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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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

(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

Biological systems activate O 2 using many mechanisms, but in nearly all cases, the activation process is regulated to assure specificity. The nature of these regulatory aspects of the reaction must be understood before the true nature of the underlying chemistry can be described with certainty. Most metal-containing oxygenases utilize amino acids in the second sphere and beyond to regulate the O 2 activation reaction. One example of this is seen in the mechanism of substrate selectivity by methane monooxygenase. The regulatory protein MMOB binds to the active site-containing MMOH and appears to create a pore sized for methane into the active site. This controls access and therefore the preferred substrate. Also, the complex appears to cause quantum tunneling to dominate in C–H bond cleavage reaction for methane, selectively increasing the rate for this substrate. Both effects can be altered by mutagenesis of MMOB, potentially broadening the substrate range of the enzyme. Second sphere effects are also important in determining the position of ring cleavage for catecholic ring cleaving dioxygenases. Intermediates throughout the catalytic cycle of homoprotocatechuate 2,3-dioxygenase can be detected by using the chromophoric substrate 4-nitrocatechol (4NC). Upon mutation of the second sphere residue histidine 200 to asparagine (H200N), the rate of reaction of the Fe-oxy intermediate is greatly slowed, allowing its detection for the first time when using either 4NC or the natural substrate 3,4-dihydroxyphenylacetate (HPCA). HPCA cleavage occurs in the usual proximal extradiol position by this mutant, but 4NC is oxidized to the quinone without ring cleavage. Use of the alternative substrate 2,3-dihydroxybenzoate results in distal extradiol cleavage for the wild type enzyme, but intradiol cleavage for the H200-phenylalanine mutant. Thus, control of the second sphere allows the enzyme to design a specific catalyst that gives only one of the four potential types of products. This insight can be used to design specific enzyme oxidation catalysts.

The hydroxylase component (MMOH) of the soluble form of methane monooxygenase (sMMO) isolated from Methylosinus trichosporium OB3b catalyzes both the O2 activation and the CH4 oxidation reactions at the oxygen-bridged dinuclear iron cluster present in its buried active site. During the reaction cycle, the diiron cluster forms a bis-mu-oxo-(Fe(IV))2 intermediate termed compound Q (Q) that reacts directly with methane. Many adventitious substrates also react with Q, most at a relatively slow rate. We have proposed that Q reacts preferentially with CH4 because the sMMO regulatory component MMOB induces a size selective pore into the MMOH active site as the two components form a complex. Support for this proposal has come through the observation of a nonlinear Arrhenius plot for the CH4 oxidation, presumably due to a shift in rate-limiting step from substrate binding at low temperature to C-H bond cleavage at high temperature. Reactions of all substrates other than CH4 fail to exhibit a break in the Arrhenius plot because binding is always rate limiting in the temperature range explored. Here we show that it is possible to induce a break in the Arrhenius plot for the ethane reaction with Q by using an MMOB mutant termed DBL2 (S109A/T111A) in which residues at the MMOH-MMOB interface are reduced in size. We hypothesize that this increases the ethane binding rate and shifts the Arrhenius breakpoint into the observable temperature range. As a result of this shift, the kinetic and activation parameters of the C-H bond breaking reaction for both methane and ethane can be observed using the DBL2 mutant. A 2H-KIE is observed for both substrate oxidation reactions when using DBL2, whereas only CH4 oxidation exhibits an effect when using wild type MMOB, consistent with the C-H bond cleaving reaction becoming at least partially rate limiting for ethane. Analysis of the temperature dependence of the 2H-KIE for ethane and methane for reactions using both mutant and wild type forms of MMOB suggests that quantum tunneling plays a significant role in methane oxidation but not ethane oxidation.

The effects of the C-terminal region of the B component (MMOB) of soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b on steady-state turnover, the transient kinetics of the reaction cycle, and the properties of the sMMO hydroxylase (MMOH) active site diiron cluster have been explored. MMOB is known to have many profound effects on the rate and specificity of sMMO. Past studies have revealed specific roles for the well-folded core structure of MMOB as well as the disordered N-terminal region. Here, it is shown that the disordered C-terminal region of MMOB also performs critical roles in the regulation of catalysis. Deletion mutants of MMOB missing 5, 8, and 13 C-terminal residues cause progressive decreases in the maximum steady-state turnover number, as well as lower apparent rate constants for formation of the key reaction cycle intermediate, compound Q. It is shown that this latter effect is actually due to a decrease in the rate constant for formation of an earlier intermediate, probably the hydroperoxo species, compound P. Moreover, the deletions result in substantial uncoupling at or before the P intermediate. It is proposed that this is due to competition between slow H(2)O(2) release from one of the intermediates and the reaction that carries this intermediate on to the next step in the cycle, which is slowed by the mutation. Electron paramagnetic resonance (EPR) studies of the hydroxylase component (MMOH) in the mixed valent state suggest that complexation with the mutant MMOBs alters the electronic properties of the diiron cluster in a manner distinct from that observed when wild-type MMOB is used. Active site structural changes are also suggested by a substantial decrease in the deuterium kinetic isotope effect for the reaction of Q with methane thought to be associated with a decrease in quantum tunneling in the C-H bond breaking reaction. Thus, the surface interactions between MMOH and MMOB that affect substrate oxidation and its regulation appear to require the complete MMOB C-terminal region for proper function.

