Soluble Methane Monooxygenase.

Aerobic life is possible because the molecular structure of oxygen (O2) makes direct reaction with most organic materials at ambient temperatures an exceptionally slow process. Of course, these reactions are inherently very favorable, and they occur rapidly with the release of a great deal of energy at high temperature. Nature has been able to tap this sequestered reservoir of energy with great spatial and temporal selectivity at ambient temperatures through the evolution of oxidase and oxygenase enzymes. One mechanism used by these enzymes for O2 activation has been studied in detail for the soluble form of the enzyme methane monooxygenase. These studies have revealed the step-by-step process of O2 activation and insertion into the ultimately stable C-H bond of methane. Additionally, an elegant regulatory mechanism has been defined that enlists size selection and quantum tunneling to allow methane oxidation to occur specifically in the presence of more easily oxidized substrates.

X-ray Crystal Structures of Methane Monooxygenase Hydroxylase Complexes with Variants of Its Regulatory Component: Correlations with Altered Reaction Cycle Dynamics.

Full activity of soluble methane monooxygenase (sMMO) depends upon the formation of a 1:1 complex of the regulatory protein MMOB with each alpha subunit of the (αßγ)2 hydroxylase, sMMOH. Previous studies have shown that mutations in the core region of MMOB and in the N- and C-termini cause dramatic changes in the rate constants for steps in the sMMOH reaction cycle. Here, X-ray crystal structures are reported for the sMMOH complex with two double variants within the core region of MMOB, DBL1 (N107G/S110A), and DBL2 (S109A/T111A), as well as two variants in the MMOB N-terminal region, H33A and H5A. DBL1 causes a 150-fold decrease in the formation rate constant of the reaction cycle intermediate P, whereas DBL2 accelerates the reaction of the dinuclear Fe(IV) intermediate Q with substrates larger than methane by three- to fourfold. H33A also greatly slows P formation, while H5A modestly slows both formation of Q and its reactions with substrates. Complexation with DBL1 or H33A alters the position of sMMOH residue R245, which is part of a conserved hydrogen-bonding network encompassing the active site diiron cluster where P is formed. Accordingly, electron paramagnetic resonance spectra of sMMOH:DBL1 and sMMOH:H33A complexes differ markedly from that of sMMOH:MMOB, showing an altered electronic environment. In the sMMOH:DBL2 complex, the position of M247 in sMMOH is altered such that it enlarges a molecular tunnel associated with substrate entry into the active site. The H5A variant causes only subtle structural changes despite its kinetic effects, emphasizing the precise alignment of sMMOH and MMOB required for efficient catalysis.

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 µM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.

Structural Studies of the Methylosinus trichosporium OB3b Soluble Methane Monooxygenase Hydroxylase and Regulatory Component Complex Reveal a Transient Substrate Tunnel.

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high-resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O2 access routes enhanced when the complex forms. Previously reported, lower-resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O2 conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O2 conduits in the M. trichosporium OB3b sMMOH:MMOB complex revealed a narrow molecular tunnel, termed the W308-tunnel. This tunnel is sized appropriately for O2 and traverses the sMMOH-MMOB interface before accessing the active site. The kinetics of reaction of O2 with the diferrous sMMOH:MMOB complex in solution show that use of the MMOB V41R variant decreases the rate constant for O2 binding >25000-fold without altering the component affinity. The location of Val41 near the entrance to the W308-tunnel is consistent with the tunnel serving as the primary route for the transfer of O2 into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-tunnel.

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

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.

Currency Devaluation as a Source of Growth in Africa: A Synthetic Control Approach.

This study examines the impact of the 1994 IMF-supported CFA franc devaluation on GDP per capita in the CFA-franc zone using the augmented synthetic control methodology. With the exception of Mali, there is no statistical evidence that GDP per capita levels rose relative to what they would have been in the absence of the IMF-supported devaluation. Three countries record statistically significant GDP per capita levels below the counterfactual following the devaluation, though these countries experienced a deterioration of their national institutional environment or were affected by external factors that offset any potential gains from the devaluation.

Filter-feeding bivalves store and biodeposit colloidally stable gold nanoparticles.

