either unmethylated or methylated. Stopped-flow fluorescence measurements were performed by mixing the DNA duplexes with C145A or R128G AGT variants. We observe a rapid, two-step, nearly irreversible binding of AGT to DNA followed by two slower steps, one of which is base-flipping. Placing 5-methylcytosine immediately 5’ to the alkylated guanosine causes a reduction in rate constant of nucleotide flipping. O(6)-POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O(6)-Me-G at the same position. A similar effect was not observed at other codons.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O(2) activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the [35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O(2), protons, and CH(4) to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O(2) access routes enhanced when the complex forms. Previously reported, lower resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O(2) conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O(2) conduits in Methylosinus trichosporium OB3b sMMOH:MMOB revealed a narrow molecular tunnel, termed the W308-Tunnel. This tunnel is sized appropriately for O(2) and traverses the sMMOH-MMOB interface before accessing the active site. Kinetics of O(2) reaction with diferrous sMMOH:MMOB in solution show that use of the MMOB V41R variant decreases the rate constant for O(2) binding >25,000-fold without altering component affinity. The location of Val41 near the entrance to the W308-Tunnel is consistent with the tunnel serving as the primary route for O(2) transfer into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-Tunnel.

Kα High-Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy (HERFD XAS) provides a powerful tool for overcoming the limitations of conventional XAS to identify the electronic structure and coordination environment of metalloprotein active sites. Herein, Fe Kα HERFD-XAS is applied to the diiron active site of soluble Methane Monooxygenase (sMMO) and to a series of high-valent diiron model complexes, including a “diamond core” [FeIV2(μ-O)2(L)2](ClO4)4] (3) and an “open core” [(O=FeIV–O–FeIV(OH)(L)2](ClO4)3 (4) (where, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) (TPA*)). Pronounced differences in the HERFD XAS pre-edge energies and intensities are observed for the open vs. closed Fe2O2 cores in the model compounds. These differences are reproduced by time-dependent density functional theory (TDDFT) calculations and allow for the pre-edge energies and intensity to be directly correlated with the local active site geometric and electronic structure. A comparison of the model complex HERFD XAS data to that of MMOHQ (the key intermediate in methane oxidation) is supportive of an open core structure. Specifically, the large pre-edge area observed for MMOHQ may be rationalized by invoking an open core structure with a terminal FeIV=O motif, though further modulations of the core structure due to the protein environment cannot be ruled out. The present study, thus, motivates the need for additional experimental and theoretical studies to unambiguously assess the active site conformation of MMOHQ.

CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of chloramphenicol (CAM). The active site of CmlI contains a (hydr)oxo- and carboxylate-bridged dinuclear iron cluster. During catalysis, a novel diferric-peroxo intermediate P is formed and is thought to directly effect oxygenase chemistry. Peroxo intermediates can facilitate at most two-electron oxidations, so the biosynthetic pathway of CmlI must involve at least three steps. Here, kinetic techniques are used to characterize the rate and/or dissociation constants for each step by taking advantage of the remarkable stability of P in the absence of substrates (decay t1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlIred) can react with NH2-CAM and O2 in either order to form a P-NH2-CAM intermediate. P-NH2-CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlIox) complex with the aryl-hydroxylamine (NH(OH)-CAM) pathway intermediate. CmlIox-NH(OH)-CAM undergoes a rapid internal redox reaction to form CmlIred -nitroso-CAM (NO-CAM) complex. O2 binding results in formation of P-NO-CAM that converts to CmlIox-CAM by enzyme-mediated oxygen atom transfer. The kinetic analysis indicates that there is little dissociation of pathway intermediates as the reaction progresses. Reactions initiated by adding pathway intermediates from solution occur much more slowly than those in which the intermediate is generated in the active site as part of the catalytic process. Thus, CmlI is able to preserve efficiency and specificity while avoiding adventitious chemistry by performing the entire six-electron oxidation in one active site.

