Towards a unified understanding of the copper sites in particulate methane monooxygenase: an X-ray absorption spectroscopic investigation

The enzymatic conversion of the greenhouse gas, methane, to a liquid fuel, methanol, is performed by methane monooxygenases (MMOs) under mild conditions. The copper stoichiometry of particulate MMO (pMMO) has been long debated, with a dicopper site previously proposed on the basis of a 2.51 Å Cu–Cu feature in extended X-ray absorption fine structure (EXAFS) data. However, recent crystallographic data and advanced electron paramagnetic resonance (EPR) characterization support the presence of only mononuclear copper sites. To reconcile these data, we have collected high-energy resolution fluorescence detected (HERFD) and partial fluorescence yield (PFY) EXAFS spectra of Methylococcus (M.) capsulatus (Bath) pMMO. Both methods reveal only monocopper sites. These data were compared to previously published pMMO PFY-EXAFS data from M. capsulatus (Bath) and Methylomicrobium alcaliphilum 20Z, supporting dicopper and monocopper sites, respectively. The FT-EXAFS feature previously attributed to a dicopper site can be reproduced by the inclusion of a metallic copper background signal. The exact position of this feature is dependent on the nature of the sample and the percentage of background contamination, indicating that visual inspection is not sufficient for identifying background metallic contributions. Additionally, an undamaged X-ray absorption spectrum was obtained, consistent with the copper oxidation-state speciation determined by EPR quantification. X-ray photodamage studies suggest that the previously observed Cu(i) XAS features are in part attributable to photodamage. This study illustrates the complex array of factors involved in EXAFS measurement and modeling of pMMO and more generally, dilute metalloproteins with multiple metal centers.

Extended X-ray absorption fine structure spectroscopic analysis of particulate methane monooxygenase reveals only monocopper sites and investigates the possible origins of the previous observed dicopper signals.     

负载型非晶态Cu／SiO2催化剂局域结构的研究

采用ＸＰＳ和ＥＸＡＦＳ方法，研究了以ｓｏｌ－ｇｅｌ法制得的超累粉体ＳｉＯ２为载体，用化学还原沉积法制备的负载型非晶态Ｃｕ／ＳｉＯ２催化剂在甲酸甲酯氢解反应前后的表面结构和局域结构。结果表明，非晶态样品中的铜原子以零价铜的形式存在，但配位数却大幅度地低于铜樯档，意味着铜原子在高比表面超细ＳｉＯ２载体上处于高分散的非晶状态，表面悬空键显著增多，表面能增大，导致配位键收缩。     

Quantum Refinement Does Not Support Dinuclear Copper Sites in Crystal Structures of Particulate Methane Monooxygenase

Particulate methane monooxygenase (pMMO) is one of the few enzymes that can activate methane. The metal content of this enzyme has been highly controversial, with suggestions of a dinuclear Fe site or mono‐, di‐, or trinuclear Cu sites. Crystal structures have shown a mono‐ or dinuclear Cu site, but the resolution was low and the geometry of the dinuclear site unusual. We have employed quantum refinement (crystallographic refinement enhanced with quantum‐mechanical calculations) to improve the structure of the active site. We compared a number of different mono‐ and dinuclear geometries, in some cases enhanced with more protein ligands or one or two water molecules, to determine which structure fits two sets of crystallographic raw data best. In all cases, the best results were obtained with mononuclear Cu sites, occasionally with an extra water molecule. Thus, we conclude that there is no crystallographic support for a dinuclear Cu site in pMMO.     

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.     

Mononuclear to polynuclear transition induced by ligand coordination: synthesis, X-ray structure, and properties of mono-, di-, and polynuclear copper(II) complexes of pyridyl-containing azo ligands

