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Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification.

Background and Aims Severely low soil nutrient status and malnutrition or "hidden hunger" are two serious global problems. The consumption of rice constitutes approximately 20% of human caloric intake. Trace elements like zinc (Zn) is essential nutrient for rice growth, and to the populations depend on rice staples. This research examines the speciation of Zn in rice, how that speciation is impacted by soil fertility and its potential effect on malnutrition. Methods The composition and Zn speciation of Cambodian rice grain is analyzed using synchrotron-based microprobe X-ray fluorescence (µ-XRF) and extended X-ray absorption fine-structure spectroscopy (EXAFS). Results Zn levels in rice grain ranged between 15–30 mg kg-1 and were not correlated to Zn availability in soils. 72%-90% of Zn in rice grains is present as Zn-phytate, generally not bioavailable, while smaller quantities of Zn are bound as labile nicotianamine complexes, Zn minerals like ZnCO3 or thiols, using EXAFS method based on coordination numbers for Zn binding to oxygen and sulfur. Conclusion Zn speciation in rice grain is affected by nutrient limitation more than previously recognized. The Zn phytate concentration in rice grain was highest for rice produced in Zn-deficient soils, consistent with increased phytate production under nutrient limitation. Phytates are generally not bioavailable, so low soil Zn fertility may not only impact grain yields, but also decrease the fraction of bioavailable Zn in grain consumers. The reduced accessibility of human zinc due to low soil nutrient levels and its exacerbation of hidden hunger may require widespread global attention.

Au nanoparticles (NP) on TiO2 have been shown to be effective catalysts for selective oxidation reactions by using molecular oxygen. In this work, we have studied the influence of support morphology on the catalytic activity of Au/TiO2 catalysts. Two TiO2 anatase supports, a nanoplatelet-shaped material with predominantly the {001} facet exposed and a truncated bipyramidal-shaped nanoparticle with predominantly the {101} facet exposed, were prepared by using a nonaqueous solvothermal method and characterized by using DRIFTS, XPS, and TEM. Au nanoparticles were deposited on the supports by using the deposition-precipitation method, and particle sizes were determined by using STEM. Au nanoparticles were smaller on the support with the majority of the {101} facet exposed. The resulting materials were used to catalyze the aerobic oxidation of benzyl alcohol and trifluoromethylbenzyl alcohol. Support morphology impacts the catalytic activity of Au/TiO2; reaction rates for reactions catalyzed by the predominantly {101} material were higher. Much of the increased reactivity can be explained by the presence of smaller Au particles on the predominantly {101} material, providing more Au/TiO2 interface area, which is where catalysis occurs. The remaining modest differences between the two catalysts are likely due to geometric effects as Hammett slopes show no evidence for electronic differences between the Au particles on the different materials.

Understanding and quantifying how the active sites in supported metal catalysts can be modified are critical for rationally designing catalysts. This problem is particularly complex for reactions t...

Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert C-H bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.

Zinc is important in neurogenesis, but excessive levels can cause apoptosis and other pathologies leading to cognitive impairments. Mast cells are present in many brain regions including the hippocampus, an area rich in vesicular zinc. Mast cells contain zinc-rich granules and a well-developed mechanism for uptake of zinc ions; both features point to the potential for a role in zinc homeostasis. Prior work using the Timm stain supported this hypothesis, as increased labile zinc was detected in the hippocampus of mast cell-deficient mice compared to wild-type mice while no differences in total zinc were found between the two genotypes in the whole brain or other tissues. The current report further examines differences in zinc homeostasis between wild-type and mast cell-deficient mice by exploring the zinc transporter ZnT3, which transports labile zinc into synaptic vesicles. The first study used immunocytochemistry to localize ZnT3 within the mossy fibre layer of the hippocampus to determine whether there was differential expression of ZnT3 in wild-type versus mast cell-deficient mice. The second study used inductively coupled plasma mass spectrometry (ICP-MS) to determine total zinc content in the whole dentate gyrus of the two genotypes. The immunocytochemical results indicate that there are higher levels of ZnT3 localized to the mossy fibre layer of the dentate gyrus of mast cell-deficient mice than in wild-type mice. The ICP-MS data reveal no differences in total zinc in dentate gyrus as a whole. The results are consistent with the hypothesis that mast cells participate in zinc homeostasis at the level of synaptic vesicles.