The final step in the biosynthesis of the antibiotic chloramphenicol is the oxidation of an aryl-amine substrate to an aryl-nitro product catalyzed by the N-oxygenase CmlI in three two-electron steps. The CmlI active site contains a diiron cluster ligated by three histidine and four glutamate residues, and activates dioxygen to perform its role in the biosynthetic pathway. It was previously shown that the active oxidant used by CmlI to facilitate this chemistry is a peroxo-diferric intermediate (CmlIP). Spectroscopic characterization demonstrated that the peroxo binding geometry of CmlIP is not consistent with the μ-1,2 mode commonly observed in nonheme diiron systems. Its geometry was tentatively assigned as μ- η2: η1 based on comparison with resonance Raman (rR) features of mixed-metal model complexes in the absence of appropriate diiron models. Here, X-ray absorption spectroscopy (XAS) and rR studies have been used to establish a refined structure for the diferric cluster of CmlIP. The rR experiments carried out with isotopically labeled water identified the symmetric and asymmetric vibrations of an Fe–O–Fe unit in the active site at 485 and 780 cm−1, respectively, which was confirmed by the 1.83-Å Fe–O bond observed by XAS. In addition, a unique Fe•••O scatterer at 2.82 Å observed from XAS analysis is assigned as arising from the distal O atom of a μ-1,1-peroxo ligand that is bound symmetrically between the irons. The (μ-oxo)(μ-1,1-peroxo)diferric core structure associated with CmlIP is unprecedented among diiron cluster-containing enzymes and corresponding biomimetic complexes. Importantly, it allows the peroxo-diferric intermediate to be ambiphilic, acting as an electrophilic oxidant in the initial N-hydroxylation of an arylamine and then becoming a nucleophilic oxidant in the final oxidation of an aryl-nitroso intermediate to the aryl-nitro product.

Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min–1).

Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. Here, the reaction of a Rieske monooxygenase, salicylate 5-hydroxylase, that does not form a cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding of salicylate and O2. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over ∼12 A and must also be very fast. However, the observed rate constant for this reaction is much slower than expected and sensitive to substrate type. This suggests that initial reaction with salicylate occurs using the same Fe(III)-superoxo-level intermediate as Rieske dioxygenases and that this reaction limits the observed rate of electron transfer. A transient intermediate (λmax = 700 nm) with an electron paramagnetic resonance (EPR) at g = 4.3 is observed after the product is formed in the active site. The use of 17O2 (I = 5/2) results in hyperfine broadening of the g = 4.3 signal, showing that gentisate binds to the mononuclear iron via its C5-OH in the intermediate. The chromophore and EPR signal allow study of product release in the catalytic cycle. Comparison of the kinetics of single- and multiple-turnover reactions shows that re-reduction of the metal centers accelerates product release ∼300-fold, providing insight into the regulatory mechanism of ROs.

The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the second-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here, the high-spin Fe(II) states of resting H200C and the H200C-homoprotocatechuate enzyme-substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin Fe(II) center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ - 8 cm(-1), E/D ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the (57)Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 Fe(II) system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation, which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well.

The diiron cluster-containing oxygenase CmlI catalyzes the conversion of the aromatic amine precursor of chloramphenicol to the nitroaromatic moiety of the active antibiotic. The X-ray crystal structures of the fully active, N-terminally truncated CmlIΔ33 in the chemically reduced Fe(2+)/Fe(2+) state and a cis μ-1,2(η (1):η (1))-peroxo complex are presented. These structures allow comparison with the homologous arylamine oxygenase AurF as well as other types of diiron cluster-containing oxygenases. The structural model of CmlIΔ33 crystallized at pH 6.8 lacks the oxo-bridge apparent from the enzyme optical spectrum in solution at higher pH. In its place, residue E236 forms a μ-1,3(η (1):η (2)) bridge between the irons in both models. This orientation of E236 stabilizes a helical region near the cluster which closes the active site to substrate binding in contrast to the open site found for AurF. A very similar closed structure was observed for the inactive dimanganese form of AurF. The observation of this same structure in different arylamine oxygenases may indicate that there are two structural states that are involved in regulation of the catalytic cycle. Both the structural studies and single crystal optical spectra indicate that the observed cis μ-1,2(η (1):η (1))-peroxo complex differs from the μ-η (1):η (2)-peroxo proposed from spectroscopic studies of a reactive intermediate formed in solution by addition of O2 to diferrous CmlI. It is proposed that the structural changes required to open the active site also drive conversion of the µ-1,2-peroxo species to the reactive form.