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated fromArthrobacter globiformisandBrevibacterium fuscumhave been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four βαβββ modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 Å), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sitestransto the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An “open” coordination sitetransto E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2during catalysis are identified and discussed in the context of current mechanistic hypotheses.

Effector proteins alter the kinetic or catalytic course of many oxygenase reactions. One of the first oxygenase effectors to be described was putidaredoxin, which serves to gate electron transfer into oxy-P450cam. In the nonheme, methane monooxygenase (MMO) system, the B-component (MMOB) serves a distinct effector function by gating substrate and oxygen into the active site of the hydroxylase component (MMOH). Here the binding parameters and binding surfaces of the MMOB–MMOH complex are determined by site-specific labeling, fluorescence titrations, chemical cross-linking, and MALDI-TOF peptide identification. Based on these data, a model for the bimolecular complex is described and a hypothesis for the structural basis for the effector function is elaborated. The bearing on the putidaredoxin effector function is discussed.

The active site of naphthalene 1,2-dioxygenase (NDO) contains a Rieske Fe-S cluster and a mononuclear non-heme iron, which are contributed by different alpha-subunits in the (alphabeta)(3) structure. The enzyme catalyzes cis-dihydroxylation of aromatic substrates, in addition to numerous other adventitious oxidation reactions. High-resolution Mims (2)H-ENDOR (electron nuclear double resonance) spectra have been recorded for the NO-ferrous center of NDO bound with the substrates d(8)-naphthalene, d(2)-naphthalene, d(8)-toluene, d(3)-toluene, and d(6)-benzene; samples were prepared in a D(2)O buffer to test for solvent-derived ligands; spectra were collected for enzymes with the Rieske diiron center in both its oxidized and reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of the substrate in a major binding geometry (denoted as A) was detected for all perdeuterated substrates. Examination of the sample prepared with 1,4-di-deutero-naphthalene shows that the signal arises from D1. Analysis of two-dimensional (2-D) orientation-selective ENDOR patterns collected for this sample defined the location of the D1 deuteron, with respect to the g-frame of the iron center and the orientation of the C-D1 bond. Consideration of the orientations of naphthalene that are permitted within the constraints of these results, as supported by a novel approach to simulations of orientation-selective, 2-D ENDOR patterns for the perdeuterated naphthalene sample, which summed contributions from D1/D2/D8, disclose the geometry of the naphthalene and the Fe-NO fragment. The two deuterons of the reactive carbons, D1 and D2, are closest to the Fe atom (r(Fe)(-)(D1) approximately 4.3 A, r(Fe)(-)(D2) approximately 5.0 A), whereas D8 is farther away (r(Fe)(-)(D8) approximately 5.3 A). Perhaps more instructive, D1-N and D2-N distances to the O(2) surrogate, NO, are approximately 2.4 and approximately 3.3 A, respectively, whereas the D8-N distance is approximately 3.7 A. The data show that benzene and the aromatic ring of toluene also sit within the substrate-binding pocket adjacent to the mononuclear Fe atom. These rings occupy a position similar to that of the "proximal" ring of naphthalene, with the closest ring deuteron being located at a distance of approximately 4.3-4.4 A from the Fe atom and with the Fe-D vector being slightly off the Fe-N(O) direction. In particular, comparison of the data for d(8)-toluene and methyl-d(3)-toluene shows that the methyl group of toluene points away from the Fe atom, despite observations that the oxidation of toluene occurs at the methyl group during catalysis. The Rieske cluster is reduced during both steady-state and single-turnover catalysis; therefore, the effect of its oxidation state on the geometry of substrate binding was examined. The spectra from the NDO-naphthalene complex also revealed a second binding conformation (denoted as B), in which the substrate is located approximately 0.5 A farther from the Fe atom. The relative populations of A- and B-sites are allosterically changed when the Rieske cluster is reduced. ENDOR of exchangeable protons shows that the water/hydroxide of Fe-NDO is retained upon binding NO.