Reactions of two hydrated cupric salts (CuCl(2).2H(2)O and Cu(ClO(4))(2).6H(2)O) with three azopyridyl ligands, viz. 2-[(arylamino)phenylazo]pyridine [aryl = phenyl (HL(1a)), p-tolyl (HL(1b)), and 2-thiomethyl phenyl (HL(1c))], 2-[2-(pyridylamino)phenylazo]pyridine (HL(2)), and 2-[3-(pyridylamino)phenylazo]pyridine (HL(3)), afford the mononuclear [CuClL(1)] (1), dinuclear [Cu(2)X(2)L(2)(2)](n)()(+) (X = Cl, H(2)O, ClO(4); n = 0, 1; 2, 3), and polynuclear [CuClL(3)](n)() (4) complexes, respectively, in high yields. Representative X-ray structures of these complexes 1-4 are reported. X-ray structure analysis of 4 reveals an infinite 1D zigzag chain that adopts a saw-tooth-like structure. Variable-temperature cryomagnetic measurements (2-300 K) on the complexes 2-4 have revealed weak magnetic interactions between the copper centers with J values -1.04, 9.88, and -1.31 cm(-1), respectively. Positive ion ESI mass spectra of the soluble complexes 1-3 are studied which provide the evidence for the integrity of the complexes also in solution. Visible range spectra of the complexes 1-3 in solution consist of intense and broad transitions in the range 700-600 nm. The solid-state spectrum of the insoluble copper complex 4, on the other hand, shows a structured band near 700 nm. The intensities of the transitions of the dinuclear complexes are much higher than those of the corresponding mononuclear copper complexes. Redox properties of the present copper complexes are reported. Notably, the dinuclear complex, 3, displays two successive redox processes: Cu(II)Cu(II) right harpoon over left harpoon Cu(II)Cu(I) right harpoon over left harpoon Cu(I)Cu(I). It catalyzes aerial oxidation of L-ascorbic acid. The catalytic cycle is most effective up to H(2)A/3 (H(2)A = L-ascorbic acid) molar ratio of 20:1.     

Geometric and electronic structure of the heme-peroxo-copper complex [(F8TPP)FeIII-(O22-)-CuII(TMPA)](ClO4)

The geometric and electronic structure of the untethered heme-peroxo-copper model complex [(F(8)TPP)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](ClO(4)) (1) has been investigated using Cu and Fe K-edge EXAFS spectroscopy and density functional theory calculations in order to describe its geometric and electronic structure. The Fe and Cu K-edge EXAFS data were fit with a Cu...Fe distance of approximately 3.72 A. Spin-unrestricted DFT calculations for the S(T) = 2 spin state were performed on [(P)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](+) as a model of 1. The peroxo unit is bound end-on to the copper, and side-on to the high-spin iron, for an overall mu-eta(1):eta(2) coordination mode. The calculated Cu...Fe distance is approximately 0.3 A longer than that observed experimentally. Reoptimization of [(P)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](+) with a 3.7 A Cu...Fe constrained distance results in a similar energy and structure that retains the overall mu-eta(1):eta(2)-peroxo coordination mode. The primary bonding interaction between the copper and the peroxide involves electron donation into the half-occupied Cu d(z)2 orbital from the peroxide pi(sigma) orbital. In the case of the Fe(III)-peroxide eta(2) bond, the two major components arise from the donor interactions of the peroxide pi*(sigma) and pi*(v) orbitals with the Fe d(xz) and d(xy) orbitals, which give rise to sigma and delta bonds, respectively. The pi*(sigma) interaction with both the half-occupied d(z)2 orbital on the copper (eta(1)) and the d(xz) orbital on the iron (eta(2)), provides an effective superexchange pathway for strong antiferromagnetic coupling between the metal centers.     

二(桥-氯)-二[氯-(N,N,N''''-三苄基乙二胺)铜(Ⅱ)]的晶体结构

0 IntroductionThe copperdim er,bis(μ鄄hydroxo)鄄bis[chloro(N,N,N',N'鄄tetram ethylethylenediam ine) copper髤] chloride(Cu(OH )Cl·TM EDA),prepared from N,N,N',N'鄄tetram鄄ethylethylenediam ine (TM EDA) and CuCl, exhibitedexcellentcatalytic activity for t     

Structure of copper sites in zeolites examined by Fourier and wavelet transform analysis of EXAFS

Copper-exchanged zeolites are a class of redox-active materials that find application in the selective catalytic reduction of exhaust gases of diesel vehicles and, more recently, the selective oxidation of methane to methanol. However, the structure of the active copper-oxo species present in zeolites under oxidative environments is still a subject of debate. Herein, we make a comprehensive study of copper species in copper-exchanged zeolites with MOR, MFI, BEA, and FAU frameworks and for different Si/Al ratios and copper loadings using X-ray absorption spectroscopy. Only obtaining high quality EXAFS data, collected at large k-values and measured under cryogenic conditions, in combination with wavelet transform analysis enables the discrimination between the copper-oxo species having different structures. The zeolite topology strongly affects the copper speciation, ranging from monomeric copper species to copper-oxo clusters, hosted in zeolites of different topologies. In contrast, the variation of the Si/Al ratio or copper loading in mordenite does not lead to significant differences in XAS spectra, suggesting that a change, if any, in the structure of copper species in these materials is not distinguishable by EXAFS.