Despite decades of intense research, the core structure of the methane C-H bond breaking diiron(IV) intermediate, Q, of soluble methane monooxygenase remains controversial, with conflicting reports supporting either a "diamond" diiron core structure or an open core structure. Early extended X-ray absorption fine structure (EXAFS) data assigned a short 2.46 Å Fe-Fe distance to Q (Shu et al. Science 1997, 275, 515 ) that is inconsistent with several theoretical studies and in conflict with our recent high-resolution Fe K-edge X-ray absorption spectroscopy (XAS) studies (Castillo et al. J. Am. Chem. Soc. 2017, 139, 18024 ). Herein, we revisit the EXAFS of Q using high-energy resolution fluorescence-detected extended X-ray absorption fine structure (HERFD-EXAFS) studies. The present data show no evidence for a short Fe-Fe distance, but rather a long 3.4 Å diiron distance, as observed in open core synthetic model complexes. The previously reported 2.46 Å feature plausibly arises from a background metallic iron contribution from the experimental setup, which is eliminated in HERFD-EXAFS due to the increased selectivity. Herein, we explore the origin of the short diiron feature in partial-fluorescent yield EXAFS measurements and discuss the diagnostic features of background metallic scattering contribution to the EXAFS of dilute biological samples. Lastly, differences in sample preparation and resultant sample inhomogeneity in rapid-freeze quenched samples for EXAFS analysis are discussed. The presented approaches have broad implications for EXAFS studies of all dilute iron-containing samples.

Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes β-hydroxylation of L-p-aminophenylalanine (L-PAPA) covalently linked to the nonribosomal peptide synthetase (NRPS) CmlP, thereby effecting the first step in the biosynthesis of chloramphenicol by Streptomyces venezuelae. CmlA houses the diiron cluster in a metallo-β-lactamase protein fold instead of the 4-helix bundle fold of nearly every other diiron monooxygenase. CmlA couples O2 activation and substrate hydroxylation via a structural change caused by formation of the L-PAPA-loaded CmlP:CmlA complex. The other new diiron family is typified by two enzymes, AurF and CmlI, which catalyze conversion of aryl-amine substrates to aryl-nitro products with incorporation of oxygen from O2. AurF from Streptomyces thioluteus catalyzes the formation of p-nitrobenzoate from p-aminobenzoate as a precursor to the biostatic compound aureothin, whereas CmlI from S. venezuelae catalyzes the ultimate aryl-amine to aryl-nitro step in chloramphenicol biosynthesis. Both enzymes stabilize a novel type of peroxo-intermediate as the reactive species. The rare 6-electron N-oxygenation reactions of CmlI and AurF involve two progressively oxidized pathway intermediates. The enzymes optimize efficiency by utilizing one of the reaction pathway intermediates as an in situ reductant for the diiron cluster, while simultaneously generating the next pathway intermediate. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis. CmlI ensures specificity by carrying out the multistep aryl-amine oxygenation without dissociating intermediate products.

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin FeIII–(hydro)-peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of FeIII–superoxo and FeIII–hydroperoxo species in nonheme Fe biochemistry.

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mossbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mossbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.

