Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases

Conspectus A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O₂, protons, electrons, and methane. To couple O₂ activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O₂ migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O₂ and methane, in the case of sMMO, to the reduced diiron center for O₂ activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of “Fe₂O₂” intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe₂O₂ intermediates by blocking reductase binding during O₂ activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze reactions very similar to those catalyzed by the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic protein complexes to accomplish this feat.National Institute of General Medical Sciences (U.S.) (GM032134

Electron Transfer Control in Soluble Methane Monooxygenase

The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.National Institutes of Health (U.S.) (Grant GM032134)Waters Corporatio

Ferromagnetic Property of Co and Ni Doped TiO 2

The Co and Ni doped diluted magnetic semiconductor nanoparticle TiO2 is prepared by sol-gel method. Ti0.97Co0.03O2, Ti0.97Ni0.03O2, Ti0.97Co0.06O2, and Ti0.97Ni0.06O2 samples were characterized by X-ray scattering techniques and high resolution transmission electron microscope. The results show that there are no other phases existing in TiO2. As to the sample of high-concentration dopant, the X-ray scattering techniques have explored the existing of CoTiO3 and NiTiO3. The ferromagnetic measurement shows that the magnetization of the sample of high-concentration dopant increases in the same external magnetic field. However, the relatively higher dopant Co and Ni may form more interstitial ions and paramagnet matters, reducing the oxygen vacancy concentration and finally leading to the decrease of remanent magnetization and coercivity of the materials

The Microstructure of Ni Layer on Single-Walled Carbon Nanotubes Prepared by an Electroless Coating Process

The single-walled carbon nanotubes (SWNTs, diameter: 2~3 nm), which were obtained in the suspension of purification solution, with Ni-P coating layers were obtained by an electroless deposition process. The SWNTs before and after coating were characterized by transmission electron microscopy (TEM) and energy dispersive spectrometry (EDS). An Ni-P layer on individual nanotube with thickness of 20 nm can be obtained after the deposition process. The X-ray diffraction (XRD) and selected area electron diffraction (SAED) analysis of Ni-P SWNTs before and after heat treatment show that the heat treatment caused the transformation of the amorphous Ni-P layer to the nanocrystalline Ni-P (crystalline Ni and Ni3P intermetallic compound) layer. The XRD pattern of SWNTs with Ni-P layers after heat treatment revealed that the crystal structures of Ni in plating layer contained: hexagonal close-packed (hcp) structure and face-centered cubic (fcc) structure. The lattice parameters of Ni (fcc) and Ni3P are larger than the bulk's, indicting that the lattice expansion has taken place. However, the lattice parameter of Ni (hcp) has no difference from the bulk's

Ab initio Investigation of Elasticity and Stability of Metal Aluminum

On the basis of the pseudopotential plane-wave(PP-PW) method in combination with the local-density-functional theory(LDFT), complete stress-strain curves for the uniaxial loading and uniaxial deformation along the [001] and [111] directions, and the biaxial proportional extension along [010] and [001] of aluminium are obtained. During the uniaxial loading, certain general behaviors of energy versus stretch and the load versus the stretch are confirmed; in each acse, there exist three special unstressed structures: f.c.c., b.c.c. and f.c.t. for [001]; f.c.c., s.c. and b.c.c. for [111]. Using stability criteria, we find that all of these state are unstable, and always occur together with shear instability, except the natural f.c.c. structure. A Bain transformation from the stable f.c.c. structure to the stable b.c.c. configuration cannot be obtained by uniaxial compression along any equivalent [001] and [111] direction. The tensile strength are similar for the two directions. For the higher energy barrier of [111] direction, the compressive strength is greater than that for the [001] direction. With increase in the ratio of the biaxial proportional extension, the stress and tensile strength increase; however, the critical strain does not change significantly. Our results add to the existing ab initio database for use in fitting and testing interatomic potentials.Comment: 9 Pages in Revtex and 11 Eps figure