The surface reactivity of TiO22 is often governed by hydroxyl groups. Fourier transform infrared (FTIR) spectroscopy is the most commonly used method to study surface hydroxyls. However, interpretation of the observed bands of powder samples is not straightforward. In this work, we propose a facet-specific assignment of the surface hydroxyls of pyrogenic TiO2 (commonly known as P25 and P90) by comparison between experimentally observed FTIR bands of P90, rutile, and anatase with calculated vibrational frequencies for well-defined surface facets using density functional theory (DFT). Titania was calcined for extended periods in extremely dry O2 to remove carbonates and water for diffuse reflectance infrared spectroscopy (DRIFTS) measurements of the most thermally stable hydroxyls remaining in the 300 – 400 °C range. Reactions of the dehydroxylated surfaces with H2(D2) provided further insight into hydroxyl formation. Theoretical assignments of hydroxyls were based on the calculated thermal stability of hyd...

This work uses multiple characterization techniques to show conclusively that the pyrogenic TiO2 photocatalysts, P90 and P25, are made of discrete rutile and anatase nanoparticles and are not composed of a core of anatase with a rutile shell as some studies have posited. Atomic pair distribution (PDF) analysis of P90 demonstrates that the technique is capable of detecting and quantifying minority phases and particle morphology in heterogeneous titania mixtures, with important implications for further studies of shape-controlled nanoparticles with well-defined surface facets.

This work characterized the surface chemistry of a number of different titania samples including four commercial anatase samples, an anatase sample that we synthesized, the pyrogenic titania samples P25 and P90, and a commercial rutile sample. X-ray photoelectron spectroscopy (XPS), inductively coupled plasma-optical emission spectroscopy (ICP-OES), and Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS), were used to identified surface species that might interfere with the acid/base properties of the surface hydroxyls. All commercial anatase samples were contaminated by sulfur, which diminished their effectiveness as metal oxide supports for heterogeneous catalysis and has implications for their utility as photocatalysts. Hydrogen-bonded surface hydroxyls remained after calcination up to 400 °C for all anatase samples, in contrast to rutile and the pyrogenic titania materials P25 and P90, in which they were eliminated. Ru(0) catalysts on titania without hydrogen-bonded surface hydroxyls showed enhanced C O hydrogenolysis selectivity in the presence of water while Ru(0) catalysts on titania with hydrogen-bonded surface hydroxyls showed diminished selectivity in water, suggesting that surface hydrophilicity is important for this reaction. Heteroepitaxy between rutile RuO2 and rutile TiO2 is not essential for the creation of small evenly-spaced supported Ru(0) nanoparticles, which are important in many catalytic reactions.

Science education research has shown that systemic racism, microaggressions, and unwelcoming or unsupportive climates disproportionally impact the ability of some individuals to flourish in chemistry. In order to help students taking general chemistry learn about the impact of systemic racism in chemistry and to provide them with a venue to discuss this issue, a special seminar-style course was created. This relatively low intensity course successfully created a space for intense conversation, reflection, increased understanding of some of the aspects of racism in chemistry, and the impetus for institutional change. A description of the course, along with student opinions, and co-facilitator reflections, are presented.

Interest in understanding the environmental distribution of the alkane monooxygenase (AlkB) enzyme led to the identification of over 100 distinct alkane monooxygenase (AlkB) enzymes containing a covalently bound, or fused, rubredoxin. The rubredoxin-fused AlkB from Dietzia cinnamea was cloned as a full-length protein and as a truncated protein with the rubredoxin domain deleted. A point mutation (V91W) was introduced into the full-length protein, with the goal of assessing how steric bulk in the putative substrate channel might affect selectivity. Based on activity studies with alkane and alkene substrates, the rubredoxin-fused AlkB oxidizes a similar range of alkane substrates relative to its rubredoxin domain-deletion counterpart. Oxidation of terminal alkenes generated both an epoxide and a terminal aldehyde. The products of V91W-mutant-catalyzed oxidation of alkenes had a higher aldehyde-to-epoxide ratio than the products formed in the presence of the wild type protein. These results are consistent with this mutation causing a structural change impacting substrate positioning.

The promoting effect of Cu on Re/SiO2 catalysts was studied for guaiacol hydrodeoxygenation. Cu(x)Re/SiO2 catalysts containing from 0 to 1.91 wt% Cu and 13 wt% of Re were prepared by successive wet impregnation and characterized using X-ray diffraction (XRD), nitrogen sorption, CO chemisorption, X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS) and surface acidity techniques. Batch reactions were carried out at 300 °C and 5 MPa of H2. The Cu(x)Re/SiO2 catalysts displayed higher activities than the Re/SiO2 catalyst with a maximum activity at 1.58 wt% Cu. At loadings above 1.5 wt%, aggregate formation and a loss of metallic Re active sites lead to a decreased activity. The increase of the activity was attributed not only to Cu increasing the Re reducibility, but also to Cu somehow increasing the metallic Re active sites favoring guaiacol conversion. All catalysts displayed the same product distribution, confirming that the Cu in the Cu(x)Re/SiO2 not change the nature of the active site in the metallic Re nanoparticles.

