Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus bath: evidence for a Diiron center

Two methane monooxygenase (MMO) systems have been identified in methanotrophic bacteria, namely, a soluble or cytoplasmic MMO and a membrane-associated or particulate MMO. The active site of the well-characterized soluble MMO contains a bis-mu-hydroxo-bridged diiron cluster. X-ray crystallographic studies of the particulate enzyme, pMMO, have identified two copper centers on the alpha subunit (pmoB) of the alphabetagamma trimer and a site at the interface of the betagamma subunits filled by a Zn, apparently from the crystallization buffer. In our hands, pMMO preparations containing 1-2 iron atoms per alphabetagamma show the highest catalytic activity. We have employed M?ssbauer spectroscopy to characterize the iron in our preparations. Interestingly, we find in pMMO a component with the same spectral properties as the antiferromagnetically coupled diiron(III) cluster in the soluble enzyme. In whole cells, we find nearly 1 diiron center per alphabetagamma of pMMO; in purified enzyme preparations, only 10% of the sites appear to be occupied. These occupancies correlate well with the measured specific activities of purified pMMO and pMMO in whole cells. We suggest that it is the "Zn site" that accommodates the diiron center in active pMMO.     

Copper-binding properties and structures of methanobactins from Methylosinus trichosporium OB3b

Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ～9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (～ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.     

The copper chelator methanobactin from Methylosinus trichosporium OB3b binds copper(I)

The oxidation state of copper bound to methanobactin, a small siderophore-like molecule from the methanotroph Methylosinus trichosporium OB3b, was investigated. Purified methanobactin loaded with Cu(II) exhibits a weak EPR signal probably due to adventitious Cu(II). The EPR signal intensity increases significantly upon addition of the strong oxidant nitric acid. Features of the X-ray absorption near edge spectrum, including a 1s --> 4p transition at 8985 eV, further indicate the presence of Cu(I). EXAFS data were best fit using a multiple scattering model generated from previously reported crystallographic parameters. These results establish definitively that M. trichosporium OB3b methanobactin binds Cu(I) and suggest that methanobactin itself reduces Cu(II) to Cu(I).     

The role of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The effect of metal ions on particulate methane monooxygenase (pMMO) was studied. The pMMO activity in the membranes was partially inhibited by ethylenediaminetetraacetic acid (EDTA), but remained more than 70% of the as-isolated membranes. The activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, copper ion stimulated the activity, indicating that copper was needed for the activity. When duroquinol and dioxygen were introduced to the EDTA-treated membranes, the electron spin resonance signal of copper did not change, suggesting that the copper cluster did not play as an electron transport and may have another function, such as active site of pMMO or regulator of the activity. On the other hand, the iron signal (g=5.98) decreased by the addition of duroquinol, dioxygen and acetylene, showing an iron atom is contained in the active site of pMMO.     

颗粒性甲烷单加氧酶的电子供体研究

从甲基弯菌M ethylosinus trichosporium IMV3011的膜中分离出颗粒性甲烷单加氧酶（Particulate MMO,Pmmo）t和NADH脱氢酶，只有当两者同时存在，并添加去垢剂解离膜组分时，NDAH才能为pMMO提供还原当量，对苯二酚能够在整细胞和膜水平代替NADH作为PMMO的电子供体，对于纯化的PMMO，对苯二分配仍是有效的电子供体，而NADH却是无效的电子供体。在NADH脱氢酶存在下，NADH可将对苯醌还原为对苯二酚，纯化过程中，采用对苯二酚作为PMMO活性分析时的电子供体，不必共纯化NADH脱氢酶，且有利于对PMMO活性中心进行深入研究。     