Genomes shed light on the evolution of Begonia, a mega‐diverse genus

Clarifying the evolutionary processes underlying species diversification and adaptation is a key focus of evolutionary biology. Begonia (Begoniaceae) is one of the most species-rich angiosperm genera with ~2,000 species, most of which are shade-adapted. Here, we present chromosome-scale genome assemblies for four species of Begonia (B. loranthoides, B. masoniana, B. darthvaderiana, and B. peltatifolia), and whole genome shot-gun data for an additional 74 Begonia representatives to investigate lineage evolution and shade adaptation of the genus. The four genome assemblies range in size from 331.75 Mb (B. peltatifolia) to 799.83 Mb (B. masoniana), and harbor 22,059 - 23,444 protein-coding genes. Synteny analysis revealed a lineage specific whole-genome duplication (WGD) that occurred just before the diversification of the Begonia. Functional enrichment of gene families retained after WGD highlight the significance of modified carbohydrate metabolism and photosynthesis possibly linked to shade-adaptation in the genus, which is further supported by expansions of gene families involved in light perception and harvesting. Phylogenomic reconstructions and genomics studies indicate that genomic introgression has also played a role in the evolution of Begonia. Overall, this study provides valuable genomic resources for Begonia and suggests potential drivers underlying the diversity and adaptive evolution of this mega-diverse clade

Elasticity, Stability and Ideal Strength of β\beta -SiC in plane-wave-based ab initio calculations

On the basis of the pseudopotential plane-wave(PP-PW) method and the local-density-functional theory(LDFT), this paper studies energetics, stress-strain relation, stability and ideal strength of $\beta$-SiC under various loading modes, where uniform uniaxial extension and tension, biaxial proportional extension are considered along directions [001] and [111]. The lattice constant, elastic constants and moduli of equilibrium state are calculated, and the results agree well with the experimental data. As the four Si-C bonds along directions [111], [$\bar{1}$11], [11$\bar{1}$] and [1$\bar{1}$1] are not the same under the loading along [111], internal relaxation and the corresponding internal displacements must be considered. We find that, at the beginning of loading, the effect of internal displacement through shuffle and glide plane diminishes the difference among the four Si-C bonds length, but will increase the difference at the subsequent loading, which will result in a crack nucleated on \{111\} shuffle plane and a subsequently cleavage fracture. Thus the corresponding theoretical strength is 50.8 GPa, which agrees well with the recent experiment value, 53.4 GPa. However, with the loading along [001], internal relaxation is not important for tetragonal symmetry. Elastic constants during the uniaxial tension along [001] are calculated. Based on the stability analysis with stiffness coefficients, we find that the spinodal and Born instabilities are triggered almost at the same strain, which agrees with the previous molecular dynamics simulation. During biaxial proportional extension, stress and strength vary proportionally with the biaxial loading ratio at the same longitudinal strain.Comment: 9 pages, 10 figure

Mechanisms of action and inhibition of [4Fe-4S] proteins IspG and IspH in isoprenoid biosynthesis

The methylerythritol phosphate pathway (also known as the non-mevalonate pathway) of isoprenoid biosynthesis is potentially an important anti-bacterial and anti-malarial drug target. However, the catalytic mechanisms of the last two enzymes in this pathway, IspG (also known as GcpE) and IspH (also known as LytB) were largely unknown, and there were no inhibitors targeting these two enzymes. These two enzymes both are [4Fe-4S] proteins with one unique iron not bonded to any cysteine residue, and catalyze 2e-/2H+ reductions. In this study, bioorganometallic mechanisms are proposed for IspG and IspH catalyses, where direct iron-carbon interactions play important roles. This is a new type of catalytic mechanism of iron-sulfur enzymes, and is supported by extensive characterizations of trapped reaction intermediates: in IspG catalysis, a reaction intermediate with Fe-C and Fe-O bonding has been identified; whereas in IspH catalysis, an alkoxide complex, a weak π-complex, and an η3-allyl complex has been discovered along the reaction pathway. No free radical intermediates were observed. In addition, the first potent inhibitors against IspG and IspH have been discovered. Based on the catalytic mechanism of IspH enzyme, alkyne diphosphate inhibitors against both enzymes were rationally designed; pyridine diphosphate inhibitors against Aquifex aeolicus IspH were discovered by compound library screening. The binding modes of both types of inhibitors have also been determined. The work reported here is of broad general interest, since it clarifies the nature of the reaction mechanisms of IspG and IspH catalyses, and opens up new routes to inhibitor design, of interest in the context of both anti-bacterial as well as anti-malarial drugs