Carminic acid and its metal derivatives have been used widely as pigments for fabrics and art, and more recently as a colorant for food. An undergraduate teaching laboratory is described in which students are instructed to design and execute experimental studies to obtain detailed information about the electronic structure, metal complex formation, redox properties, and photochemical stability of extracted carminic acid. The lab maintains room for student innovation and is well-suited to upper-level undergraduates in an advanced spectroscopy lab. Students are invited to apply knowledge previously gained through highly directed experiments to the analysis of an unfamiliar, complex, and relevant problem. The laboratory lends itself to flexibility in implementation but is designed to deepen students’ understanding of UV–vis spectroscopy, fluorescence spectroscopy, IR spectroscopy, electrochemistry, pH and metal spectrophotometric titrations, and experimental determination of the kinetic behavior of UV-induce...

The toxicity of lead, one of the most ubiquitous toxic metals, is well known. Some of its pathological effects are related to its preference for the sulfhydryl groups of proteins. Metallothioneins (MT) are a particular family of metalloproteins characterized by their high Cys content that, among other functions, are linked to the detoxification of heavy metals. In mammals, 4 MT isoforms have been found. The MT3 isoform, also called “neuronal growth inhibitory factor”, is mainly synthesized in the brain and contains several structural differences that may contribute to important functional differences between it and other MT isoforms. The abilities of recombinant MT3 and its individual αMT3 and βMT3 fragments to bind Pb(II) have been investigated here, under different pH conditions, by means of spectroscopy, mass spectrometry and isothermal titration calorimetry. The results obtained show that the binding of Pb(II) to the intact MT3 protein is relatively unaffected by pH, while the individual domains interact with Pb(II) in a pH-sensitive manner. The mass spectrometry data reveal the evolution with time of the initially formed Pb-MT complexes. In the case of the full length protein, Pb(II) remains bound for a long period of time. With the isolated fragments, the lead is eventually released. The Pb-species formed depend on the amount of Pb(II) present in solution. The thermodynamic data recorded, as measured by ITC, for the replacement of Zn(II) by Pb(II) in reactions with Zn-MT3, Zn-αMT3 and Zn-βMT3 are all similar, and in all cases, the displacement of Zn(II) by Pb(II) is thermodynamically favorable. Zn-Replete and Pb-replete MT3 have distinctive circular dichroism spectra, suggestive of structural differences with different metallation status.

Childhood lead poisoning is a costly and largely preventable public health problem that lowers IQs, decreases attention spans, and leads to the development of other childhood intellectual disabilities. Furthermore, recent evidence links developmental lead poisoning with the etiology of disorders that appear much later in life, such as Alzheimer's disease, Parkinson's disease, and schizophrenia. Little is known about how lead influences the onset of these disorders. This paper reviews the evidence that lead substitution for zinc in zinc-finger proteins contributes to the development of Alzheimer's disease, Parkinson's disease, and schizophrenia. The zinc-finger proteins potentially impacted by lead include DNA methyltransferase 1 (DNMT1) and Presenilin 1 and 2 (PSEN1/2) in Alzheimer's disease, the dopamine receptor in Parkinson's disease, and the NMDA receptor, zinc-finger protein 804A (ZNF804A), and disrupted-in-schizophrenia 1 (DISC1)-binding zinc-finger (DBZ) in schizophrenia.

Catalytic reduction of pyrolyzed biomass is required to remove oxygen and produce transportation fuels, but limited knowledge of how hydrodeoxygenation (HDO) catalysts work stymies the rational design of more efficient and stable catalysts, which in turn limits deployment of biofuels. This work reports results from a novel study utilizing both isotopically labeled phenol (which models the most recalcitrant components of biofuels) with D2O and DFT calculations to provide insight into the mechanism of the highly efficient HDO catalyst, Ru/TiO2. The data point to the importance of interface sites between Ru nanoparticles and the TiO2 support and suggest that water acts as a cocatalyst favoring a direct deoxygenation pathway in which the phenolic OH is replaced directly with H to form benzene. Rather than its reducibility, we propose that the amphoteric nature of TiO2 facilitates H2 heterolysis to generate an active site water molecule that promotes the catalytic C–O bond scission of phenol. This work has cle...

