Cryptic stereospecificity of methane monooxygenase

Priestley, Nigel D.; Floss, Heinz G.; Froland, Wayne A.; Lipscomb, John D.; Williams, Philip G.; Morimoto, Hiromi

doi:10.1021/ja00045a037

Cited by 157 publications

(155 citation statements)

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“…In the first step of this mechanism a homolytic cleavage of a CH bond of substrate alkane leads to a radical intermediate, and in the second step the resultant alkyl radical moves to the newly formed OH group, resulting in the formation of an alcohol complex. 9 However, Newcomb, Lippard, and co-workers have suggested from radical-clock experiments that a measured lifetime of a putative radical species in the MMOH catalysis is shorter than ca. 150 fs, which is inconsistent with the formation of a discrete radical species.…”

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confidence: 99%

Quantum Chemical Studies on Dioxygen Activation and Methane Hydroxylation by Diiron and Dicopper Species as well as Related Metal–Oxo Species

Yoshizawa

2013

Bulletin of the Chemical Society of Japan

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Quantum chemical studies by the author and co-workers on dioxygen activation and methane hydroxylation by diiron and dicopper enzyme species as well as related metaloxo species are reviewed. The activation of the OO bond of dioxygen at the mono-and dimetal sites of metalloenzymes is an essential process in the initial catalytic stages of naturally occurring oxygenation reactions. A way of orbital thinking about dioxygen activation at diiron and dicopper enzymes is developed at the extended Hückel level of theory. Two different reaction pathways that lead from dimetal peroxo complexes with¯-η , and CuO + are systematically analyzed on the basis of density functional theory (DFT) calculations. An important feature in the reaction is the spin crossover between the highspin and low-spin potential energy surfaces in particular in the CH activation process. The spin inversion from the highspin state to the low-spin state effectively decreases the barrier height of CH activation. Another feature in the reaction is that no radical species is involved in the course of the hydroxylation because the methyl species formed as a result of the CH bond cleavage can directly coordinate to the metal active site. The coupling of the OH and CH 3 ligands occurs to produce methanol at the metal active center. The reaction profiles obtained from DFT calculations are similar to those of the Gif chemistry proposed by D. H. R. Barton, in particular the involvement of the HOMCH 3 species as an intermediate in the hydroxylation of methane. The nonradical mechanism is extended to methane hydroxylation by the diiron and dicopper species of methane monooxygenase (MMO). This mechanism is possible when the metal active center is coordinatively unsaturated to have a space for the coordination of the OH and CH 3 groups as ligands. Although compelling discussion to support the involvement of oxygen-and carbon-centered radicals is provided, the nonradical mechanism still seems to be applicable for methane hydroxylation from the viewpoint of reaction selectivity. Kinetic isotope effects (KIEs) in the CH activation process by the bare FeO + complex and diiron and dicopper enzyme models are compared with respect to the radical and nonradical mechanisms by using transition state theory.

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Quantum Chemical Studies on Dioxygen Activation and Methane Hydroxylation by Diiron and Dicopper Species as well as Related Metal–Oxo Species

Yoshizawa

2013

Bulletin of the Chemical Society of Japan

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“…Later radical clock studies revealed no radical rearrangement for the M. capsulatus (Bath) system and very little for the enzyme from M. trichosporium OB3b (19,24). Hydroxylations of chiral tritiated alkanes proceeded with ϳ30% inversion of stereochemistry, an amount corresponding to putative radical lifetimes (Ͻ100 fs) too short to be attributed to formation of a discrete radical intermediate (25,26).The possibility exists that substrate-dependent reactivity might invalidate the conclusions of research based on diagnostic substrate probes. In recent work, 3 we obtained evidence that an intermediate other than Q, the active high valent diiron species that effects methane hydroxylation (1-3), is competent to oxidize olefins.…”

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Oxidation of Ultrafast Radical Clock Substrate Probes by the Soluble Methane Monooxygenase from Methylococcus capsulatus(Bath)