The structure of copper-oxo species hosted in zeolites of various topology has been examined using wavelet and Fourier transform analysis of Cu K-edge EXAFS spectra.     

The X-ray absorption spectroscopic model of the copper(II) imidazole complex ion in liquid aqueous solution: a strongly solvated square pyramid

Cu K-edge extended X-ray absorption fine structure (EXAFS) and Minuit X-ray absorption near-edge structure (MXAN) analyses were combined to evaluate the structure of the copper(II) imidazole complex ion in liquid aqueous solution. Both methods converged to the same square-pyramidal inner coordination sphere [Cu(Im)(4)L(ax)](2+) (L(ax) indeterminate) with four equatorial nitrogen atoms at EXAFS, 2.02 ± 0.01 ?, and MXAN, 1.99 ± 0.03 ?. A short-axial N/O scatterer (L(ax)) was found at 2.12 ± 0.02 ? (EXAFS) or 2.14 ± 0.06 ? (MXAN). A second but very weak axial Cu-N/O interaction was found at 2.9 ± 0.1 ? (EXAFS) or 3.0 ± 0.1 ? (MXAN). In the MXAN fits, only a square-pyramidal structural model successfully reproduced the doubled maximum of the rising K-edge X-ray absorption spectrum, specifically excluding an octahedral model. Both EXAFS and MXAN also found eight outlying oxygen scatterers at 4.2 ± 0.3 ? that contributed significant intensity over the entire spectral energy range. Two prominent rising K-edge shoulders at 8987.1 and 8990.5 eV were found to reflect multiple scattering from the 3.0 ? axial scatterer and the imidazole rings, respectively. In the MXAN fits, the imidazole rings took in-plane rotationally staggered positions about copper. The combined (EXAFS and MXAN) model for the unconstrained cupric imidazole complex ion in liquid aqueous solution is an axially elongated square-pyramidal core, with a weak nonbonded interaction at the second axial coordination position and a solvation shell of eight nearest-neighbor water molecules. This core square-pyramidal motif has persisted through [Cu(H(2)O)(5)](2+), [Cu(NH(3))(4)(NH(3),H(2)O)](2+), (1, 2) and now [Cu(Im)(4)L(ax))](2+) and appears to be the geometry preferred by unconstrained aqueous-phase copper(II) complex ions.     

Determination by X-ray absorption spectroscopy of the Fe-Fe separation in the oxidized form of the hydroxylase of methane monooxygenase alone and in the presence of MMOD

The diiron active site in the hydroxylase of Methylococcus capsulatus (Bath) methane monooxygenase (MMOH) has been studied in the oxidized form by X-ray absorption spectroscopy (XAS). Previous investigations by XAS and X-ray crystallography have identified two different distances (3.0 and 3.4 angstroms) between the two Fe atoms in the dinuclear site. The present study has employed a systematic extended X-ray absorption fine structure (EXAFS) fitting methodology, utilizing known and simulated active site and relevant model structures, to determine unambiguously the Fe-Fe separation in the oxidized form of MMOH. Consistent and unique fits were only possible for an Fe-Fe distance of 3.0 angstroms. This methodology was then applied to study potential changes in the active site local structure in the presence of MMOD, a protein of unknown function in multicomponent MMO. Fe K-edge and EXAFS analyses revealed negligible changes in the diiron site electronic and geometric structure upon addition of MMOD to oxidized MMOH.     

Molecular scale speciation of first-row transition elements bound to ligneous material by using X-ray absorption spectroscopy

To develop a solid scientific basis for maintaining soil quality and formulating effective remediation strategies, it is critical to determine how environmentally-important trace metals are sequestered in soils at the molecular scale. The speciation of Mn, Fe and Cu in soil organic matter has been determined by synchrotron-based techniques: extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES). We show the structural similarity between the surface complexes of Mn(II), Fe(III) and Cu(II). These cations are bound to the surface through oxygen atoms. Each one presents a more or less tetragonal-distorted octahedral geometry. The use of X-ray absorption spectroscopy provides a relevant method for determining trace-metal speciation in both natural and contaminated environmental materials.     