Rieske dearomatizing dioxygenases utilize a Rieske iron-sulfur cluster and a mononuclear Fe(II) located 15 Å across a subunit boundary to catalyze O2-dependent formation of cis-dihydrodiol products from aromatic substrates. During catalysis, O2 binds to the Fe(II) while the substrate binds nearby. Single-turnover reactions have shown that one electron from each metal center is required for catalysis. This finding suggested that the reactive intermediate is Fe(III)-(H)peroxo or HO-Fe(V)═O formed by O-O bond scission. Surprisingly, several kinetic phases were observed during the single-turnover Rieske cluster oxidation. Here, the Rieske cluster oxidation and product formation steps of a single turnover of benzoate 1,2-dioxygenase are investigated using benzoate and three fluorinated analogues. It is shown that the rate constant for product formation correlates with the reciprocal relaxation time of only the fastest kinetic phase (RRT-1) for each substrate, suggesting that the slower phases are not mechanistically relevant. RRT-1 is strongly dependent on substrate type, suggesting a role for substrate in electron transfer from the Rieske cluster to the mononuclear iron site. This insight, together with the substrate and O2 concentration dependencies of RRT-1, indicates that a reactive species is formed after substrate and O2 binding but before electron transfer from the Rieske cluster. Computational studies show that RRT-1 is correlated with the electron density at the substrate carbon closest to the Fe(II), consistent with initial electrophilic attack by an Fe(III)-superoxo intermediate. The resulting Fe(III)-peroxo-aryl radical species would then readily accept an electron from the Rieske cluster to complete the cis-dihydroxylation reaction.

Inspired by the remarkable chemistry of the family of Rieske oxygenase enzymes, nonheme iron complexes of tetradentate N4 ligands have been developed to catalyze hydrocarbon oxidation reactions using H2O2 in the presence of added carboxylic acids. The observation that the stereo- and enantioselectivity of the oxidation products can be modulated by the electronic and steric properties of the acid implicates an oxidizing species that incorporates the carboxylate moiety. Frozen solutions of these catalytic mixtures generally afford two S = ½ intermediates, a highly anisotropic g2.7 subset (gmax = 2.58 to 2.78 and Δg = 0.85 – 1.2) that we assign to an FeIII–OOAc species and the less anisotropic g2.07 subset (g = 2.07, 2.01, and 1.96 and Δg ~ 0.11) we associate with an FeV(O)(OAc) species. Kinetic studies on the reactions of iron complexes supported by the TPA (tris(pyridyl-2-methyl)amine) ligand family with H2O2/AcOH or AcOOH at −40 °C reveal the formation of a visible chromophore at 460 nm, which persists in a steady state phase and then decays exponentially upon depletion of the peroxo oxidant with a rate constant that is substrate independent. Remarkably, the duration of this steady state phase can be modulated by the nature of the substrate and its concentration, which is a rarely observed phenomenon. A numerical simulation of this behavior as a function of substrate type and concentration affords a kinetic model in which the two intermediates exist in a dynamic equilibrium that is modulated by the electronic properties of the supporting ligands. This notion is supported by EPR studies of the reaction mixtures. Importantly, these studies unambiguously show that the g2.07 species, and not the g2.7 species, is responsible for substrate oxidation in the (L)FeII/H2O2/AcOH catalytic system. Instead the g2.7 species appears to be off-pathway and serves as a reservoir for the g2.07 species. These findings will be helpful not only for the design of regio- and stereo-specific nonheme iron oxidation catalysts but also for providing insight into the mechanisms of the remarkably versatile oxidants formed by nature’s most potent oxygenases.

Isotope effects of four broad and overlapping categories have been applied to the study of the mechanisms of chemical reaction and regulation of nonheme diiron cluster-containing oxygenases. The categories are: (a) mass properties that allow substrate-to-product conversions to be tracked, (b) atomic properties that allow specialized spectroscopies, (c) mass properties that impact primarily vibrational spectroscopies, and (d) bond dissociation energy shifts that permit dynamic isotope effect studies of many types. The application of these categories of isotope effects is illustrated using the soluble methane monooxygenase system and CmlI, which catalyzes the multistep arylamine to arylnitro conversion in the biosynthetic pathway for chloramphenicol.