Valentine

LeTadic-Biadatti

Toy

et al. 1999

Journal of Biological Chemistry

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Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenylcyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.The soluble methane monooxygenase (sMMO) 1 from Methylococcus capsulatus (Bath), the hydroxylase component of which contains a dinuclear nonheme iron center, effects the mixed function oxidation of substrate,Equation 1 (1-3). In methanotrophic bacteria from which sMMO is isolated, methane is the natural substrate, providing both carbon and energy for the growth of the cell. The kinetically difficult selective transformation of methane to methanol is carried out at the active site of a 251-kDa hydroxylase enzyme (MMOH) (4 -7), the structure of which has been determined by x-ray crystallography. Electrons are supplied from NADH via a 38.5-kDa reductase protein (MMOR) (4,8), and the reactivity of the system is modulated by a 15.8-kDa coupling protein (MMOB) (9, 10). 2In addition to methane, sMMO can oxidize a wide variety of substrates (12)(13)(14)(15). This property has allowed the use of radical clock substrate probes to derive mechanistic information about the hydroxylation step in the enzyme mechanism. Radical clocks were first described in 1980 (16) and calibrated (17) for use in mechanistic studies of proteins (18,19) and related model compounds (20,21). Specifically, these substrates can reveal whether radical intermediates are formed during a reaction and, if so, can establish their lifetime and the rate constant for radical rebound. Fig. 1 depicts the oxidation of a radical clock substrate probe, which uses the ring opening of a cyclopropylcarbinyl radical. If hydrogen atom abstraction from the probe occurs, the resulting radical can either rebound to form the unrearranged alcohol or undergo ring opening before rebound to afford the rearranged alcohol. If the ring opening rate is known (17), the rebound rate, k oxidation , can be calculated according to Equation 2. k oxidation ϭ k ring opening ͑unrearranged alcohol / rearranged alcohol) (Eq. 2)The hypothesis that substrate radical intermediates form in the sMMO s...

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“…One involves the radical mechanism, wherein an activated oxygen atom species abstracts a hydrogen atom from the substrate methane molecule, followed by radical rebound chemistry of the methyl radical with the hot hydroxyl radical to form the product (19). The other mechanism invokes anchoring of the methane molecule at an activated metal cluster and concerted oxenoid or oxene insertion across one of the C-H bonds via the formation of a four-or three-centered transition state (20,21).…”

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“…When the hydroxylation chemistry mediated by sMMO from Methylosinus trichosporium OB3b (19) and Methylococcus capsulatus (Bath) (22) was investigated by the use of cryptically chiral ethanes, stereochemical analysis of the products revealed partial racemization of the chiral ethanol formed. These results have been interpreted in terms of the formation of a short-lived alkyl radical (19), or one that is so short-lived that the mechanism is essentially concerted yet non-synchronous (22,23). This conclusion seems to be corroborated by stopped flow experiments designed to delineate the kinetic steps of the hydroxylation chemistry in the case of the M. trichosporium (OB3b) enzyme (24).…”

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The Stereospecific Hydroxylation of [2,2-2H2]Butane and Chiral Dideuteriobutanes by the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)

Chen

et al. 2003

Journal of Biological Chemistry

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Experiments on cryptically chiral ethanes have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of configuration at the carbon center attacked. This result would seem to rule out a radical mechanism for the hydroxylation chemistry, at least as mediated by this enzyme. The interpretation of subsequent experiments on n-propane, n-butane, and n-pentane has been complicated by hydroxylation at both the pro-R and pro-S secondary C-H bonds, where the hydroxylation takes place. It has been suggested that these results merely reflect presentation of both the pro-R and pro-S C-H bonds to the hot "oxygen atom" species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of configuration. In the present work, we have augmented these earlier studies with experiments on [2,2-2 H 2 ]butane and designed d,l form chiral dideuteriobutanes. Essentially equal amounts of (2R)-[3,3-2 H 2 ]butan-2-ol and (2R)-[2-2 H 1 ]butan-2-ol are produced upon hydroxylation of [2,2-2 H 2 ]butane. The chemistry is stereospecific with full retention of configuration at the secondary carbon oxidized. In the case of the various chiral deuterated butanes, the extent of configurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of configuration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet oxene insertion across the C-H bond similar to that previously noted for singlet carbene insertion (Kirmse, W., and Ö zkir, I. S. (1992) J. Am. Chem. Soc. 114, 7590؊7591). Finally, we discuss how even the oxene insertion mechanism, with "spin crossover" in the transition state, could lead to small amounts of radical rearrangement products, if and when such products are observed. A scheme is described that unifies the two extreme mechanistic limits, namely the concerted oxene insertion and the hydrogen abstraction radical rebound mechanism within the same over-arching framework.

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Cryptic stereospecificity of methane monooxygenase

Cited by 157 publications

References 0 publications

Quantum Chemical Studies on Dioxygen Activation and Methane Hydroxylation by Diiron and Dicopper Species as well as Related Metal–Oxo Species

Quantum Chemical Studies on Dioxygen Activation and Methane Hydroxylation by Diiron and Dicopper Species as well as Related Metal–Oxo Species

Oxidation of Ultrafast Radical Clock Substrate Probes by the Soluble Methane Monooxygenase from Methylococcus capsulatus(Bath)

The Stereospecific Hydroxylation of [2,2-2H2]Butane and Chiral Dideuteriobutanes by the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)

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