Biosynthesis of methanol from methane by <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Methanol has recently attracted significant interest in the energetic field. Current technology for the conversion of methane to methanol is based on energy intensive endothermic steam reforming followed by catalytic conversion into methanol. The one-step method performed at very low temperatures (35°C) is methane oxidation to methanol via bacteria. The aim of this work was to examine the role of copper in the one-step methane oxidation to methanol by utilizing whole cells of Methylosinus trichosporium OB3b bacteria. From the results obtained it was found that copper concentration in the medium influences the rate of bacterial biomass growth or methanol production during the process of methane oxidation to methanol. The presented results indicate that the process of methane oxidation to methanol by Methylosinus trichosporium OB3b bacteria is most efficient when the mineral medium contains 1.0 × 10⁻⁶ mol dm⁻³ of copper. Under these conditions, a satisfactory growth of biomass was also achieved. Presented at the 35th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 26–30 May 2008.     

Sur les carbonates basiques de fer (III): II. Préparation,radiocristallographie et spectres IR. du (NH4)2Fe2(OH)4(CO3)2 · H2O

The obtention of the crystalline basic carbonate of iron (III) and ammonium, (NH₄)₂Fe₂(OH)₄(CO₃)₂ · H₂O, is described and its formula is established by chemical analysis and infrared spectroscopy. The powder X-ray diagram could be indexed tetragonally leading to a body centred elementary cell with a = 12,04 ± 0,02 Å and c = 6,62 ± 0,01 Å. The infrared spectra show that in the CO groups either one oxygen atom is linked to one iron atom or, rather, two oxygen atoms are linked to two iron atoms. The symmetry of the NH groups is lower than C_3v. The OH-groups are linked by hydrogen bonds of 2,75 Å. Two sorts of OH-groups can be distinguished, with a radius of approximately 1,34 Å and 1, 48 Å, respectively. The iron atoms are octahedrally coordinated by oxygen atoms, but either the octahedra are deformed or the iron atoms are in part coordinated tetrahedrally.     

Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.     

甲烷氧化菌素-铜配合物催化过氧化氢氧化对苯二酚

为了探讨甲烷氧化菌素(Mb)-铜配合物(Mb-Cu)模拟过氧化物酶的可行性, 利用HP20大孔树脂、 Supelco LC-C₁₈固相萃取和固定化金属亲和层析从甲基弯菌IMV3011中分离纯化得到Mb. 铬天青比色法显示Mb具有铜亲和性. 通过液相色谱-飞行时间质谱联用仪、 紫外光谱和荧光光谱对Mb结构进行了表征. 使用Mb-Cu配合物作为过氧化物酶模拟物, 利用紫外-可见分光光度法研究了Mb-Cu催化过氧化氢氧化对苯二酚的动力学. 考察了体系温度、 Mb-Cu添加量及过氧化氢浓度对催化反应的影响, 发现Mb-Cu符合生物催化剂条件影响的一般规律, 但比生物酶具有更高的热稳定性. 研究结果表明, Mb-Cu可作为催化氧化对苯二酚的过氧化物酶模拟酶.     

Synthesis, crystal structure and DFT analysis of a new trinuclear complex of copper

The new trinuclear complex [Cu₂(μ-L)₂CuCl₂] has been synthesized and characterized by elemental analysis, IR, UV-Vis and X-ray spectroscopy, where L is a dianionic tetradentate Schiff base ligand with N₂O₂ donor atoms. The molecular structure of [Cu₂(μ-L)₂CuCl₂] was determined by X-ray crystallography. In the complex, the most remarkable aspect of the trinuclear complex is that it adopts a bent structure for the three copper atoms, with a Cu1Cu3Cu2 intramolecular angle of approximately 90.62(2)°. All three copper atoms are five coordinate, with a slightly distorted square pyramidal geometry. In the two terminals moieties, the basal plane of the square pyramidal is formed by two oxygen atoms and two nitrogen atoms of the Schiff base ligand, and the apical position at the Cu atom is occupied by the bridging Cl1 anion. The Cu1Cl1Cu2 angle is 110.51(5)°. The central copper atom also has a five-coordinate, slightly distorted square pyramidal geometry, with four phenolato oxygens belonging to the Schiff base ligands from Cu(salpn) units describing the square planar base and the Cl anions being apical. The optimized structure of the complex has been studied using the B3LYP/6-31G(d)/LanL2TZf level of theory. The calculation shows that all the copper atoms are five coordinate with distorted square pyramidal structures, which is consistent with experimental data.     