A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3.

Substituted phenols are the most recalcitrant oxygenates in conventional pyrolysis oils and the dominant oxygenates in lower-oxygen content, formate-assisted pyrolysis oils (FAsP). Ru catalysts with a wide range of dispersion on carbon, silica, alumina, and titania supports were synthesized, characterized and evaluated for hydrodeoxygenation (HDO) activity using phenol as a model compound. Metal content, phase, and particle size were determined with ICP-OES, EXAFS/XANES, and CO pulse chemisorption, respectively. High dispersion of ruthenium on the supports converts more phenol to products. The majority of catalysts predominantly catalyze the hydrogenation (HYD) route typical of noble metal catalysts. A highly dispersed Ru/TiO2 catalyst shows unusually high selectivity toward direct deoxygenation (DDO) and outstanding activity. We suggest that the DDO pathway on titania involves a bifunctional catalyst, where hydrogen creates reduced titania sites, created by hydrogen spillover, that interact strongly with the phenol hydroxyl group.

An alkane hydroxylase from the marine organism Alcanivorax borkumensis ( Ab AlkB) was purified. The purified protein retained high activity in an assay with purified rubredoxin (AlkG), purified maize ferredoxin reductase, NADPH, and selected substrates. The reaction mechanism of the purified protein was probed using the radical clock substrates bicyclo[4.1.0]heptane (norcarane), bicyclo[3.1.0]hexane (bicyclohexane), methylphenylcyclopropane and deuterated and non-deuterated cyclohexane. The distribution of products from the radical clock substrates supports the hypothesis that purified Ab AlkB hydroxylates substrates by forming a substrate radical. Experiments with deuterated cyclohexane indicate that the rate-determining step has a significant C H bond breaking character. The products formed from a number of differently shaped and sized substrates were characterized to determine the active site constraints of this AlkB. Ab AlkB can catalyze the hydroxylation of a large number of aromatic compounds and linear and cyclic alkanes. It does not catalyze the hydroxylation of alkanes with a chain length longer than 15 carbons, nor does it hydroxylate sterically hindered C H bonds.

Summary Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

A series of mesoporous silica supported cobalt catalysts for the Fischer–Tropsch reaction with pore diameters from 3 to 22 nm was synthesized and characterized. Detailed X-ray diffraction measurements were used to determine the composition and particle diameters of the metal fraction, analyzed as a three-phase system containing Co fcc , Co hcp and CoO. Catalyst properties were determined at three stages of catalyst history: (1) after the initial calcination step to thermally decompose the catalyst precursor into Co 3 O 4 , (2) after the hydrogen reduction step to activate the catalyst to Co and (3) after the FT reaction. Small Co hcp particles were detected in all reduced catalysts contributing significantly to the Co surface area. The reaction rate increased with the pore diameter of the silica support. The results indicate the importance of careful catalyst characterization in determining the factors that contribute to reactivity. In addition, the identification of significant quantities of two cobalt metal phases suggests that further study of the intrinsic activity of each phase, as well as the structural features of the supports that determine the distribution of cobalt phases and particle sizes is warranted.

Isothermal titration calorimetry (ITC) was used to quantify the thermodynamics of Pb(2+) and Zn(2+) binding to metallothionein-3 (MT-3). Pb(2+) binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (ΔG) and enthalpy (ΔH). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extracted in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodynamics. Metal binding is entropically favoured, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb(2+) binding to both apo MT-3 and Zn7MT-3 is thermodynamically favourable, and implicate MT-3 in neuronal lead biochemistry.

SummaryMechanistically informative chemical probes are used to characterize the activity of functional alkane hydroxylases in whole cells. Norcarane is a substrate used to reveal the lifetime of radical intermediates formed during alkane oxidation. Results from oxidations of this probe with organisms that contain the two most prevalent medium-chain-length alkane-oxidizing metalloenzymes, alkane ω-monooxygenase (AlkB) and cytochrome P450 (CYP), are reported. The results—radical lifetimes of 1–7 ns for AlkB and less than 100 ps for CYP—indicate that these two classes of enzymes are mechanistically distinguishable and that whole-cell mechanistic assays can identify the active hydroxylase. The oxidation of norcarane by several recently isolated strains (Hydrocarboniphaga effusa AP103, rJ4, and rJ5, whose alkane-oxidizing enzymes have not yet been identified) is also reported. Radical lifetimes of 1–3 ns are observed, consistent with these organisms containing an AlkB-like enzyme and inconsistent with their employing a CYP-like enzyme for growth on hydrocarbons.