The regulatory component MMOB of soluble methane monooxygenase (sMMO) has been hypothesized to control access of substrates into the active site of the hydroxylase component (MMOH) through formation of a size specific channel or region of increased structural flexibility tuned to methane and O(2). Accordingly, a decrease in the size of four MMOB residues (N107G/S109A/S110A/T111A, the Quad mutant) was shown to accelerate the reaction of substrates larger than methane with the reactive MMOH intermediate Q [Wallar, B. J., and Lipscomb, J. D. (2001) Biochemistry 40, 2220-2233]. Here, this hypothesis is tested by construction of single and double mutations involving the residues of the Quad mutant. It is shown that mutations of residues that extend into the core structure of MMOB alter many aspects of the MMOH catalyzed reaction but do not mimic the effects of the Quad mutant. In contrast, the MMOB residues that are thought to form part of the interface in the MMOH-MMOB complex increase active site accessibility as observed for the Quad mutant. In particular, the mutant T111A mimics most of the effects of the Quad mutant; thus, Thr111 is proposed to most directly control access. Unexpectedly, mutation of Thr111 to the larger Tyr greatly increases the rate constant for the reaction of larger substrates such as ethane, furan, and nitrobenzene with Q while decreasing the rate constant for the reaction with methane. Other steps in the cycle are dramatically slowed, the regiospecificity for nitrobenzene oxidation is altered, and 10-fold more T111Y than wild-type MMOB is required to maximize the rate of turnover. Thus, T111Y appears to make a more extensive change in local interface structure that allows hydrocarbons at least as large as ethane to bind and react with Q similarly. As a result, the bond cleavage rates for methane, ethane, and their deuterated analogues are shown for the first time to correlate with bond strength in accord with a mechanism in which C-H bond cleavage occurs during reaction of substrates with Q.

Naphthalene 1,2-dioxygenase (NDOS) catalyzes the NAD(P)H and O(2)-dependent oxidation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDOS consists of three protein components: a flavo-[2Fe-2S] reductase (NDR), a ferredoxin electron transfer protein (NDF), and an (alphabeta)(3) oxygenase (NDO) containing a mononuclear iron site and a Rieske-type [2Fe-2S] cluster in each alpha-subunit. The active site is built across a subunit-subunit boundary, and each subunit contributes one type of metal center. Our previous studies have shown that NDO with both metal centers reduced is capable of an O(2)-coupled single turnover to yield the correct cis-diol product in the absence of the NDR and NDF components (Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953). It is shown here that addition of H(2)O(2) to NDO allows reaction with naphthalene to rapidly yield the correct product in a "peroxide shunt" reaction that does not require a reduced Rieske cluster. The mononuclear Fe(2+) center is oxidized during turnover, while the Rieske cluster remains in the oxidized state. Peroxide shunt turnover in the presence of (18)O-labeled H(2)O(2), H(2)O, or O(2) shows that both oxygen atoms in the product derive primarily from H(2)O(2). The peroxide shunt halts after one turnover despite the presence of excess H(2)O(2) and naphthalene, but this is not the result of enzyme inactivation. Rather, it appears that the product cannot be released when the mononuclear iron is in the Fe(3+) state, blocking a second turnover. This work supports the hypotheses that the cis-dihydroxylation activity of NDOS requires only the NDO component, that a peroxo intermediate is formed during normal catalysis, and that product release requires an additional reducing equivalent beyond those necessary for the first turnover.

Methane monooxygenase (MMO) catalyzes the NADH and O2 coupled oxidation of CH4 to yield CH3OH. The enzyme consists of three components: MMOH (hydroxylase), MMOB (effector), and MMOR (reductase). We have structurally characterized MMOH and MMOB. The active site is located in MMOH and contains a bis-μ-hydroxo-bridged diiron cluster that is responsible for O2 activation and substrate oxidation. Transient kinetic studies have revealed many reaction cycle intermediates and allowed some of them to be characterized. The unique intermediate Q contains a bis-μ-oxo-Fe(IV)Fe(IV) cluster and can directly oxidize CH4. Large deuterium isotope effects are observed for the reaction of Q with methane but, remarkably, not ethane. Arrhenius plots are nonlinear for the CH4 reaction with Q but not for other substrates. We propose that the Q reaction with substrates occurs in two steps, substrate binding and CH bond cleavage, with the latter rate limiting only for CH4. Mutagenesis of MMOB shows that it affects the kinetics throughout the reaction cycle. One mutant dramatically alters the rates of Q decay and product release according to substrate size. It is proposed that MMOB serves to gate substrates into the active site of MMOH, thereby allowing it to select methane from more readily oxidized substrates.

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence. ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen. In addition to substrate and cosubstrate, ACCO requires the activator CO(2) for continuous turnover. NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism. Resting ACCO/Fe(II) is coordinatively saturated (six-coordinate). In the presence of CO(2), one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme. The open coordination position allows rapid O(2) activation for the oxidation of both substrates. In the absence of CO(2), ACC binding alone converts the site to five-coordinate, which would react with O(2) in the absence of ascorbate and quickly deactivate the enzyme. These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO(2) as stabilizing the six-coordinate ACCO/Fe(II)/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.

Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe(3+) to activate O2 and catecholic substrates for reaction. The inability of Fe(3+) to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe(3+) species, and the anhydride-Fe(3+) intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe(2+)-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe(2+) intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.

Research during the 54 years since the discovery of non-heme iron oxygenase enzymes has revealed a remarkable diversity in the strategies used for O2 activation. Many of these are represented in dioxygenase enzymes that introduce oxygen atoms from O2 into aromatic compounds (1). These include direct oxygen activation by external electron transfer to the redox-active mononuclear iron in Rieske dioxygenases, Fe(II)-mediated internal electron transfer from the substrate to O2 in extradiol dioxygenases, and Fe(III)-mediated spin inversion during internal electron transfer from substrate to O2 in intradiol dioxygenases. Recently, the exploration of these mechanisms has moved forward through the combined use of transient kinetics, spectroscopic characterizations, high level computations, and X-ray crystallographic techniques that allow the visualization of discrete intermediates. For each enzyme class, catalysis is found to depend on an elegant balance between the Lewis acidity, redox, and ligand exchange properties of the metal, the traditional structural and catalytic properties of the second sphere residues of the active site, and the less-appreciated contributions from structural dynamics that encompass the entire enzyme. Together, these mediate not only specific catalysis, but just as importantly, regulated catalysis that mitigates against the activation of O2 without attendant substrate oxygenation.

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O 2 , CO 2 , and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme⋅(substrate or cofactor)⋅NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O 2 complexes. It is shown here that the ternary Fe(II)ACCO⋅ACC⋅NO complex is readily formed, but no Fe(II)ACCO⋅ascorbate⋅NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15 N or 17 O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the α-amino and α-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O 2 bind to the iron as a step in O 2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O 2 , thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

The reactive oxy intermediate of the catalytic cycle of extradiol aromatic ring-cleaving dioxygenases is formed by binding the catecholic substrate and O2 in adjacent ligand positions of the active site metal [usually Fe(II)]. This intermediate and the following Fe(II)-alkylperoxo intermediate resulting from oxygen attack on the substrate have been previously characterized in a crystal of homoprotocatechuate 2,3-dioxygenase (HPCD). Here a subsequent intermediate in which the O-O bond is broken to yield a gem diol species is structurally characterized. This new intermediate is stabilized in the crystal by using the alternative substrate, 4-sulfonylcatechol, and the Glu323Leu variant of HPCD, which alters the crystal packing.

The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17. Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3, 4-PCD.PCA) [Orville, A. M., Lipscomb, J. D.,Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacteriumfuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at approximately 1320 cm-1 which are diagnostic of a HPCA. Fe(III) chelate complex. 18O labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of these data suggests that the Fe(III)-HPCAO4 bond is shorter than the Fe(III)-HPCAO3 bond. This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(Oeta) (108beta). This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2Fo - Fc electron density and suggests that this is the conformation required for catalysis.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.

The soluble methane monooxygenase system (MMO), consisting of a hydroxylase (MMOH), a reductase, and component B (MMOB), catalyzes the NADH and O2 dependent hydroxylation of methane and many other hydrocarbons. The binuclear non-heme ferrous active site cluster of the hydroxylase−component B (MMOH−MMOB) complex in the presence of substrate and small molecules has been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. CD studies reveal that addition of the alternative substrate, trans-1,2-dichloroethylene, or inhibitor, tetrachloroethylene, induces a conformational change in the active site pocket only in the presence of MMOB. Complementary MCD data indicate that this conformational change does not result in a direct change in the ligation of the iron atoms. Comparison of the CD/MCD data with the crystal structure of MMOH allows a tentative correlation between the perturbations observed and the iron atoms affected. The binding of MMOB to MMOH distorts the ligand fi...

The conversion from methane to methanol is catalyzed by methane monooxygenase (MMO) in methanotrophic bacteria. Earlier work on the crystal structures of the MMO hydroxylase component (MMOH) from Methylococcus capsulatus (Bath) at 4 °C and –160 °C has revealed two different core arrangements for the diiron active site. To ascertain the generality of these results, we have now carried out the first structural characterization on MMOH from Methylosinustrichosporium OB3b. Our X-ray absorption spectroscopic (XAS) analysis suggests the presence of two Fe-Fe distances of about 3 A and 3.4 A, which are proposed to reflect two populations of MMOH molecules with either a bis(μ-hydroxo)(μ-carboxylato)- or a (μ-hydroxo)(μ-carboxylato)diiron(III) core structure, respectively. The observation of these two different core structures, together with the crystallographic results of the MMOH from Methylococcus capsulatus (Bath), suggests the presence of an equilibrium that may reflect a core flexibility that is required to accommodate the various intermediates in the catalytic cycle of the enzyme. XAS studies on the binding of component B (MMOB) to the hydroxylase component show that MMOB does not perturb either this equilibrium or the gross structure of the oxidized diiron site in MMOH.