Cu K-edge EXAFS characterisation of copper(I) arenethiolate complexes in both the solid and liquid state: detection of Cu-Cu coordination

Herein we describe a structural characterisation with EXAFS of copper(I) arenethiolate complexes in both the solid and liquid state. Previously noted difficulties in the detection of the Cu-Cu interaction have been attributed to anti-phase behaviour of different Cu-Cu neighbour contributions. A data analysis procedure solely based on EXAFS parameters is presented which resolves these problems. A careful analysis of the individual coordination shells and the use of different k-weightings during the data analysis are shown to be an absolute necessity to obtain reliable results. During R-space fitting, the difference file technique is used to separate, examine and compare the individual contributions. Using this technique their statistical significance and correctness can be determined. Anti-phase behaviour can be detected and accounted for in this way. An additional mixed organocopper aggregate [Cu4(SAr)2(Mes)2] with different Cu sites is analysed, which proves the value of the analysis procedure described above. Moreover, this newly developed EXAFS data analysis procedure is applicable to any other EXAFS spectrum obtained. The structural analysis of these organocopper complexes with EXAFS provides information about their actual structure and dynamic behaviour in solution. The technique can now be used to obtain insights into the reactivity of these complexes and the way in which they form catalytic reaction intermediates.     

The quest for the particulate methane monooxygenase active site

Particulate methane monooxygenase is a copper-containing, membrane-bound metalloenzyme that converts methane to methanol in Nature. How pMMO accomplishes this difficult reaction under ambient conditions is one of the major unsolved problems in bioinorganic chemistry. Despite considerable research efforts in the past 20 years, the active site of the enzyme remains unknown. We recently solved the first crystal structure of pMMO to 2.8 è resolution, revealing the overall structure, oligomerization state, subunit ratio, and composition and location of the metal centers. Almost none of the key structural features were predicted. In this Perspective, we review the state of knowledge before and after the structure determination, emphasizing elucidation of the pMMO active site.     

In situ Fe K-edge X-ray absorption fine structure of a nitrosyl adduct of iron phthalocyanine irreversibly adsorbed on a high area carbon electrode in an acidic electrolyte

Key aspects of the microenvironment surrounding the Fe center in the nitrosyl adduct of iron phthalocyanine, [Fe(Pc)(NO)], have been elucidated from the analysis of the Fe K-edge extended X-ray absorption fine structure (EXAFS) of the material adsorbed on the surface of a high area carbon electrode recorded in situ, in 0.5 M H(2)SO(4). Statistical best fits to the EXAFS data place the Fe center in a five-coordinated square pyramidal configuration shifted away from the Pc plane toward the axially bound NO bent at an angle of ca. 40 degrees with respect to the normal to the Pc plane. This environment is analogous to that of Fe in the nitrosyl adduct of crystalline [Fe(TPP)], where TPP = meso-tetraphenylporphyrinato(2-), determined from X-ray diffraction.     

Creation of a type 1 blue copper site within a de novo coiled-coil protein scaffold

Type 1 blue copper proteins uniquely coordinate Cu(2+) in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu(2+) coordination scaffold composed of a four-stranded α-helical coiled-coil structure. Two His residues and one Cys residue were situated to form the trigonal planar geometry and to coordinate the Cu(2+) in the hydrophobic core of the scaffold. The protein bound Cu(2+), displayed a blue color, and exhibited UV-vis spectra with a maximum of 602-616 nm, arising from the thiolate-Cu(2+) ligand to metal charge transfer, depending on the exogenous axial ligand, Cl(-) or HPO(4)(2-). The protein-Cu(2+) complex also showed unresolved small A(∥) values in the electron paramagnetic resonance (EPR) spectral analysis and a 328 mV (vs normal hydrogen electrode, NHE) redox potential with a fast electron reaction rate. The X-ray absorption spectrum revealed that the Cu(2+) coordination environment was identical to that found in natural type 1 blue copper proteins. The extended X-ray absorption fine structure (EXAFS) analysis of the protein showed two typical Cu-N(His) at around 1.9-2.0 ?, Cu-S(Cys) at 2.3 ?, and a long Cu-Cl at a 2.66 ?, which are also characteristic of the natural type 1 blue copper proteins.     