The synthesis and characterization of a novel catalyst for the photodecomposition of carbaryl (1-naphthyl, N-methylcarbamate) is reported. In the absence of a catalyst, but in the presence of UV light, a 30 ppm solution of carbaryl decomposes with a first-order rate constant of (5.6 ± 0.3) × 10-5 s-1 (298 K) and a quantum efficiency of 4.8 × 10-3 molecules/photon. In the presence of the Ag−zeolite Y catalyst with 2.42% Ag by weight, the photodecomposition rate becomes 80 times faster. The addition of Suwannee River natural organic matter (NOM), which can inactivate photocatalysts, has a minimal effect on this system. In the presence of three different concentrations of NOM and 30 ppm carbaryl, our results indicate that the NOM increases or decreases the catalytic photodecomposition rate by only a factor of 3 at most.

Should iron and copper be added to the environment to stimulate the natural bioremediation of marine oil spills? The key enzymes that catalyze the oxidation of alkanes require either iron or copper, and the concentration of these ions in seawater is vanishingly low. Nevertheless, the dependence of alkane oxidation activity on external metal concentrations remains unclear. This perspective will summarize what is known about the co-regulation of alkane oxidation and metal acquisition and pose a series of critical questions to which, for the most part, we do not yet have answers. The paucity of answers points to the need for additional studies to illuminate the cellular biology connecting microbial growth on alkanes to the acquisition of metal ions.

Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu-oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH(-) is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.

Dicyanoaurate and dicyanoargentate ions doped in A-type zeolite were prepared and analyzed spectroscopically. Data from luminescence and Raman spectroscopies along with extended Huckel calculations indicate the formation of [M(CN)2-]n oligomers (M = Ag, Au) in the zeolite host. Variations in luminescent properties as a function of excitation wavelength, dopant concentration inside the zeolite host, and temperature facilitate assignment of cluster structures. The data obtained from a comparison of Ag- and Au-doped silica and γ-alumina show that the [M(CN)2-]n clusters form in the channels of the zeolite rather than on the surface. The [M(CN)2-]n clusters in A zeolite show strong catalytic activities toward the photodecomposition of carbaryl in aqueous solution. The photodecomposition of carbaryl at room temperature shows different decomposition products in the presence and absence of the M(CN)2- doped A zeolite catalyst. In addition, the decomposition rate constants increase as the metal content increases....

Bringing environmental topics into undergraduate curricula connects students with complex real-world issues

Specifically deuteriated samples of toluene, anisole, chlorobenzene, α,α,α-trifluoromethylbenzene, naphthalene and quinoline have been synthesised and used as substrates for dioxygenase-catalysed asymmetric dihydroxylation studies to yield the corresponding cis-dihydrodiols as major bioproducts. Phenols were also detected as minor metabolites in some cases. Dehydration of the deuterium-labelled cis-dihydrodiol metabolites, under thermal conditions, in all cases, resulted in phenol formation accompanied by the NIH Shift. A comparison of NIH Shift results, obtained when phenols are produced by aromatisation of chemically synthesised deuteriated arene cis- and trans-dihydrodiols (dehydration) and arene oxides (isomerisation), suggests that this phenomenon may be associated with both monooxygenase- and dioxygenase-catalysed aromatic hydroxylations.