Scientific Reports 7: Article number: 44628; published online: 16 March 2017; updated: 21 June 2017. In this Article, Henry N. Chapman is incorrectly listed as being affiliated with ‘Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA’ and an additional affiliation was omitted.

Isotope effects of four broad and overlapping categories have been applied to the study of the mechanisms of chemical reaction and regulation of nonheme diiron cluster-containing oxygenases. The categories are: (a) mass properties that allow substrate-to-product conversions to be tracked, (b) atomic properties that allow specialized spectroscopies, (c) mass properties that impact primarily vibrational spectroscopies, and (d) bond dissociation energy shifts that permit dynamic isotope effect studies of many types. The application of these categories of isotope effects is illustrated using the soluble methane monooxygenase system and CmlI, which catalyzes the multistep arylamine to arylnitro conversion in the biosynthetic pathway for chloramphenicol.

During my third week of high school chemistry, our instructor left in the middle of class in obvious pain and passed away shortly thereafter. This sad event could have spelled disaster for a budding career in chemistry, except for the fact that it occurred at Brandywine High School near Wilmington, Delaware, where 2,500 Ph.D. chemists lived in the school district. These included by father, Robert D. Lipscomb, who like so many of his Dupont Central Research & Development colleagues was a product of the Roger Adams/Reynold Fuson/Carl "Speed" Marvel/John Bailar, Jr. era at the University of Illinois. Chemistry class was taken over in principle by a substitute, who was an excellent football coach but knew little chemistry. In fact, it became a learning experiment, fed vicariously by a community of nearly unparalleled depth of expertise, and as I came to experience for the first time, an inbred commitment to understanding over learning. Our fathers and mothers taught us every night and we taught each other in class. We learned to hear how others interpreted the principles of chemistry and to evaluate quickly how this fit into our own framework for understanding. I have used these lessons, both the importance of listening and the commitment to the profession, continuously in the 50 years that have since passed. I have encountered them in one form or another in each of my mentors. My graduate school advisor I. C. "Gunny" Gunsalus said it very concisely: Science is about "Learning to Listen". Learn to listen to what your collaborators and students say beneath the words and to what experiments tell you beyond your expectations.

Extradiol catechol ring-cleaving dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase (HPCD) are summarized with the objective of showing how Nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active site metals, introducing active site amino acid substituted variants, and using substrates with different electron donating capacities. While each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic and computational analysis of the various intermediates shed light on how catalytic efficiency can be achieved.

Aldehyde-deformylating oxygenase (ADO) catalyzes O2-dependent release of the terminal carbon of a biological substrate, octadecanal, to yield formate and heptadecane in a reaction that requires external reducing equivalents. We show here that ADO also catalyzes incorporation of an oxygen atom from O2 into the alkane product to yield alcohol and aldehyde products. Oxygenation of the alkane product is much more pronounced with C9-10 aldehyde substrates, so that use of nonanal as the substrate yields similar amounts of octane, octanal, and octanol products. When using doubly-labeled [1,2-13C]-octanal as the substrate, the heptane, heptanal and heptanol products each contained a single 13C-label in the C-1 carbons atoms. The only one-carbon product identified was formate. [18O]-O2 incorporation studies demonstrated formation of [18O]-alcohol product, but rapid solvent exchange prevented similar determination for the aldehyde product. Addition of [1-13C]-nonanol with decanal as the substrate at the outset of the reaction resulted in formation of [1-13C]-nonanal. No 13C-product was formed in the absence of decanal. ADO contains an oxygen-bridged dinuclear iron cluster. The observation of alcohol and aldehyde products derived from the initially formed alkane product suggests a reactive species similar to that formed by methane monooxygenase (MMO) and other members of the bacterial multicomponent monooxygenase family. Accordingly, characterization by EPR and Mössbauer spectroscopies shows that the electronic structure of the ADO cluster is similar, but not identical, to that of MMO hydroxylase component. In particular, the two irons of ADO reside in nearly identical environments in both the oxidized and fully reduced states, whereas those of MMOH show distinct differences. These favorable characteristics of the iron sites allow a comprehensive determination of the spin Hamiltonian parameters describing the electronic state of the diferrous cluster for the first time for any biological system. The nature of the diiron cluster and the newly recognized products from ADO catalysis hold implications for the mechanism of C-C bond cleavage.

