O-O bond cleavage and alkane hydroxylation in methane monooxygenase

Several new aspects of the O-O bond cleavage and alkane hydroxylation mechanisms have been studied by hybrid density functional theory in this reinvestigation of methane monooxygenase. As concerning key intermediates in these reactions, a new important low-lying state is found, described either as Fe2(III,V) or as Fe2(III,IV)O. A fully optimized transition state for O-O bond cleavage has been determined. It is suggested that the large difference in optimal size (as determined in gas phase) of the complex, before and after the O-O bond cleavage, leads to an additional driving force for the reaction, not considered previously. The strain of the enzyme is estimated to lead to a driving force in the forward direction of about 5 kcal/mol, which could explain some of the pH dependence found in recent experiments. For the hydroxylation reaction, a clean hydrogen abstraction transition state leading to a substrate radical is again found, in contrast to interpretations of radical clock experiments. An explanation, based on new results, is suggested that could account for both the experimental and theoretical results.     

Substrate binding and C-H bond activation in the soluble methane monooxygenase hydroxylase

 The selective oxidation of CH₄ to CH₃OH is a conceptually simple, yet functionally difficult, chemical transformation. In nature, this reaction is performed by methane monooxygenases, the soluble class of which employ carboxylate-bridged dinuclear iron centers to activate dioxygen. The process by which small molecules access the active site of the sMMO hydroxylase, the structures of intermediates in the catalytic reaction cycle, and mechanistic details about the attack on the C–H bond are subjects of intense investigation. In this commentary, we present our current views on exogenous ligand binding and dioxygen activation at the active site and the mechanism of alkane hydroxylation. Received: 20 October 1997 / Accepted: 26 February 1998     

Purification and properties of the hydroxylase component of methane monooxygenase.

Methane monooxygenase from Methylobacterium sp. strain CRL-26 which catalyzes the oxygenation of hydrocarbons was resolved into two components, a hydroxylase and a flavoprotein. An anaerobic procedure was developed for the purification of the hydroxylase to homogeneity. The molecular weight of the hydroxylase as determined by gel filtration was 220,000, and that determined by sedimentation equilibrium analysis was about 225,000. The purified hydroxylase contained three nonidentical subunits with molecular weights of about 55,000, 40,000, and 20,000, in equal amounts as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is an alpha 2 beta 2 gamma 2 protein. Optical absorption spectra revealed peaks near 408 and 280 nm, and fluorescence spectra revealed emission peaks at 490 and 630 nm. The purified hydroxylase contained 2.8 +/- 0.2 mol of iron and 0.5 +/- 0.1 mol of zinc per mol of protein but negligible amounts of acid-labile sulfide. The antisera prepared against the hydroxylase showed cross-reactivity with hydroxylase components in soluble extracts from other methanotrophs.     

Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O(2)- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (alphabetagamma)(2) complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the alpha subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.     

X-ray absorption spectroscopic studies of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b

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 Methylosinus trichosporium OB3b. Our X-ray absorption spectroscopic (XAS) analysis suggests the presence of two Fe-Fe distances of about 3?Å and 3.4?Å, 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.     

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

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 catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O-O bond cleavage

The oxidation of phenols to ortho-quinones, catalyzed by tyrosinase, has been studied using the hybrid DFT method B3LYP. Since no X-ray structure exists for tyrosinase, information from the related enzymes hemocyanin and catechol oxidase were used to set up a chemical model for the calculations. Previous studies have indicated that the direct cleavage of O(2) forming a Cu(2)(III,III) state is energetically very unlikely. The present study therefore followed another mechanism previously suggested. In this mechanism, dioxygen attacks the phenolate ring which is then followed by O[bond]O cleavage. The calculations give a reasonable barrier for the O(2) attack of only 12.3 kcal/mol, provided one of the copper ligands is able to move substantially away from its direct copper coordination. This can be achieved with six histidine ligands even if these ligands are held in their positions by the enzyme, but can also be achieved if one of the coppers only has two histidine ligands and the third ligand is water. The next step of O[bond]O cleavage has a computed barrier of 14.4 kcal/mol, in reasonable agreement with the experimental overall rate for the catalytic cycle. For the other steps of the mechanism, only a preliminary investigation was made, indicating a few problems which require future QM/MM studies.     

Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): Implications for substrate gating and component interactions

The crystal structure of the nonheme iron-containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 Å resolution. The enzyme is composed of two copies each of three subunits (α₂β₂γ₂), and all three subunits are almost completely α-helical, with the exception of two β hairpin structures in the α subunit. The active site of each α subunit contains one dinuclear iron center, housed in a four-helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4°C, a bridging acetate ion, which is replaced at −160°C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural difference identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position. Proteins 29:141–152, 1997. © 1997 Wiley-Liss, Inc.     

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.     

Ligation of the diiron site of the hydroxylase component of methane monooxygenase. An electron nuclear double resonance study.

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.     

Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B

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.     

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by M?ssbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.     