Hydrocarbons are ubiquitous compounds of both natural and anthropogenic origin. Natural sources are derived via biosynthesis reactions in bacteria, phytoplankton, plants and fungi (Ladygina et al., 2006), as well as diagenesis and catagenesis (Horsfield and Rullkotter, 1994). As a result, hydrocarbons are environmentally distributed in microbial mats (Green and Jahnke, 2010), deep subsurface oil reservoirs (Sephton and Hazen, 2013), coalbeds (Strąpoc et al., 2011), and natural oil seeps (Kvenvolden and Cooper, 2003). Hydrocarbons are energy-rich biological substrates. However, accessing this energy requires novel enzymes. The metabolic ability of microorganisms to transform hydrocarbons is not surprising given that microbes have been exposed to these compounds throughout the course of evolution. The coupling of microbial processes and hydrocarbon transformation have important implications with regard to our understanding of biogeochemical cycling, biotransformation of environmental pollutants, subsurface production and oxidation of methane, and host/pathogen relationships. This special issue highlights recent advances in our understanding of the enzymes that govern hydrocarbon transformation in both microbial and environmental systems. From a microbe's metabolic perspective, the largest thermodynamic benefit of hydrocarbon metabolism is derived from the oxidation of hydrocarbons with molecular oxygen. Harnessing the free energy inherent in oxygen reactivity requires an organism to have evolved mechanisms of activating oxygen and processing the reactive intermediates in ways that lead to the selective oxidation of the desired substrate, without incurring indiscriminate reactions within the organism itself. To date, the mechanisms involved in aerobic hydrocarbon oxidation are well-described, and the most prevalent feature of these processes is the dual role of oxygen as a physiological requirement and as a reactant (Berthe-Corti and Fetzner, 2002; National Research Council, 2003). Several articles in this issue review these aerobic mechanisms and the requisite enzymes. Specifically, these articles address aerobic enzymes involved in alkane oxidation (Ji et al., 2013; Wang and Shao, 2013) and new insights to the characterization of the model methanotroph, Methylosinus trichosporium OB3b (Matsen et al., 2013; Yang et al., 2013). In the absence of oxygen, anaerobic and facultative microorganisms use alternative electrons acceptors, such as sulfate, nitrate, iron, manganese, and carbon dioxide to oxidize hydrocarbons. The last 30 years have yielded new insights into the novel biochemistry of anaerobic microorganisms with respect to hydrocarbon activation and degradation. Several pathways have emerged as common themes among physiologically diverse microorganisms utilizing a range of hydrocarbon substrates under a myriad of terminal electron accepting conditions (Heider and Schuhle, 2013). Among recent discoveries are new mechanisms and enzymes involved in anaerobic alkane oxidation. In this issue, we have included review articles by Callaghan (2013) and Agrawal and Gieg (2013) outlining the current state of knowledge regarding anaerobic methane and non-methane alkane oxidation and methods for the in situ detection of anaerobic alkane biodegradation. In addition to hydrocarbon metabolism, which is widespread among microbial taxa, some microorganisms synthesize hydrocarbons, and the purpose of these biosynthesized hydrocarbons is not well-elucidated. For example, the microalga Botryococcus braunii can synthesize up to 75% of its dry weight in alkanes (Banerjee et al., 2002). In some organisms, the alkanes constitute protective coatings, whereas in other microbes, they may serve as a strategy to store excess energy when other required nutrients are not available for metabolism and protein synthesis. Protective coatings, however, can serve as substrates for antagonistic microorganisms, and the strategies employed to oxidize hydrocarbons on the outside of cells appear to be different than those employed to utilize hydrocarbon stores within cells. The article by Pedrini et al. (2013) provides detailed evidence for microbial oxidation of alkanes in a host/pathogen relationship. Given the tragedy of the Deepwater Horizon oil spill, the role of microorganisms in mitigating natural and anthropogenic hydrocarbon inputs to marine and terrestrial ecosystems has been brought to the forefront of scientific inquiry. Three papers in this issue focus on microbial activity in oil-impacted environments. Porter and Young (2013) investigate the distribution and potential use of bamA as a biomarker for anaerobic aromatic hydrocarbon degradation in environmental samples. Kimes et al. (2013) evaluate the use of metabolite profiling coupled with metagenomics to investigate the response of sediment microbial communities to the Deepwater Horizon oil spill. Finally, Bertrand et al. (2013) elucidate the identity and mechanisms of alkane hydroxylases found in organisms isolated from deep-sea vents, an environment in which the natural release of chemosynthetic alkanes may serve as an important fuel source. While the flux of hydrocarbons through anthropogenic combustion is much larger than the flux via microbial processing and tends to dominate the public's attention due to concerns about global climate change, the selectivity and diversity of microbial transformations of hydrocarbons remains a powerful reminder of the unrivaled elegance of molecular evolution. The future, in which we continue to isolate novel organisms, harness significant advances in sequencing technology, and develop new environmental monitoring tools will undoubtedly unveil exciting and unexpected aspects of the fundamental roles that microbial metabolism plays in hydrocarbon transformation.