A family of dinuclear iron cluster-containing oxygenases that catalyze β-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Münck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 Å X-ray crystal structure of the archetypal enzyme from the family, CmlA, is reported. CmlA catalyzes β-hydroxylation of l-p-aminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of CmlA is unlike any previously reported, but the C-terminal domain has the αββα fold of the metallo-β-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of O2 activation.

Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD–HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII–O2 ·− species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the MIII–O2 ·− species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD–4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD–ligand complexes that bind NO are also active in normal O2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of FeII and MnII. Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover. Accordingly, past studies have shown that intermediate FeIII species are observed for these mutant enzymes.

The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor L-para-aminophenylalanine (L-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster which is reduced to the diferrous state (WTR) to initiate O2 activation. However, rapid O2 activation only occurs when WTR is bound to CmlP, the NRPS to which L-PAPA is covalently attached. Here the X-ray crystal structure of WTR is reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the WTR cluster ligand structure as well as the structures of WTR in complex with a functional CmlP variant (CmlPAT) with and without L-PAPA attached. It is found that formation of the active WTR-CmlPAT~L-PAPA complex converts at least one iron of the cluster from six- to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of WTR is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O2 in the presence or absence of CmlPAT~L-PAPA, showing loss of regulation. However this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O2 reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases.

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O2, CmlI forms a long-lived peroxo intermediate, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under (18)O2 show that both oxygen atoms in the nitro function of CAM derive from O2. However, when the single-turnover reaction between (18)O2-P and NH2-CAM is carried out in an (16)O2 atmosphere, CAM nitro groups contain both (18)O and (16)O, suggesting that P can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric CmlI yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O2 binding, while itself being oxidized to NO-CAM. Finally, the reformed P oxidizes NO-CAM to CAM with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.

Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O(2) bind in adjacent ligand sites of the active site Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the Fe(II) to O(2) gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases k(cat) by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F(Int1)(HPCA) and Y257F(Int2)(HPCA), accumulate during the Y257F-HPCA + O(2) reaction prior to formation of the ring-cleaved product. Y257F(Int1)(HPCA) is colorless and is formed as O(2) binds reversibly to the HPCA−enzyme complex. Y257F(Int2)(HPCA) forms spontaneously from Y257F(Int1)(HPCA) and displays a chromophore at 425 nm (ε(425) = 10 500 M(−1) cm(−1)). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain Fe(II). The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of Fe(II), and the low O(2) affinity suggest that Y257F(Int1)(HPCA) is an HPCA-Fe(II)-O(2) complex with little electron delocalization onto the O(2). In contrast, the intense spectrum of Y257F(Int2)(HPCA) suggests the intermediate is most likely an HPCA quinone-Fe(II)-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O(2).

Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD−HPCA and H200N−HPCA, respectively) with O(2). A blue intermediate forms within 20 ms of mixing of O(2) with H200N−HPCA (H200N(Int1)(HPCA)). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin Fe(III) (S = 5/2) antiferromagnetically coupled to a radical (S(R) = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200N(Int1)(HPCA) decays over the next 2 s, possibly through an Fe(II) intermediate (H200N(Int2)(HPCA)), to yield the product and the resting Fe(II) enzyme. Reaction of FeHPCD−HPCA with O(2) results in rapid formation of a colorless Fe(II) intermediate (FeHPCD(Int1)(HPCA)). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCD(Int1)(HPCA) suggests it is an intermediate occurring after O(2) activation and attack. The similar Mössbauer parameters for FeHPCD(Int1)(HPCA) and H200N(Int2)(HPCA) suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O(2) via the iron does occur, leading to aromatic ring cleavage.