Mechanistic insights into C-H activation from radical clock chemistry: oxidation of substituted methylcyclopropanes catalyzed by soluble methane monooxygenase from Methylosinus trichosporium OB3b

The soluble methane monooxygenase (MMO) system isolated from Methylosinus trichosporium OB3b catalyzes the adventitious oxidation of alkyl substituted methylcyclopropanes. If the chemical mechanism of C-H activation by MMO involves formation of a radical or carbocation intermediate at the methyl C-H of these 'radical clock' substrates, then cyclopropyl ring opened alcohols may appear in the product mixture due to rearrangement of the intermediate. The lifetime of radical intermediates can be determined from known rearrangement rate constants, k(r). Rearrangement was observed during the oxidation of 1,1,2,2-tetramethylcyclopropane (k(r)=1.7-17. 5x10(8) s(-1), 30 degrees C) but not for cis- or trans-1, 2-dimethylcyclopropane (k(r)=1.2-6.4x10(8) s(-1), 30 degrees C) or the very fast radical clock, trans-2-phenylmethylcyclopropane (k(r)=3.4x10(11) s(-1), 30 degrees C). The results show that the occurrence of rearranged products fails to correlate with either the chemical nature of the C-H bond being broken, which is very similar for all of the methylcyclopropanes studied here, or the magnitude of the radical k(r) value. This study suggests that the steric properties of the substrate play an important role in determining the outcome of the reaction. Substrates with bulky substituents near the C-H bond that is attacked appear to yield intermediates with sufficient lifetimes to rearrange. In contrast, substrates with less steric bulk are postulated to be able to approach the reactive oxygen species in the MMO active site more closely so that intermediates are either rapidly quenched or undergo subsequent interaction with the dinuclear iron cluster of MMO that prevents rearrangement.     

Cofactor-independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode.

Soluble methane monooxygenase (sMMO) is a three-component enzyme that catalyses dioxygen- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Oxygenation occurs at the binuclear iron active centre in the hydroxylase component (MMOH), to which electrons are passed from NAD(P)H via the reductase component (MMOR), along a pathway that is facilitated and controlled by the third component, protein B (MMOB). We previously demonstrated that electrons could be passed to MMOH from a hexapeptide-modified gold electrode and thus cyclic voltammetry could be used to measure the redox potentials of the MMOH active site. Here we have shown that the reduction current is enhanced by the presence of catalase or if the reaction is performed in a flow-cell, probably because oxygen is reduced to hydrogen peroxide, by MMOH at the electrode surface and the hydrogen peroxide then inactivates the enzyme unless removed by catalase or a continuous flow of solution. Hydrogen peroxide production appears to be inhibited by MMOB, suggesting that MMOB is controlling the flow of electrons to MMOH as it does in the presence of MMOR and NAD(P)H. Most importantly, in the presence of MMOB and catalase, the electrode-associated MMOH oxygenates acetonitrile to cyanoaldehyde and methane to methanol. Thus the electochemically driven sMMO showed the same catalytic activity and regulation by MMOB as the natural NAD(P)H-driven reaction and may have the potential for development into an economic, NAD(P)H-independent oxygenation catalyst. The significance of the production of hydrogen peroxide, which is not usually observed with the NAD(P)H-driven system, is also discussed.     

Oxygen activation in neuronal NO synthase: resolving the consecutive mono-oxygenation steps

The vital signalling molecule NO is produced by mammalian NOS (nitric oxide synthase) enzymes in two steps. L-arginine is converted into NOHA (Nω-hydroxy-L-arginine), which is converted into NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor BH4 (tetrahydrobiopterin) activates dioxygen at the haem site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochrome P450, a haem-iron oxo species may be formed, or direct reaction between a haem-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. In the present paper we analyse the two reaction steps using the G586S mutant of nNOS (neuronal NOS), which introduces an additional hydrogen bond in the active site and provides an additional proton source. In the mutant enzyme, BH4 activates dioxygen as in the wild-type enzyme, but an interesting intermediate haem species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-arginine) indicating that the extra hydrogen bond enables it to discriminate between the two mono-oxygenation steps. This implies that the two steps follow different chemical mechanisms.     

Protein motions and the activation of the CH bond catalyzed by dihydrofolate reductase

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Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.     

Role of the C-terminal region of the B component of Methylosinus trichosporium OB3b methane monooxygenase in the regulation of oxygen activation

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.     

Probing the mechanism of C-H activation: oxidation of methylcubane by soluble methane monooxygenase from Methylosinus trichosporium OB3b.

The soluble form of methane monooxygenase (MMO) isolated from methanotrophic bacteria catalyzes the O2-dependent conversion of methane to methanol, as well as the adventitious oxidation of many other hydrocarbons. In past studies, it was reported that the oxidation reaction of methylcubane, a radical clock substrate, catalyzed by MMO from Methylococcus capsulatus (Bath) gave only cubylmethanol as the product rather than methylcubanol(s) or rearranged products characteristic of a radical formed on the methyl group [Choi, S.-Y., Eaton, P. E., Hollenberg, P. F., Liu, K. E., Lippard, S. J., Newcomb, M., Putt, D. A., Upadhyaya, S. P., and Xiong, Y. (1996) J. Am. Chem. Soc. 118, 6547-6555]. Such a substrate radical intermediate would be expected if the mechanism of MMO involves hydrogen atom abstraction as indicated by many previous mechanistic studies. Here it is shown that the reaction of methylcubane with the reconstituted MMO system from Methylosinus trichosporium OB3b yields both cubylmethanol and methylcubanols, with methyl hydroxylation favored over cubyl hydroxylation. This unexpected regioselectivity indicates steric effects on the reaction in agreement with past product distribution studies. In addition, the apparent majority product of the reaction is tentatively assigned as one of the possible rearranged products for this radical probe, on the basis of gas chromatography and mass spectrometry data. This result suggests the formation of a radical intermediate in the reaction, thus supporting a radical-based mechanism for this form of MMO.