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Time-resolved resonance Raman vibrational spectroscopy was used to study the mechanism of soluble methane monooxygenase and obtain structural information on the key reaction cycle intermediate, compound Q , which contains a unique dinuclear Fe IV cluster that breaks the strong C-H bond of methane and inserts an oxygen atom (from O 2 ) to form methanol. Greenhouse gas to liquid fuel via Q Using time-resolved resonance Raman vibrational spectroscopy, Rahul Banerjee et al . have determined the structure of 'compound Q', a key transient intermediate from the soluble methane monooxygenase (sMMO) system found in methanotrophic bacteria. Q is the strongest known biological oxidant and catalyses cleavage of the ultimately stable C–H bond of methane with insertion of oxygen to form the liquid fuel methanol. With a better understanding of the structure and mechanism of action of Q it might be possible to synthesize small molecule enzyme mimetics that could convert naturally occurring methane to methanol, thereby converting a damaging greenhouse gas into a potentially important source of liquid fuel and chemicals. Methane monooxygenase (MMO) catalyses the O 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 IV cluster that reacts with methane to break an exceptionally strong 105 kcal mol −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 , 3 , 4 , 5 , 6 , 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 (TR 3 ). TR 3 permits fingerprinting of intermediates by their unique vibrational signatures through extended signal averaging for short-lived species. We report unambiguous evidence that Q possesses a bis-μ-oxo diamond core structure and show that both bridging oxygens originate from O 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 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.

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

We report the structures of three intermediates in the O₂ activation and insertion reactions of an extradiol ring-cleaving dioxygenase. A crystal of Fe²⁺-containing homoprotocatechuate 2,3-dioxygenase was soaked in the slow substrate 4-nitrocatechol in a low O₂ atmosphere. The x-ray crystal structure shows that three different intermediates reside in different subunits of a single homotetrameric enzyme molecule. One of these is the key substrate-alkylperoxo-Fe²⁺ intermediate, which has been predicted, but not structurally characterized, in an oxygenase. The intermediates define the major chemical steps of the dioxygenase mechanism and point to a general mechanistic strategy for the diverse 2-His-1-carboxylate enzyme family.

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 Fe2+/Fe2+ state and a cisμ-1,2(η1:η1)-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(η1:η2) 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(η1:η1)-peroxo complex differs from the μ-η1:η2-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.

Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe ³⁺ to activate O ₂ and catecholic substrates for reaction. The inability of Fe ³⁺ to directly bind O ₂ 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 O ₂ 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. Significance Vast quantities of aromatic compounds enter the environment due to the natural breakdown of lignin as well as industrial and agricultural pollution. Intradiol aromatic ring-cleaving dioxygenases play a pivotal role in the biodegradation of these aromatics. Despite exhaustive study, the mechanism of intradiol dioxygenases has remained elusive because the reaction cycle intermediates in which O ₂ is activated and inserted into the aromatic are too fleeting to be trapped and characterized. Here the intradiol dioxygenase reaction is carried out in a crystal, allowing the two reaction cycle intermediates that most clearly define the mechanism to be trapped and their structures solved.

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.

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

The uncatalyzed reactions of O₂ (S = 1) with organic substrates (S = 0) are thermodynamically favorable but kinetically slow because they are spin-forbidden and the one-electron reduction potential of O₂ is unfavorable. In nature, many of these important O₂ reactions are catalyzed by metalloenzymes. In the case of mononuclear non-heme iron enzymes, either FeII or FeIII can play the catalytic role in these spin-forbidden reactions. Whereas the ferrous enzymes activate O₂ directly for reaction, the ferric enzymes activate the substrate for O₂ attack. The enzyme-substrate complex of the ferric intradiol dioxygenases exhibits a low-energy catecholate to FeIII charge transfer transition that provides a mechanism by which both the Fe center and the catecholic substrate are activated for the reaction with O₂. In this Perspective, we evaluate how the coupling between this experimentally observed charge transfer and the change in geometry and ligand field of the oxidized metal center along the reaction coordinate can overcome the spin-forbidden nature of the O₂ reaction.

Biological O₂ activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-to-O₂ 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 O₂ 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 KM and Vmax 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.

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_M^O_ 2 \\) and kcat. 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.