The extradiol-cleaving catechol 2,3-dioxygenase (2,3-CTD) isolated from Pseudomonas putida mt-2 and its catechol and ternary E.S.NO complexes are characterized by X-ray absorption spectroscopy (XAS). The intensities of the 1s-->3d transitions in the pre-edge spectra of the uncomplexed enzyme and its substrate complex show that the Fe(II) center is five-coordinate in both complexes, in agreement with earlier magnetic circular dichroism studies [Mabrouk, P. A., Orville, A. M., Lipscomb, J. D., & Solomon, E. I. (1991) J. Am. Chem. Soc. 113, 4053-4061]. Analysis of the EXAFS region of uncomplexed 2,3-CTD shows five N/O ligand atoms 2.09 A from the active site Fe(II). In the 2,3-CTD.catechol complex, one N/O atom is located at 1.93 A and four N/O type ligands are at 2.10 A. By comparison with [FeII-(6TLA)(DBCH)](ClO4), the first well-characterized mononuclear Fe(II).catechol model complex, the 1.93 A scatterer is proposed to be the oxygen from the deprotonated hydroxyl group of the coordinated catecholate monoanion. Nitric oxide binds to the Fe(II) center in the enzyme.catechol complex without displacing the existing ligands, resulting in the formation of a six-coordinate complex, as indicated by the addition of a new N/O type scatterer at 1.74 A. Bond valence sum (BVS) analysis of the bond lengths derived from the EXAFS fits gives values that correspond to the iron oxidation states established for these complexes, thus lending credence to the coordination environment deduced for the iron center in those complexes. The present study provides the first evidence for a monoanionic substrate binding mode in an extradiol dioxygenase, which is distinct from the dianionic binding mode proposed for intradiol dioxygenases. We speculate that this difference in binding mode may have important ramifications for the site of aromatic ring cleavage in the subsequent oxygen insertion reactions.