Nanoparticles resistant to salt-induced aggregation are continually being developed for biomedical and industrial applications. Because of their colloidal stability these functionalized nanoparticles are anticipated to be persistent aquatic contaminants. Here, we show that Corbicula fluminea, a globally distributed clam that is a known sentinel of aquatic ecosystem contamination, can uptake and biodeposit bovine serum albumin (BSA) stabilized gold nanoparticles. Nanoparticle clearance rates from suspension were dictated by diameter and concentration, with the largest particles cleared most quickly on a mass basis. Particle capture facilitates size-selective 'biopurification' of particle suspensions with nanoscale resolution. Nanoparticles were retained either within the clam digestive tract or excreted in feces. Our results suggest that biotransformation and biodeposition will play a significant role in the fate and transport of persistent nanoparticles in aquatic systems.

Biotransformation of the insensitive munition constituents 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN) by aerobic methane-oxidizing consortia and pure cultures.

We present the first report of biotransformation of 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN), replacements for the explosives 1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT), respectively, by methane-oxidizing cultures under aerobic conditions. Two consortia, dominated by Methylosinus spp., degraded both compounds with transient production of reduced NTO products, and non-stoichiometric production of reduced DNAN products. No release of inorganic nitrogen was observed with either compound, indicating that NTO and DNAN may be utilized as nitrogen sources by these consortia. The pure culture Methylosinus trichosporium OB3b also degraded both compounds. Degradation was observed in the presence of acetylene (a known inhibitor of methane monooxygenase; MMO) when methanol was supplied, indicating that MMO was not involved. Furthermore, studies with purified soluble MMO (sMMO) from OB3b indicated that neither compound was a substrate for sMMO. Degradation was inhibited by 2-iodosobenzoic acid, but not by dicoumarol, suggesting involvement of an oxygen- and dicoumarol-insensitive (nitro)reductase. These results indicate methanotrophs can aerobically degrade NTO and DNAN via one or more (nitro)reductases, with sMMO serving a supporting role deriving reducing equivalents from methane. This finding is important because methanotrophic bacteria are widely dispersed, and may represent a previously unrecognized route of NTO and DNAN biotransformation in aerobic environments.

Comparison of chemical lithography using alkanethiolate self-assembled monolayers on GaAs (001) and Au.

We have investigated the efficiency of bifunctional pattern formation in alkanethiolate self-assembled monolayers (SAMs) adsorbed on GaAs (001) and Au, using time-of-flight secondary ion mass spectrometry. Two patterning techniques were employed: electron beam lithography and UV photopatterning. Previous work has always assumed that complete degradation of the SAM was necessary for the formation of well-defined multifunctional patterned surfaces, requiring large electron doses or long UV irradiation times. We demonstrate that well-defined multifunctional patterned surfaces can be produced on GaAs (001) with only partial degradation of the SAM, allowing greatly reduced electron beam doses and UV irradiation times to be used. Using electron beam lithography we observe that sharp well-defined patterns can form after an electron dose as low as 450 microC cm(-2). We also demonstrate that only 50% of the monolayer must be photooxidized in UV photopatterning, reducing the exposure time needed by a factor of 3. In contrast, patterning of alkanethiolate SAMs adsorbed on Au requires much higher electron doses (> or = 1250 microC cm(-2)) and photooxidation times (2 h). The substantial differences observed on these two substrates appear to arise from differences in the SAM structure on GaAs and Au. These results suggest that alkanethiolate SAM resists may be a suitable technology for nanometer scale lithography of GaAs and possibly other semiconductors.

Molecular orientation in octanedithiol and hexadecanethiol monolayers on GaAs and Au measured by infrared spectroscopic ellipsometry.

Infrared spectroscopic ellipsometry was used for determination of molecular orientation and for lateral homogeneity studies of organic monolayers on GaAs and Au, the organic layer being either octanedithiol or hexadecanethiol (HDT). The laterally resolved measurements were performed with the infrared mapping ellipsometer at the synchrotron storage ring BESSY II. The molecular orientation within the monolayers was determined by optical model simulations of the measured ellipsometric spectra. Different tilt angles were obtained for the monolayers of HDT and octanedithiol on GaAs: 19 degrees and >30 degrees , respectively. The tilt angle of the methylene chains for HDT on Au substrate (22 degrees ) is similar to the 19 degrees tilt which was obtained for the HDT monolayers on GaAs, thus suggesting similar molecular ordering of the thiolates on both substrates.