Direct determination of copper and iodine in milk and milk powder in alkaline solution by flow injection inductively coupled plasma mass spectrometry

The development and evaluation of a flow injection inductively coupled plasma mass spectrometric (FI-ICP-MS) method for the determination of copper and iodine in milk and milk powder has been described. The sample preparation is based simply on the dilution of the sample by an alkaline solution containing 0.05 mol/l potassium hydroxide and 0.07 mol/l tetramethylammonium hydroxide. Possible matrix interferences on the determination of copper were alleviated by the use of standard addition calibration. Detection limits (3s) were 0.94 gl^-1 and 0.45 gl^-1 for copper and iodine, respectively. Four different certified milk powder reference materials were analysed and the concentrations found were in a good agreement with the certified values indicating that the method is unbiased. Due to the simplicity of the method a high sample throughput is possible, approximately 90 samples can be analysed in one day. More than 100 samples of Danish raw milk were analysed and median values of 0.050 mgkg^-1 of Cu and 0.084 mgkg^-1 of I were found.     

大肠杆菌异源表达pMMO活性中心pmoB及生物催化研究

颗粒甲烷单加氧酶（pMMO）是甲烷氧化菌中催化甲烷氧化生成甲醇的一种酶．Methylococcus capsulatus IMV 3021的pMMO活性位点是pmoB亚基,该亚基是一种可溶性蛋白．我们研究将pmoB亚基进行异源表达及生物催化活性的验证．当培养基中烟酰胺腺嘌呤二核苷酸（NADH）浓度为5 mmol／L时,可以观察到异源表达pmoB亚基具有催化甲烷氧化成甲醇活性,生成甲醇浓度为1．04 mmol／L．研究pMMO活性对于开发能直接将甲烷转化成甲醇的新型、环保催化剂有非常重要意义．     

Direct Decomposition of Nitrous Oxide to Nitrogen by In Situ Oxygen Removal with a Perovskite Membrane

Direct decomposition of N ₂ O to N ₂ using perovskite hollow fiber membranes is achieved by combination with in situ oxygen removal (see picture). A coupled partial methane oxidation allows N₂‐free synthesis gas to be obtained. This sustainable process combines N₂O removal with the simultaneous production of valuable chemicals.

     

Crystal Structure of Copper(II) and Cobalt(II) Hexafluoroacetylacetonate Complexes with 2,2,5,5-Tetramethyl-4-phenyl-3-imidazoline-1-oxyl-3-oxide

The crystal structures of the copper(II) and cobalt(II) hexafluoroacetylacetonate complexes with the nitroxide 2,2,5,5-tetramethyl-4-phenyl-3-imidazoline-1-oxyl-3-oxide (PhImOx) Cu(hfac)₂(PhImOx), Co(hfac)₂(PhImOx), and Co(hfac)₂(PhImOx)₂ have been determined. The former two are chain type complexes, whereas the latter has a molecular structure. The coordination polyhedra of the copper and cobalt atoms are tetragonally distorted octahedra. In the two chain type compounds, the octahedra are formed from the hfac anion, nitroxyl, and N-oxide O atoms; in the molecular complex, the O atoms of the NO groups are not coordinated to the metal atoms. In the structure of Cu(hfac)₂(PhImOx), F...F contacts equal to 2.805 have been found.     

