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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 O2 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.

(Cell Chemical Biology 24, 1259–1275; October 19, 2017) During the cropping of Figure 2H, the image of the p62 row was deleted in error and then remaining labels were shifted down by one row and were therefore out of registration with the images. Figure 2H has now been corrected in the article online and in print; the corrected Figure 2H is also shown below. The authors apologize for this labeling error.

Transient kinetic studies of sMMO have revealed eight reaction cycle intermediates, thereby providing the most comprehensive description of enzymatic [O.sub.2] activation and C-H bond oxidation currently available for any di-iron [...], Methane monooxygenase (MMO) catalyses the [O.sub.2]-dependent conversion of methane to methanol in methanotrophic bacteria, thereby preventing the atmospheric egress of approximately one billion tons of this potent greenhouse gas annually. The key reaction cycle intermediate of the soluble form of MMO (sMMO) is termed compound Q (Q). Q contains a unique dinuclear [Fe.sup.IV] cluster that reacts with methane to break an exceptionally strong 105 kcal [mol.sup.-1] C-H bond and insert one oxygen atom (1,2). No other biological oxidant, except that found in the particulate form of MMO, is capable of such catalysis. The structure of Q remains controversial despite numerous spectroscopic, computational and synthetic model studies (2-7). A definitive structural assignment can be made from resonance Raman vibrational spectroscopy but, despite efforts over the past two decades, no vibrational spectrum of Q has yet been obtained. Here we report the core structures of Q and the following product complex, compound T, using time-resolved resonance Raman spectroscopy (TR3). TR3 permits fingerprinting of intermediates by their unique vibrational signatures through extended signal averaging for shortlived species. We report unambiguous evidence that Q possesses a bis-p-oxo diamond core structure and show that both bridging oxygens originate from [O.sub.2]. This observation strongly supports a homolytic mechanism for O-O bond cleavage. We also show that T retains a single oxygen atom from [O.sub.2] as a bridging ligand, while the other oxygen atom is incorporated into the product (8). Capture of the extreme oxidizing potential of Q is of great contemporary interest for bioremediation and the development of synthetic approaches to methane-based alternative fuels and chemical industry feedstocks. Insight into the formation and reactivity of Q from the structure reported here is an important step towards harnessing this potential.

[Fe.sup.III]-[O.sub.2.sup.*-] intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear [Fe.sup.II] enzyme family. Many steps in the [O.sub.2] activation and reaction cycle of [Fe.sup.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.sub.2] to the H200N-4NC complex is trapped and characterized using EPR and Mossbauer (MB) spectroscopies. Int-1 is a high-spin ([S.sub.1] = 5/2) [Fe.sup.III] antiferromagnetically (AF) coupled to an [S.sub.2] = 1/2 radical (J [approximately equal to] 6 [cm.sup.-1] in H = [JS.sub.1]*[S.sub.2]). 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 [sup.17][O.sub.2] hyperfine interactions ([A17.sub.O] [approximately equal to] 180 MHz). Thus, Int-1 is an AF-coupled [Fe.sup.III]-[O.sub.2.sup.*-] species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound [O.sub.2]. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled [Fe.sup.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 [sup.17][O.sub.2] ( oxygen activation | oxygenase | spectroscopy | superoxide doi/ 10.1073/pnas.1010015107

Near-infrared magnetic circular dichroism (MCD) and variable temperature, variable field (VTVH) MCD are applied to naphthalene 1,2-dioxygenase (NDO) in order to describe the coordination geometry and electronic structure of the catalytic ferrous site in the resting and substrate-bound forms with both oxidized and reduced Rieske 2Fe2S clusters. The studies have shown the correlation with the crystal structures of NDO and other ROs and have provided molecular level mechanistic insights for this class of enzymes.

A combination of studies is employed to analyze the thiolate bonding to [{FeNO}.sup.2]/[{[FeO.sub.2]}.sup.8] complexes of isopencillin N synthase (IPNS). The determination of oxidase substrate versus the oxygenase activity in nonheme Fe enzymes shows that the single center reaction in IPNS allows it to avoid the oxygen bridged binding completely.

The disordered C-terminal region of the B component (MMOB) of soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b performs a critical role in the regulation of catalysis. Electron paramagnetic resonance (EPR) studies of MMOH active site diiron cluster show that 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.

A study based is used to demonstrate that in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, and the catechol binds to the Fe(III) as a dianion. It can be concluded that the basis of the respective regiospecificities of intradiol and extradiol dioxydenase linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex.

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, [Mn.sup.2+] and [Fe.sup.2+], 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 super-family); each monomer has four [beta][alpha][beta][beta][beta] 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, H[214.sup.NE1] and E[267.sup.OE1]; one axial ligand, H[155.sup.NE1]; 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 [Angstrom]), 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 sites trans to 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 site trans to E267 is the likely binding site for [O.sup.2]. 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 [O.sup.2] during catalysis are identified and discussed in the context of current mechanistic hypotheses.

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. [ABSTRACT FROM AUTHOR]

Research has been conducted on Pseudomonas putida benzoate 1,2-dioxygenase system. The use of the two-component Rieske dioxygenase system in demonstrating the ability of its oxygenase component to leave both metal centers in the oxidized states is described.

Research has been conducted on the Fe (super)II active site in 1- aminocyclopropane-1-carboxylic acid oxidase. The geometric and electronic structure of this active site in aminocyclopropane-1-carboxylic acid oxidase and its interactions with the substrate aminocyclopropane-1-carboxylic acid, cosubstrate ascorbate and activator CO (sub)2 have been examined and are described.

Research has been conducted on the methane monooxygenase, a non-heme-iron-containing enzyme. The interactions between this monooxygenase and the oxidized states of a methane monooxygenase hydroxylase have been investigated via NMR spectroscopy.

Research has been conducted on the binding of the component B to the hydroxylase component. The identification of the amino acid residues of the component B that affect the interconversion of the methane monooxygenase cycle intermediates has been conducted and results are presented.