Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

Froland, Wayne A.; Andersson, Karin; Lee, Sang‐Kyu; Liu, Yi; Lipscomb, John D.

doi:10.1016/s0021-9258(19)37083-8

Cited by 146 publications

(157 citation statements)

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“…Protein B is a 16 kDa polypeptide with no metal or prosthetic groups . It has been implicated in several roles, including the coupling of electron transfer by the reductase with hydroxylation of substrate and affecting the rate and regioselectively of substrate oxidation Fox et al 1991;Froland et al 1992). Protein B has also been shown to shift the redox potential values of the hydroxylase (Liu & Lippard 1991;Lee et al 1993;Paulsen et al 1994;Liu et al 1995a;Kazlauskaite et al 1996).…”

Section: The Regulatory/effector Protein B (Mmob)mentioning

confidence: 99%

“…Such a role may be achieved through cyclic association and dissociation of the reductase-hydroxylase complex as the hydroxylase oscillates between redox states during catalysis. Binding of protein B has been detected by electron paramagnetic resonance (EPR) spectroscopy for all three oxidation states of the hydroxylase (Fox et al 1991;Froland et al 1992;Davydov et al 1997Davydov et al , 1999. As B binds, the negative shift in the mid-point potential of the hydroxylase suggests that this allows the diferric hydroxo-bridged diiron cluster of A to be reduced by the NADHcoupled reductase to lower its redox potential, thus making it bind to oxygen more easily.…”

Section: The Regulatory/effector Protein B (Mmob)mentioning

confidence: 99%

“…The reductase component of sMMO from M. trichosporium OB3b has been shown to bind to the b subunit of the hydroxylase (Fox et al 1991), and, in addition to its role as a supplier of electrons, recent evidence suggests that the reductase could also have a regulatory function given that: (i) it causes a shift in product distribution (Froland et al 1992); (ii) the reductase can bind to the hydroxylase at different sites and with different affinities (Fox et al 1991); and (iii) the redox potential of the hydroxylase is altered by the reductase (Paulsen et al 1994;Liu et al 1997).…”

Section: The Reductase (Mmor)mentioning

confidence: 99%

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The Leeuwenhoek Lecture 2000 The natural and unnatural history of methane-oxidizing bacteria

Dalton

2005

Phil. Trans. R. Soc. B

116

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Methane gas is produced from many natural and anthropogenic sources. As such, methane gas plays a significant role in the Earth's climate, being 25 times more effective as a greenhouse gas than carbon dioxide. As with nearly all other naturally produced organic molecules on Earth, there are also micro-organisms capable of using methane as their sole source of carbon and energy. The microbes responsible (methanotrophs) are ubiquitous and, for the most part, aerobic. Although anaerobic methanotrophs are believed to exist, so far, none have been isolated in pure culture. Methanotrophs have been known to exist for over 100 years; however, it is only in the last 30 years that we have begun to understand their physiology and biochemistry. Their unique ability to use methane for growth is attributed to the presence of a multicomponent enzyme system-methane monooxygenase (MMO)-which has two distinct forms: soluble (sMMO) and membrane-associated (pMMO); however, both convert methane into the readily assimilable product, methanol. Our understanding of how bacteria are capable of effecting one of the most difficult reactions in chemistry-namely, the controlled oxidation of methane to methanol-has been made possible by the isolation, in pure form, of the enzyme components.The mechanism by which methane is activated by sMMO involves abstraction of a hydrogen atom from methane by a high-valence iron species (FeIV or possibly FeV) in the hydroxylase component of the MMO complex to form a methyl radical. The radical combines with a captive oxygen atom from dioxygen to form the reaction product, methanol, which is further metabolized by the cell to produce multicarbon intermediates. Regulation of the sMMO system relies on the remarkable properties of an effector protein, protein B. This protein is capable of facilitating component interactions in the presence of substrate, modifying the redox potential of the diiron species at the active site. These interactions permit access of substrates to the hydroxylase, coupling electron transfer by the reductase with substrate oxidation and affecting the rate and regioselectivity of the overall reaction. The membrane-associated form is less well researched than the soluble enzyme, but is known to contain copper at the active site and probably iron. From an applied perspective, methanotrophs have enjoyed variable successes. Whole cells have been used as a source of single-cell protein (SCP) since the 1970s, and although most plants have been mothballed, there is still one currently in production. Our earlier observations that sMMO was capable of inserting an oxygen atom from dioxygen into a wide variety of hydrocarbon (and some non-hydrocarbon) substrates has been exploited to either produce value added products (e.g. epoxypropane from propene), or in the bioremediation of pollutants such as chlorinated hydrocarbons. Because we have shown that it is now possible to drive the reaction using electricity instead of expensive chemicals, there is promise that the system could be exploited as a s...