The performance of density functional and wavefunction‐based methods for 2D and 3D structures of Au10

The transition from 2D to 3D structures in small gold clusters occurs around 10 atoms. Density functional theory predicts a planar structure for in contrast to recent second‐order Møller–Plesset perturbation theory calculations, which predict a 3D arrangement. The validity of the use of single‐reference second‐order Møller–Plesset theory for near metallic systems remains, however, questionable. On the other hand, it is less than clear how well density functional approximations perform for such clusters. We, therefore, decided to carry out quantum chemical calculations for using a variety of different density functionals as well as wavefunction‐based methods including coupled cluster theory to compare the different energetically low lying 2D and 3D cluster isomers. The results are perhaps not encouraging showing that most computational methods do not predict correctly the energetic sequence of isomers compared to coupled cluster theory. As perturbative triple corrections in the coupled cluster treatment change the order in cluster stability, the onset of 2D to 3D transition in these gold clusters remains elusive. As expected, second‐order Møller–Plesset theory is not suitable for correctly describing such systems.     

The Application of Copper/Iron Cocatalysis in Cross‐Coupling Reactions

For conventional cross‐couplings in organic chemistry, precious metal (such as Pd or Rh) complexes have been the preferable choices as catalysts. However, their high cost, toxicity, and potential contamination of products limit their massive applications on some occasions, particularly in the pharmaceutical industry, where close monitoring of the metal contamination of products is required. Therefore, the use of metals that are less expensive and less toxic than Pd or Rh can be greatly advantageous and earth abundant metal (such Fe or Cu) catalysts have shown promise for replacing the precious metals. Interestingly, a certain copper catalyst combined with an iron catalyst displays higher catalytic efficiency than itself in various coupling reactions. Notably, ligand‐free conditions make such protocols more useful and practical in many cases. In this account, we summarize the recent progress made in this increasingly attractive topic by describing successful examples, including our own work in the literature, regarding effective copper/iron cocatalysis. In addition, a few examples involving a magnetic and readily recyclable CuFe₂O₄ nanoparticle cocatalyst are also included.

     

Synthesis and Structure of Trinuclear Iron Acetate [Fe3O(CH3COO)6(H2O)3][AuCl4]·6H2O

Iron acetate of composition [Fe₃O(CH₃COO)₆(H₂O)₃][AuCl₄]·6H₂O (I) was synthesized and investigated by X-ray diffraction analysis and Mössbauer spectroscopy. The [Fe₃O(CH₃COO)₆(H₂O)₃]⁺ complex cation has a structure typical for ₃-O bridged trinuclear ferric compounds with iron atoms lying at the vertices of a regular triangle with an oxygen atom at the center. The iron atoms are each coordinated by 4 oxygen atoms of the four bridging carboxylic groups, the ₃-O bridging atom, and the coordinated water molecule in the trans-position to the latter. In the trinuclear cation, the Fe(III) ions are coupled by antiferromagnetic exchange interactions with the exchange parameter J = -29.0 cm ^–1 (HDVV model for D _3h symmetry). The specific role of the solvate water molecules in structure formation is discussed.     

Molecular structure of the dimeric complex of copper (II) chloride with triphenyl-N-[6-methylpyridyl-2]phosphinimine

The complex of copper(II) dichloride with triphenyl-N-(6-methylpyridyl-2)phosphinimine has been studied by X-ray diffraction. This compound has a dimeric structure with two ₂-bridge chlorine atoms. The coordination of copper atoms by three chlorine atoms and the N atom of the heterocycle is of a strongly distorted planar-square type. A peculiar feature of this structure is the presence of intramolecular contacts Cu...N (2.504 Å and 2.492 Å) involving the imine nitrogens of the P,N-containing ligands which are responsible for strong additional coordination of the copper atoms. In methanol the P,N-containing ligand forms a complex with Cu(II) where the metal-to-ligand ratio is equal to 12. The ESR data point to the bidentate nature of the ligand in solution, where all four nitrogen atoms, contributed by both of the ligands, are located in the plane of the metal atom.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 337–341, February, 1993.