Homoprotocatechuate 2,3-dioxygenase (FeHPCD) utilizes an active site FeII to activate O2 in a reaction cycle that ultimately results in aromatic ring cleavage. Here, the roles of the conserved active site residue Tyr257 are investigated by solving the X-ray crystal structures of the Tyr257-to-Phe variant (Y257F) in complex with the substrate homoprotocatechuate (HPCA) and the alternative substrate 4-nitrocatechol (4NC). These are compared with structures of the analogous wild type enzyme complexes. In addition, the oxy intermediate of the reaction cycle of Y257F-4NC + O2 is formed in crystallo and structurally characterized. It is shown that both substrates adopt a previously unrecognized, slightly non-planar, strained conformation affecting the geometries of all aromatic ring carbons when bound in the FeHPCD active site. This global deviation from planarity is not observed for the Y257F variant. In the Y257F-4NC-oxy complex, the O2 is bound side-on to the FeII, while the 4NC is chelated in two adjacent sites. The ring of the 4NC in this complex is planar, in contrast to the equivalent FeHPCD intermediate, which exhibits substantial local distortion of the substrate hydroxyl moiety (C2-O−) that is hydrogen bonded to Tyr257. We propose that Tyr257 induces the global and local distortions of the substrate ring in two different ways. First, van der Waals conflict between the Tyr257-OH substituent and the substrate C2 carbon is relieved by adopting the globally strained structure. Second, Tyr257 stabilizes the localized out-of-plane position of the C2-O− by forming a stronger hydrogen bond as the distortion increases. Both types of distortion favor transfer of one electron out of the substrate to form a reactive semiquinone radical. Then, the localized distortion at substrate C2 promotes formation of the key alkylperoxo intermediate of the cycle resulting from oxygen attack on the activated substrate at C2, which becomes sp3 hybridized. The inability of Y257F to promote the distorted substrate structure may explain the observed 100-fold decrease in the rates of the O2 activation and insertion steps of the reaction. Homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum catalyzes the fission of the O-O bond of O2 with incorporation of both atoms into the aromatic substrate (HPCA) resulting in ring cleavage as shown in Scheme 1.1–5 The mechanism shown in Scheme 1 begins with O2 and HPCA binding in adjacent coordination sites of the active site FeII.4,6–10 Electron transfer from HPCA to the O2 via the iron would give both substrates radical character, allowing facile recombination to form an alkylperoxo intermediate from which O-O bond fission and aromatic ring cleavage could ensue. Both the initial intermediate, in which each substrate has radical character, and the alkylperoxo intermediate resulting from oxygen attack have been structurally characterized by X-ray crystallography after a crystal of the enzyme soaked with the slow substrate 4-nitrocatechol (4NC) was exposed to low concentrations of O2. 3 While the chemical nature of the alkylperoxo species is apparent from the structure, the diradical character of the initial intermediate was surmised from the pronounced lack of planarity observed for the aromatic substrate. It was proposed that the non-planar ring results from a localized semiquinone radical (SQ•) on the ring C2 (See Scheme 1 for numbering system used here) where the attack by oxygen (at the level of O2•−) will occur. Accordingly, in the alkylperoxo intermediate, this carbon in observed to be fully sp3 hybridized and 4 coordinate. Scheme 1 Proposed reaction mechanism for extradiol dioxygenases. In the case of FeHPCD, R is –CH2COO− (optimal substrate HPCA) or –NO2 (alternative substrate 4NC), B(H) is His200. The ring carbon numbering system shown is adopted for both ... The structural studies from our laboratory and others have shown that there are several second sphere active site residues that may contribute to catalysis (e.g. Figure 1).3,5,9–11 Extensive investigations of an active site His (His200 in the case of FeHPCD) have shown that it plays many roles including stabilization of the Fe-bound O2 via hydrogen bonding and charge interaction, acid-base catalysis of the alkylperoxo intermediate formation and subsequent oxygen insertion chemistry, and possibly, stabilization of the bound oxygen in the side-on orientation properly aligned to attack the aromatic substrate.3,12–15 Intermediates trapped from solution reactions of FeHPCD and its variants at His200 show that iron converts transiently to the FeIII state in the absence of H200, but it is always observed in the FeII state in its presence. 13,14 Thus, when His200 is present, one or two electrons appear to pass from the aromatic substrate to the oxygen with either no change in iron oxidation state or a transient change that persists for much less than the freezing time for rapid freeze quench (RFQ) experiments (~10 ms).14 This suggests that there is a considerable driving force for electron transfer that is not apparent from the redox potentials of the isolated aromatic substrate and O2. Figure 1 Active site environment of FeHPCD in complex with the optimal substrate HPCA (PDB 4GHG, subunit C). Atom color code: gray, carbon (enzyme); yellow, carbon (substrate); blue, nitrogen; red, oxygen; bronze, iron. Red dashed lines show hydrogen bonds (A). ... Another second sphere amino acid, Tyr257, is hydrogen bonded to the deprotonated C2-O− of bound HPCA (Figure 1). The orientation of Tyr257 is such that this interaction might stabilize the non-planar structure of the substrate in the reaction cycle intermediate. In the accompanying report,16 it is shown that mutation of Tyr257 to Phe (Y257F) results in a significant decrease in the rate constants for many steps with reduction approaching 100-fold in the oxygen binding and insertion steps despite the presence of the acid-base catalyst His200. The results suggest that the putative HPCA SQ•-FeII-O2•− is either not formed or is unreactive, leading to formation of an observed HPCA quinone-FeII-(H)peroxo species that then slowly yields the correct ring cleaved product. Here, structural studies of the Y257F variant, its complexes with HPCA and 4NC, and the intermediate that results from exposure of the Y257F-4NC complex to O2 are reported. The results give insight into the structural basis for the observed decreases in rate constants and the change in the chemical nature of the reactive intermediate as well as the strategy used by aromatic dioxygenases to simultaneously maintain high rates and high fidelity in ring cleaving reactions.

Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea catalyzes the oxidation of NH2OH to NOz-. The enzyme contains eight hemes per subunit (63 ma) which participate in catalytic function and electron transport. In the resting ferric state of the enzyme, we find that these hemes can be categorized on the basis of quantitative integer-spin and multifrequency EPR data as follows: (a) Four hemes are noninteracting, ferric C-type hemes. (b) Two hemes are low-spin and weakly spin-coupled through an interaction which is best described as dipolar on the basis of frequency-dependent g values. (c) The final two hemes are exchange-coupled; this cluster is the origin of a new integer-spin EPR signal which can be quantitatively associated with the reactive Pm heme of HAO. Accordingly, treatments that affect the optical properties of Pm and catalysis of the enzyme, such as substrates, CN-, or H202, all caused the integer-spin resonance to vanish. For reduced HAO, a second integer-spin EPR signal is observed which derives from one high-spin ferrous heme; previous Mossbauer studies have shown that the only such heme is Pm. An analysis of potential electronic models for the exchange-coupled cluster indicated that the interacting spins are both S = l/2. It thus appears that the active site of HA0 contains a pair of low-spin hemes which are coupled in a manner not previously observed in proteins. The structure of the diheme cluster and in the presence of substrates or inhibitors, and its bearing on catalysis by HA0 is discussed.

X-ray absorption and resonance Raman spectroscopies show that CmlA, the β-hydroxylase of the chloramphenicol biosynthetic pathway, contains a (μ-oxo)-(μ-1,3-carboxylato)diiron(III) cluster with 6-coordinate iron centers and 3 – 4 His ligands. This active site is found within a unique β-lactamase fold and is distinct from those of soluble methane monooxygenase and related enzymes that utilize a highly conserved diiron cluster with a 2-His-4-carboxylate ligand set within a 4-helix bundle motif. These structural differences may have an impact on the nature of the activated oxygen species of the reaction cycle.