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Section: The Regulatory/effector Protein B (Mmob)mentioning

confidence: 99%

Section: The Regulatory/effector Protein B (Mmob)mentioning

confidence: 99%

Section: The Reductase (Mmor)mentioning

confidence: 99%

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The Leeuwenhoek Lecture 2000 The natural and unnatural history of methane-oxidizing bacteria

Dalton

2005

Phil. Trans. R. Soc. B

116

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“…Additionally, sequence alignment and structural modelling show that all residues directly lining the active site cavity of BMOH are the same as those in the methane monooxygenase hydroxylase (MMOH). Despite the similarities, several biochemical observations indicate that sBMO represents a class distinct from the sMMO family: (i) methanol accumulation ceases during methane oxidation once~20-50 mM has been reached (Halsey et al, 2006), whereas sMMO can continue to accumulate methanol; (ii) sBMO is predominantly a terminal hydroxylator of intermediate-chain-length alkanes (Dubbels et al, 2007), whereas sMMO forms secondary alcohols (Froland et al, 1992); (iii) product regioselectivity is minimally altered by the presence of BMOB (Dubbels et al, 2007), unlike sMMO (Froland et al, 1992); (iv) catalase is required to maintain hydroxylase activity during steady-state turnover (Dubbels et al, 2007) but not to maintain sMMO activity; and (v) the use of peroxide in the 'peroxide shunt' mechanism of substrate oxidation is three orders of magnitude less efficient in sBMO than in sMMO (Dubbels et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

et al. 2009

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Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C 2 -C 9 alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C 1 -C 5 alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by k cat : K m ratios). Relative rates of oxidation for C 1 -C 5 alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K m values. The low micromolar K m for linear C 2 -C 5 alkanes and the millimolar K m for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar K m and k cat values to those of methane. This inability to kinetically discriminate between the C 1 alkane and C 1 alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C 2 -C 5 do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C 3 -C 5 alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes ¢C 2, despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.

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“…22,23 The reductive activation of dioxygen is a complicated procedure that is difficult to control under artificial experimental conditions. Therefore, the use of peroxides (the so-called shunt path) 24 was the main avenue of research during the early stages of research on metal-oxo complexes. However, oxidation reactions using peroxides to form metal-oxo complexes as the active species have a problem: low selectivity toward products as a result of the formation of undesirable radical species.…”

Section: Introductionmentioning

confidence: 99%

Oxidation of Organic Substrates with RuIV=O Complexes Formed by Proton-Coupled Electron Transfer

Ishizuka

Ohzu

Kojima

2014

Synlett

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We describe our recent progress in mechanistic investigations on substrate oxidation reactions with Ru IV =O complexes having pyridylamine ligands. The Ru IV =O complexes are synthesized from the corresponding Ru II -OH 2 complexes either through a proton-coupled electron-transfer procedure using a cerium(IV) complex as an oxidant or by bulk electrolysis. We have verified the presence of an adduct-formation equilibrium between the Ru IV =O complex and a substrate in water, which strongly affects the reaction mechanism of the substrate oxidation. We have also shown that the reaction rates of the substrate oxidation in water are independent of the spin states of the Ru IV centers and of the bond-dissociation enthalpy of the C-H bond of the substrate. Additionally, we have applied the Ru II -OH 2 complexes as catalysts for photocatalytic substrate oxidations and have observed very efficient catalytic activity.

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Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

Cited by 146 publications

References 46 publications

The Leeuwenhoek Lecture 2000 The natural and unnatural history of methane-oxidizing bacteria

The Leeuwenhoek Lecture 2000 The natural and unnatural history of methane-oxidizing bacteria

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

Oxidation of Organic Substrates with RuIV=O Complexes Formed by Proton-Coupled Electron Transfer

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