The genotoxic environmental contaminant 1-nitropyrene is metabolised in mammalian systems by pathways more complex than the straightforward nitroreduction which accounts for most of its biological activity in bacteria. In order to evaluate the role of O-acetyltransferase (OAT) activity in generation of genotoxic intermediates from 1-nitropyrene, the mutagenicity of the major primary oxidised metabolites of 1-nitropyrene was characterised in the Ames Salmonella typhimurium plate incorporation assay with strain TA98, and with variants of TA98 deficient (TA98/1,8-DNP6) or enhanced (YG1024) in O-acetyltransferase. 1-Nitropyren-3-ol was more mutagenic in the absence than in the presence of S9, while 1-nitropyren-4-ol, 1-nitropyren-6-ol and 1-nitropyren-8-ol required S9 for maximum expression of mutagenicity. 1-Nitropyren-4-ol (176 rev/nmol without S9, 467 rev/nmol with S9 in TA98) and 1-nitropyren-6-ol (13 rev/nmol without S9, 266 rev/nmol with S9 in TA98) were overall the most potent nitropyrenol isomers assayed. 1-Acetamidopyren-8-ol and 1-acetamidopyrene 4,5-quinone were only minimally active. 1-Acetamidopyren-3-ol exhibited direct-acting mutagenicity. 1-Acetamidopyren-6-ol, previously shown to be a major contributor to mutagenicity in the urines of rats dosed with 1-nitropyrene (Ball et al., 1984b), was confirmed as a potent (359 rev/nmol) S9-dependent mutagen. Both the direct-acting and the S9-dependent mutagenicity of all the compounds studied was enhanced in the OAT-overproducing strain and much diminished (though not always entirely lost) in the OAT-deficient strain, showing that OAT amplifies expression of the genotoxicity of these compounds. 1-Acetamidopyren-6-ol required both S9 and OAT activity in order to exhibit any mutagenicity; this finding strongly implicates N-hydroxylation followed by O-esterification, as opposed to further S9-catalyzed ring oxidation, as a major route of activation for urinary metabolites of 1-nitropyrene.

A series of (oxoferryl)porphyrin pi-cation radicals generated from porphyrins substituted at the meso positions with highly electron-withdrawing aryl groups has been characterized: tetrakis-5,10,15,20-(2,6-dichlorophenyl)-, 5-(2-chloro-6-nitrophenyl)-10,15,20-tris(2,6-dichlorophenyl)-, and 5-(2,6-dinitrophenyl)-10,15,20-tris(2,6-dichlorophenyl)porphyrins (porphyrins 1-3, respectively). The physical-chemical properties of the oxidized complexes of 1-3 are compared to those of two (oxoferryl)porphyrin pi-cation radical complexes substituted with electron-releasing aryl groups: tetramesitylporphyrin (TMP) and 2-iodotetramesitylporphyrin (2-iodoTMP). While all of the complexes examined show close correspondance in a number of spectroscopic parameters, some significant differences were observed. In contrast to observations for the oxidized complexes of TMP and 2-iodoTMP, the resonance Raman marker bands nu(2) and nu(11), which are indicators of symmetry state of porphyrin pi-cation radicals of 1-3, do not show the expected downfrequency shifts for oxidation to compound I analogs in a(2u) symmetry states. The upfield hyperfine NMR shifts of the pyrrole beta-proton signals of the compound I analogs of 1-3 are much larger than those for TMP and 2-iodoTMP. These data may be explained by admixture of some a(1u) character into the ground state of radical cations of 1-3, consistent with the hypothesis that electron-withdrawing meso substituents lower the energy of the a(2u) molecular orbital, favoring an a(1u) admixture.

The synthesis of a complete series of substituted 4,5,6,7-tetrahydroindenes (4,5,6,7-tetrahydroindene, 1; 1-methyl-, 1,3-dimethyl- and 1,2,3-trimethyl-4,5,6,7-tetrahydroindene, 2-4) is reported. The synthesis of ferrocenes (8-11) from these substituted cyclopentadienes is described. The electrochemistry of these ferrocenes indicates that these complexes are more readily oxidized than ferrocene and that the effect of methyl or alkyl substituents on the ease of oxidation is additive. A single crystal X-ray structure of two of the ferrocenes, bis(1,3-dimethyl-4,5,6,7-tetrahydroindenyl)iron(II), 10, and bis(1,2,3-trimethyl-4,5,6,7-tetrahy-droindenyl)iron(II), 11, indicates that steric hindrance causes the alkyl substituents to be bent away from the plane of the cyclopentadienyl ring. However, the structures differ in that the cyclopentadienyl rings in 10 are staggered wheras those in 11 are eclipsed. The synthesis of the cyclopentadienyltricarbonylmethyl compounds of molybdenum and tungsten from 3 and 4 is also described.