A combination of circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies has been used to probe the geometric and electronic structure of the binuclear Fe(II) active site of the reduced hydroxylase component of methane monooxygenase (MMOH). Excited-state data provide the numbers and energies of d→d transitions which are interpreted in terms of ligand field calculations to estimate the geometry of each iron. Variable-temperature variable-field (VTVH) MCD data are analyzed by using a non-Kramers doublet model to obtain the zero field splitting (ZFS) and g∥ value of the ground state and the excited sublevel energies. These results are further interpreted in terms of a spin Hamiltonian which includes the ZFS of each Fe 2+ combined with the exchange coupling between iron centers

Soluble methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of three components: hydroxylase, reductase, and component B. The active-site diiron cluster of the hydroxylase has been studied with Mossbauer, ANDOR, and APR spectroscopies. Mossbauer spectra of the oxidized cluster show that the two high-spin irons are antiferromagnetically coupled in accord with our preliminary study (Fox et al. J. Biol. Chem. 1988, 263, 10553-10556). Mossbauer studies also reveal the presence of two cluster conformations at pH 9. The excited-state S=2 multiplet of the exchange-coupled cluster (Fe 3+ -Fe 3+ ) gives rise to an integer-spin EPR signal near g=8; this is the first quantitative study of such a signal from any system

The active site Fe3+ of protocatechuate (PCA) 3,4-dioxygenase can be nonenzymatically reduced to Fe2+, to give a colorless and EPR-silent enzyme (Er). Nitric oxide (NO) binds to Er to yield a species with EPR (S = 3/2; g = 4.341, 3.693, 1.984; E/D = 0.055) and optical absorption (lambda max = 430 nm, epsilon approximately 1870 M-1 cm-1/iron) spectra. Addition of NO to a preformed Er. PCA complex results in a new species (EPR: S = 3/2; g = 4.920, 2.988, 1.846; E/D = 0.175; optical: lambda max = 404 nm, epsilon approximately 3930 M-1 cm-1/iron). Hyperfine broadening from the substrates [17O]PCA or [17O]homoprotocatechuate (HPCA) is observed in the EPR spectra of Er.substrate.NO complexes only when the 17O (I = 5/2) is placed in the carbon-4 OH group, suggesting that only this group binds to the iron when NO is bound. Previous studies (Orville, A. M., and Lipscomb, J. D. (1989) J. Biol. Chem. 261, 8791-8801) showed that both OH groups of HPCA can bind to the Fe3+ of the oxidized enzyme. Thus, the NO may compete with the substrate carbon-3 OH group for a binding site on the Fe2+. In contrast, when either PCA or HPCA is added to a preformed Er.NO complex, no substrate binding to the Fe2+ is detected. At 2.3 K, white light photodissociates NO from the Er.NO and Er.PCA.NO complexes. The Er.NO complex is photodissociated to a greater extent than the Er.PCA.NO complex, and different NO rebinding kinetics are observed showing that the substrate strongly influences the photodissociation/reassociation process. Photodissociation of each complex results in the formation of some Fe3+, suggesting that the nitrosyl complex has at least partial Fe(3+)-NO- character. In solution at 5-10 degrees C, white light promotes conversion of preformed Er.NO plus PCA to the Er.PCA.NO complex, suggesting that formation of the latter complex requires dissociation of NO. It is proposed that initial NO binding blocks the single site for exogenous ligand binding on the iron, thereby inhibiting PCA association. In contrast, PCA binding before NO appears to evoke an enzyme conformational change that allows simultaneous NO binding in another ligand site. These results are consistent with the current model for the mechanism of intradiol dioxygenases in which a PCA-induced conformational change allows substrate to bind as an Fe3+ chelate and O2 reacts initially with the PCA rather than the Fe3+.

Resonance Raman studies of the protocatechuate 3,4-dioxygenase (PCD) from Brevibacterium fuscum have been carried out to take advantage of the high iron-site homogeneity of this enzyme. Native uncomplexed PCD exhibits individual resonance-enhanced nu CO and delta CH vibrations for the two tyrosinates coordinated to the active site iron center, which can be assigned to a particular residue by their excitation profiles. Of the two nu CO features observed at 1254 and 1266 cm-1, only the latter is upshifted (to 1272 cm-1) when H2O is replaced by D2O. Similarly the 1254-cm-1 feature is unaffected, while the 1266-cm-1 feature is shifted to approximately 1290 cm-1 when inhibitors such as phenolates or terephthalate bind to the active site. These observed shifts can be rationalized by the presence of hydrogen-bonding interactions with solvent in the active site cavity, which are modulated by D2O and eliminated upon inhibitor binding. Examination of the PCD crystal structure suggests that the axial tyrosine can be hydrogen bonded in the uncomplexed enzyme to water molecules present in the substrate binding pocket. The equatorial tyrosine may also be hydrogen bonded but to solvent molecules which are trapped in a pocket inaccessible to bulk solvent. These studies allow for the first time the association of particular Raman spectroscopic features, i.e., the nu CO's at 1254 and 1266 cm-1, with the equatorial and axial tyrosine residues in the PCD active site, respectively; they lay the groundwork for further Raman studies on catalytically important species to determine the roles these tyrosine residues may play in the PCD reaction cycle.