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). Whereas Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently, exhibiting significantly higher $$ K_{\text{M}}^{{{\text{O}}_{ 2} }} $$ and k cat. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-HPCD, Mn-HPCD, and Co-HPCD shows that metal substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady-state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe-HPCD and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O2 to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O2 activation mechanism associated with the extradiol dioxygenase family.

intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear Fe II enzyme family. Many steps in the O 2 activation and reaction cycle of Fe II -containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O 2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin ( S 1 = 5/2) Fe III antiferromagnetically (AF) coupled to an S 2 = 1/2 radical ( J ≈ 6 cm -1 in ). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by hyperfine interactions ( A 17 O ≈ 180 MHz). Thus, Int-1 is an AF-coupled species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O 2 . Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III -radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by (III -4NC semiquinone radical species. Our results demonstrate facile electron transfer between Fe II , O 2 , and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.

The biosynthesis of chloramphenicol requires a β-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O 2 is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.

Biological O 2 activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-to-O 2 electron transfer results in a reactive M(III)-superoxo species. For the extradiol aromatic ring-cleaving dioxygenases, we have proposed a different model where an electron is transferred from substrate to O 2 via the M(II) center to which they are both bound, thereby obviating the need for an integral change in metal redox state. This model is tested by using homoprotocatechuate 2,3-dioxygenases from Brevibacterium fuscum (Fe-HPCD) and Arthrobacter globiformis (Mn-MndD) that share high sequence identity and very similar structures. Despite these similarities, Fe-HPCD binds Fe(II) whereas Mn-MndD incorporates Mn(II). Methods are described to incorporate the nonphysiological metal into each enzyme (Mn-HPCD and Fe-MndD). The x-ray crystal structure of Mn-HPCD at 1.7 Å is found to be indistinguishable from that of Fe-HPCD, while EPR studies show that the Mn(II) sites of Mn-MndD and Mn-HPCD, and the Fe(II) sites of the NO complexes of Fe-HPCD and Fe-MndD, are very similar. The uniform metal site structures of these enzymes suggest that extradiol dioxygenases cannot differentially compensate for the 0.7-V gap in the redox potentials of free iron and manganese. Nonetheless, all four enzymes exhibit nearly the same K M and V max values. These enzymes constitute an unusual pair of metallo-oxygenases that remain fully active after a metal swap, implicating a different way by which metals are used to promote oxygen activation without an integral change in metal redox state.

Oxidase and oxygenase enzymes allow the use of relatively unreactive O2 in biochemical reactions. Many of the mechanistic strategies employed in nature for this key reaction are represented within the 2-His-1-carboxylate facial triad family of non-heme Fe(II) containing enzymes. The open face of the metal coordination sphere opposite the three endogenous ligands participates directly in the reaction chemistry. Here, data from several studies are presented showing that reductive O2 activation within this family is initiated by substrate (and in some cases co-substrate or cofactor) binding, which then allows coordination of O2 to the metal. From this starting point, both the O2 activation process and the reactions with substrates diverge broadly. The reactive species formed in these reactions have been proposed to encompass four oxidation states of iron and all forms of reduced O2 as well as several of the reactive oxygen species that derive from O–O bond cleavage.

Near-IR MCD and variable temperature, variable field (VTVH) MCD have been applied to Naphthalene 1,2-dioxygenase (NDO) to describe the coordination geometry and electronic structure of the mononuclear non-heme ferrous catalytic site in the resting and substrate-bound forms with the Rieske 2Fe2S cluster oxidized and reduced. The structural results are correlated with the crystallographic studies of NDO and other related Rieske non-heme iron oxygenases to develop molecular level insights into the structure/function correlation for this class of enzymes. The MCD data for resting NDO with the Rieske center oxidized indicate the presence of a six-coordinate high-spin ferrous site with a weak axial ligand which becomes more tightly coordinated when the Rieske center is reduced. Binding of naphthalene to resting NDO (Rieske oxidized and reduced) converts the six-coordinate sites into five-coordinate (5c) sites with elimination of a water ligand. In the Rieske oxidized form the 5c sites are square pyramidal, but transform to a 1:2 mixture of trigonal bipyramial/square pyramidal sites when the Rieske center is reduced. Thus the geometric and electronic structure of the catalytic site in the presence of substrate can be significantly affected by the redox state of the Rieske center. The catalytic ferrous site is primed for the O2 reaction when substrate is bound in the active site in the presence of the reduced Rieske site. These structural changes insure that two electrons and the substrate are present before the binding and activation of O2, which avoids the uncontrolled formation and release of reactive oxygen species.