A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d(4), afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

The hydrodeoxygenation of 2-methoxyphenol (guaiacol) has been studied over a MoS 2 catalyst supported on two activated carbons with marked differences in porosity and oxygen surface functionality. The catalysts were prepared by rotary evaporator impregnation and characterized by BET surface area and XPS, while the supports were characterized by TPD, PZC, FTIR, BET and XPS techniques. The reaction was studied in a batch reactor at 300 °C and 5 MPa. Activity differences on the carbon supports are correlated with the dispersion of MoS 2 . Greater abundance of surface oxygen (mainly carboxylic, quinonic and lactonic groups) on the support resulted in lower catalyst dispersion and lower HDO activity. The dispersion, textural properties and surface chemistry did not affect the nature of the MoS 2 active sites.

In order to study structural influences on the interaction of Fe(IV) (S=1) and porphyrin cation radical (S′=1/2) in high-valent iron porphyrin complexes of the type ¦X-(TMP)Fe=O¦+(Cl−), X=I, Br2, Br4 were generated by mCPBA oxidation of corresponding Fe(III) porphyrins. The halogen substitution at the peripheral positions of the porphyrin leads to distortion of the planar porphyrin ring of ¦(TMP)Fe=O¦+. The new species have beeen investigated by temperature-dependent EPR and field-dependent Mossbauer spectroscopy; for the evaluation of spectra, we adopted the spin-Hamiltonian formalism including exchange interaction explicitly. As in ¦(TMP)Fe=O¦+, strong ferromagnetic spin coupling was observed with|J0|D=0.9–1 and a zero-field spltting ofD∼32 cm−1. For consistent parametrization of EPR and Mossbauer results, anisotropic coupling had to be introduced. Compared to ¦(TMP)Fe=O¦+ [1], analysis of the spectroscopic data shows that zero-field splitting and spin coupling is only slightly affected by the halogen distortion of the porphyrin structure.

This review examines the metalloenzymes that catalyze the oxidation of alkanes in the environment. The focus of the review is on what is known about the relative abundances of these metalloenzymes, their metal ion requirements, and their reaction mechanisms. The relative significance of these reactions in the global transformation of alkanes is discussed.

Specifically deuteriated samples of toluene, anisole, chlorobenzene, α,α,α-trifluoromethylbenzene, naphthalene and quinoline have been synthesised and used as substrates for dioxygenase-catalysed asymmetric dihydroxylation studies to yield the corresponding cis-dihydrodiols as major bioproducts. Phenols were also detected as minor metabolites in some cases. Dehydration of the deuterium-labelled cis-dihydrodiol metabolites, under thermal conditions, in all cases, resulted in phenol formation accompanied by the NIH Shift. A comparison of NIH Shift results, obtained when phenols are produced by aromatisation of chemically synthesised deuteriated arene cis- and trans-dihydrodiols (dehydration) and arene oxides (isomerisation), suggests that this phenomenon may be associated with both monooxygenase- and dioxygenase-catalysed aromatic hydroxylations.

The X-ray structures of the β-substituted-pyrrole, its nickel(II) complex (NiTMOBP), and the nickel(II) derivative are reported. The synthesis of the free base H 2 TPFPOBP is also described. All these molecules are nonplanar, displaying saddle-shaped conformations

Three structurally analogous radical clock substrates with a 100-fold span in their rearrangement rates are hydroxylated by the diiron oxygenase AlkB to afford similar amounts of rearranged and unrearranged products. Such a result is predicted by a mechanistic scheme by which radical rebound competes with cage escape of the geminate substrate radical. The results show that radical clocks can measure both the radical life-time and the kinetics of cage escape.

Rieske dioxygenases catalyze the cis-dihydroxylation of a wide range of aromatic compounds to initiate their biodegradation. The archetypal Rieske dioxygenase naphthalene 1,2-dioxygenase (NDOS) catalyzes dioxygenation of naphthalene to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDOS is composed of three proteins: a reductase, a ferredoxin, and an α3β3 oxygenase (NDO). In each α subunit, NDO contains a Rieske Fe2S2 cluster and a mononuclear iron site where substrate dihydroxylation occurs. NDOS also catalyzes monooxygenase reactions for many substrates. The mechanism of the reaction is unknown for either the mono- or dioxygenase reactions but has been postulated to involve direct reaction of either a structurally characterized Fe(III)−hydroperoxy intermediate or the electronically equivalent Fe(V)−oxo−hydroxo intermediate formed by O−O bond cleavage before reaction with substrate. The reaction for the former intermediate is expected to proceed through cationic intermediates, while the latter i...

Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximately 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximately 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximately 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.