Isopenicillin N synthase (IPNS) catalyzes double ring closure of the tripeptide (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of penicillin-type antibiotics. Our previous spectroscopic study using IPNS from Cephalosporium acremonium expressed in Escherichia coli [Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Munck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681] indicated that a thiolate enters the coordination of the essential active site Fe2+ when ACV binds to IPNS. The presence of an Fe-S bond in the IPNS.ACV complex is confirmed by EXAFS data presented in the preceding paper [Scott, R. A., Wang, S., Eidsness, M. K., Kriauciunas, A., Frolik, C. A. & Chen, V. J. (1992) Biochemistry (preceding paper in this issue)]. However, these studies leave unclear whether the coordinating thiolate derives from ACV or an endogenous cysteine. Here, we examine the spectroscopic properties of three genetically engineered variants of IPNS in which the only two endogenous cysteines are individually and collectively replaced by serine. The EPR, Mossbauer, and optical spectra of the mutant enzymes and their complexes with ACV, NO, or both ACV and NO are found to be essentially the same as those of wild-type IPNS, showing that the endogenous cysteines are not Fe2+ ligands in any of these complexes. Spectral quantitations show that the double Cys----Ser mutation decreases the affinity of the enzyme for ACV by about 6-fold, suggesting that the endogenous cysteines influence the structure of the substrate binding pocket remote from the iron. Thiolate complexation of the Fe2+ is also examined using ACV analogues. All ACV analogues examined in which the cysteinyl thiol moiety is unaltered are found to bind to the IPNS.NO complex to give optical and EPR spectra very similar to those of the ACV complex. In contrast, analogues in which the cysteinyl moiety of ACV is replaced with serine or cysteic acid fail to elicit the characteristic EPR and optical features despite the fact that they are bound with reasonable affinity to the enzyme. These results demonstrate that the thiolate of ACV coordinates the Fe2+. The EPR spectra of both the IPNS.NO and IPNS.ACV.NO complexes are broadened for samples prepared in 17O-enriched water, showing that water (or hydroxide) is also an iron ligand in each case. Thus, the Fe2+ coordination of the IPNS.ACV.NO complex accommodates at least three exogenous ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron nuclear double resonance (ENDOR) spectroscopy is used to probe the coordination of the mixed valence (Fe(II).Fe(III)) diiron cluster of the methane monooxygenase hydroxylase component (MMOH-) isolated from Methylosinus trichosporium OB3b. ENDOR resonances are observed along the principal axis directions g1 = 1.94 and g3 = 1.76 from at least nine different protons and two different nitrogens. The nitrogens are strongly coupled and appear to be directly coordinated to the cluster irons. The ratio of their superhyperfine coupling constants is roughly 4:7, which equals the ratio of the spin expectation values of the Fe(II) and Fe(III) in the ground state and suggests that at least one nitrogen is coordinated to each iron of the mixed valence cluster. Moreover, the superhyperfine and quadrupole coupling constants assigned to the Fe(III) site (AN = 13.6 MHz, PN = 0.7 MHz) are comparable with those observed for semimethemerythrin sulfide (AN = 12.1 MHz, PN = 0.7 MHz), for which the nitrogen ligands are histidines. At least three of the coupled protons exchange slowly when MMOH- is incubated in D2O, and 2H ENDOR resonances are subsequently observed. These observations are also consistent with histidine ligation of the iron cluster. On addition of the inhibitor dimethyl sulfoxide (Me2SO) to MMOH- the EPR spectrum sharpens and shifts dramatically. Only one set of 14N ENDOR resonances is observed with frequencies equal to those assigned to the Fe(III)-histidine resonances of uncomplexed MMOH- suggesting that the nitrogen coordination to the Fe(II) site is altered or possibly lost in the presence of Me2SO. 2H ENDOR resonances are observed in the presence of d6-Me2SO indicating that the inhibitor Me2SO binds near or possibly to the diiron cluster. In contrast, no 2H ENDOR resonances are observed from d4-methanol upon addition to MMOH-. Thus, the changes observed in the EPR spectrum of MMOH- upon addition of methanol may result from binding to a site away from the diiron cluster or from bulk solvent effects on the protein structure.