The uncatalyzed reactions of O 2 (S = 1) with organic substrates (S = 0) are thermodynamically favorable but kinetically slow because they are spin-forbidden and the one-electron reduction potential of O 2 is unfavorable. In nature, many of these important O 2 reactions are catalyzed by metalloenzymes. In the case of mononuclear non-heme iron enzymes, either Fe II or Fe III can play the catalytic role in these spin-forbidden reactions. Whereas the ferrous enzymes activate O 2 directly for reaction, the ferric enzymes activate the substrate for O 2 attack. The enzyme–substrate complex of the ferric intradiol dioxygenases exhibits a low-energy catecholate to Fe III charge transfer transition that provides a mechanism by which both the Fe center and the catecholic substrate are activated for the reaction with O 2 . In this Perspective, we evaluate how the coupling between this experimentally observed charge transfer and the change in geometry and ligand field of the oxidized metal center along the reaction coordinate can overcome the spin-forbidden nature of the O 2 reaction.

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio- and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogues designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analogue compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogues lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.

Intermediates in the reaction cycle of an oxygenase are usually very informative with respect to the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.

Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species.

CmlA is a tailoring enzyme from the nonribosomal peptide synthetase (NRPS)-based biosynthetic pathway for chloramphenicol expressed in Streptomyces venezuelae. The enzyme is a monooxygenase that catalyzes the β-hydroxylation of l-para-aminophenylalanine (l-PAPA) covalently tethered to the NRPS CmlP. Spectroscopic and X-ray crystallographic studies reveal an oxo-bridged dinuclear iron cluster in the active site that binds and activates O2. The structure shows that the subunits of the homodimeric CmlA are composed of a 248 residue N-terminal domain with a novel fold and a 284 residue C-terminal domain with a metallo-β-lactamase (MBL) fold where the diiron cluster is bound. Oxygenase activity has not been previously observed for a diiron cluster in an MBL fold and no other diiron cluster enzyme is known to catalyze β-hydroxylation. In the diferrous state, the diiron cluster is reactive with O2, but the reaction is very slow in the absence of the CmlP–l-PAPA complex. Thus, CmlA is regulated so that O2 is activated only when substrate is bound. CmlA is the first diiron cluster-containing enzyme known to be active in an NRPS pathway, but a gene search reveals numerous homologs of CmlA that are likely to catalyze similar reactions during biosynthesis of other natural products. 3D Structure Schematic representation of the CmlA dimer from Streptomyces venezuelae. The left monomer is depicted with the secondary structure highlighted in blue for α-helices and orange for β-strands. The second monomer is colored maroon. Iron ions are depicted as brown spheres. The structure was generated from PDB structure 4JO0. All figures shown herein were generated using PyMOL Molecular Graphics System, Version 1.5.0.4, Schrodinger, LLC. Keywords: monooxygenase; dinuclear iron cluster; oxygen; nonribosomal peptide synthetase (NRPS); β-hydroxylation; antibiotic; natural product biosynthesis; spectroscopy; C–H bond activation; chloramphenicol

Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O(2) to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t(1/2) = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an (18)O(2)-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η(1):η(1)) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O-O) that is at least 60 cm(-1) lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ-η(1):η(1)-peroxo ligand; we propose that a μ-η(1):η(2)-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.