A trigonal planar mu 3-fluorido coinage metal complex from a dicationic (diphosphinomethane)copper(I) dimer: syntheses,structures, and bonding

The triflate and hexafluorophosphate salts of [Cu2(mu-dtbpm)2]2+ (1(2+)) [dtbpm = bis(di-tert-butylphosphino)methane, tBu2PCH2PtBu2] and of [Cu3(mu 3-F)(mu-dtbpm)3]2+ (2(2+)) were synthesized and characterized. Coordination of solvent or counterions to 1(2+) is observed neither in solution nor in the solid state. The two copper(I) centers in 1(2+) indicate weak d10-d10 closed-shell interactions. 1(2+) reacts slowly with PF6- anions in acetone or KF in CH2Cl2 to yield the mu 3-fluorido complex 2(2+) with idealized D3 symmetry, containing a trigonal planar Cu3F core, as shown by single-crystal X-ray diffraction. Distinct structural differences are observed compared to monocationic bicapped, trinuclear copper(I) dppm halide complexes [dppm = bis(diphenylphosphino)methane, Ph2PCH2PPh2]. The average Cu-Cu, Cu-F, and Cu-P distances and the P-Cu-P' angle in 2(2+) are 3.85, 2.22, and 2.28 A and 144.3 degrees, respectively. The P2Cu units are twisted out of the Cu3F plane by an average angle of 18.4 degrees. DFT calculations (BPW91/LANL2DZ) for the model [Cu3(mu 3-F)(mu-dhpm)3]2+ (dhpm = diphosphinomethane, H2PCH2PH2) are used to explain the formation, structure, and bonding pattern of 2(2+).     

Role of iron and copper in particulate methane monooxygenase of Methylosinus trichosporium OB3b

The role of iron and copper in particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b is described, and an overview of the enzyme's properties is presented. The pMMO from M. trichosporium OB3b was solubilized in the detergent n-dodecyl--D-maltoside and purified by chromatographic techniques. The enzyme consists of 0.9 iron atoms and 12.8 copper atoms per molecule. The iron site in pMMO may be mononuclear non-heme iron. Copper exists as either copper ion coupled to four nitrogen atoms and/or trinuclear copper cluster wherein copper ions are ferromagnetically coupled.     

Synthesis and characterization of a new Schiff base derived from 2,3-diaminopyridine and 5-methoxysalicylaldehyde and its Ni(II), Cu(II) and Zn(II) complexes. Electrochemical and electrocatalytical studies

We describe the synthesis and characterization of a new tetradentate Schiff base ligand obtained from 2,3-diaminopyridine and 5-methoxysalicylaldehyde. This ligand (H₂L) reacted with nickel(II), copper(II), and zinc(II) acetates to give complexes. The ligand and its metal complexes were characterized using analytical, spectral data (UV–vis, IR, and mass spectroscopy), and cyclic voltammetry (CV). The crystal structure of the copper complex was elucidated by X-ray diffraction studies. The electrochemical behavior of these compounds, using CV, revealed that metal centers were distinguished by their intrinsic redox systems, e.g. Ni(II)/Ni(I), Cu(II)/Cu(I), and Zn(II)/Zn(I). Moreover, the electrocatalytic reactions of Ni(II) and Cu(II) complexes catalyze the oxidation of methanol and benzylic alcohol.     

Intact and fragmented triosmium clusters on MgO: characterization by X-ray absorption spectroscopy and high-resolution transmission electron microscopy

Oxidative fragmentation of the clusters Os(3)(CO)(12) adsorbed on MgO powder was investigated by X-ray absorption spectroscopy and scanning transmission electron microscopy (STEM). Exposure of the clusters to air leads to their fragmentation, oxidation of the osmium, and formation of ensembles consisting of three Os atoms. X-ray absorption near-edge spectra demonstrate the oxidative nature of the fragmentation process. Extended X-ray absorption fine structure (EXAFS) spectra indicate an average Os-Os distance of 3.33 Angstrom and an Os-Os coordination number of 2, consistent with the formation of ensembles of three Os atoms on the support. STEM images confirm the presence of such trinuclear ensembles, and the diameters of the observed scattering centers (6.0 Angstrom) match that indicated by the EXAFS results.     

Investigation of the copper binding site and the role of histidine as a ligand in riboflavin binding protein

Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 A, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O 3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.