Developmental changes in bone mineral structure demonstrated by extended X-ray absorption fine structure (EXAFS) spectroscopy

EXAFS spectra have been recorded above the calcium K edge from bones of mice aged 3 days, 1 week, 1 month, 2 months and 7 months. Spectra indicated that the calcium ion environment in bone mineral changes during development. Results were compared with those obtained from amorphous calcium phosphate and a poorly crystalline hydroxyapatite matured from this amorphous calcium phosphate in the presence of water. Spectra from the older mice closely resembled those of the matured product but those from the younger mice were more like those from the freshly prepared amorphous calcium phosphate.     

Interactions of Cu (II) and Fe (III) with mangal humic substances studied by synchronous fluorescence spectroscopy and potentiometric titration

Humic (HA) and fulvic (FA) acids isolated from mangrove sediments of Sundarban, the largest delta on earth in the estuarine phase of the river Ganges, were studied and attempts were made to characterize their binding sites by quenching of Synchronous fluorescence (SyF) bands with Fe (III) and Cu (II). A modified Stern-Volmer relationship applicable for static quenching was applied for the determination of conditional stability constants and the data were compared with those determined by potentiometric titration. In the excited state HA and FA showed different acidity constant compared to the ground state. Values of the conditional stability constant (log K_c) for Fe (III) and Cu (II) indicated that binding sites were bidentate in nature. FA were better chelators than the HA fractions. High energy binding sites of both FA & HA were occupied by Fe(III) and the low energy binding sites, mainly responsible for mobilization and immobilization of metal, were occupied by Cu(II).     

Secondary structure and Pd(II) coordination in S-layer proteins from Bacillus sphaericus studied by infrared and X-ray absorption spectroscopy

The S-layer of Bacillus sphaericus strain JG-A12, isolated from a uranium-mining site, exhibits a high metal-binding capacity, indicating that it may provide a protective function by preventing the cellular uptake of heavy metals and radionuclides. This property has allowed the use of this and other S-layers as self-assembling organic templates for the synthesis of nanosized heavy metal cluster arrays. However, little is known about the molecular basis of the metal-protein interactions and their impact on secondary structure. We have studied the secondary structure, protein stability, and Pd((II)) coordination in S-layers from the B. sphaericus strains JG-A12 and NCTC 9602 to elucidate the molecular basis of their biological function and of the metal nanocluster growth. Fourier transform infrared spectroscopy reveals similar secondary structures, containing approximately 35% beta-sheets and little helical structure. pH-induced infrared absorption changes of the side-chain carboxylates evidence a remarkably low pK < 3 in both strains and a structural stabilization when Pd((II)) is bound. The COO(-)-stretching absorptions reveal a predominant Pd((II)) coordination by chelation/bridging by Asp and Glu residues. This agrees with XANES and EXAFS data revealing oxygens as coordinating atoms to Pd((II)). The additional participation of nitrogen is assigned to side chains rather than to the peptide backbone. The topology of nitrogen- and carboxyl-bearing side chains appears to mediate heavy metal binding to the large number of Asp and Glu in both S-layers at particularly low pH as an adaptation to the environment from which the strain JG-A12 has been isolated. These side chains are thus prime targets for the design of engineered S-layer-based nanoclusters.     

Sulfur K-edge extended X-ray absorption fine structure spectroscopy of homoleptic thiolato complexes with Zn(II) and Cd(II)

The sulfur K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy is applied to homoleptic thiolato complexes with Zn(II) and Cd(II), (Et(4)N)[Zn(SAd)(3)] (1), (Et(4)N)(2)[{Zn(ScHex)(2)}(2)(mu-ScHex)(2)] (2), (Et(4)N)(2)[{Cd(ScHex)(2)}(2)(mu-ScHex)(2)] (3), (Et(4)N)(2)[{Cd(ScHex)}(4)(mu-ScHex)(6)] (4), [Zn(mu-SAd)(2)](n) (5), and [Cd(mu-SAd)(2)](n) (6) (HSAd=1-adamantanethiol, HScHex=cyclohexanethiol). The EXAFS results are consistent with the X-ray crystal data of 1-4. The structures of 5 and 6, which have not been determined by X-ray crystallography, are proposed to be polynuclear structures on the basis of the sulfur K-edge EXAFS, far-IR spectra, and elemental analysis. Clear evidences of the S...S interactions (between bridging atoms or neighboring sulfur atoms) and the S...C(far) interactions (in which C(far) atom is next to carbon atom directly bonded to sulfur atom) were observed in the EXAFS data for all complexes and thus lead to the reliable determination of the structures of 5 and 6 in combination with conventional zinc K-edge EXAFS analysis for 5. This new methodology, sulfur K-edge EXAFS, could be applied for the structural determination of in vivo metalloproteins as well as inorganic compounds.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Novel structural variation of silver(I)-pyridine complexes in nitromethane as studied by X-ray absorption spectroscopy

The XAFS spectra were measured at around the Ag K-edge of the Ag(I) ion in nitromethane (NM) with a variety of concentrations of pyridine (PY). In NM without PY, the Ag(I) ion is tetrahedrally solvated by four NM molecules similar to those in most solvents. The Ag-O bond length in NM solvent is longer than that in aqueous solution, indicating the low donating ability of NM. The mono-, bis-, tris-, and tetrakis-pyridine complexes are formed in NM by the addition of PY. The EXAFS analyses reveal that the structure of the formed PY complex in NM is linear for Ag(py)(nm)⁺, linear for , triangular for , and tetrahedral for . The longer Ag-O bond length for Ag(py)(nm)⁺ than that for and the release of bound NM molecules at the formation of Ag(py)(nm)⁺ are interpreted to be due to the strong σ donating property of PY. The Ag-N bond length (220 pm) for is intermediate between 216 pm for and 228 pm for . The formation equilibria of and are analyzed on the basis of the changeover of EXAFS spectra as a function of the total concentrations of Ag⁺ and PY in NM.     

pH-dependent local structure of ferricytochrome c studied by x-ray absorption spectroscopy

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

5 K extended X-ray absorption fine structure and 40 K 10-s resolved extended X-ray absorption fine structure studies of photolyzed carboxymyoglobin

A previous extended X-ray absorption fine structure (EXAFS) study of photolyzed carboxymyoglobin (MbCO) [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829; Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523] has provoked much discussion on the heme structure of the photoproduct (MbCO). The EXAFS interpretation that the Fe-CO distance increases by no more than 0.05 A following photodissociation has been regarded as inconsistent with optical, infrared, and magnetic susceptibility studies [Fiamingo, F. G., & Alben, J. O. (1985) Biochemistry 24, 7964-7970; Sassaroli, M., & Rousseau, D. L. (1986) J. Biol. Chem. 261, 16292-16294]. The present experiment was performed with well-characterized dry film samples in which MbCO molecules were embedded in a poly(vinyl alcohol) matrix [Teng, T. Y., & Huang, H. W. (1986) Biochim. Biophys. Acta 874, 13-18]. The sample had a high protein concentration (12 mM) to yield adequate EXAFS signals but was very thin (40 micron) so that complete photolysis could be easily achieved by a single flash from a xenon lamp. Although the electronic state of MbCO resembles that of deoxymyoglobin (deoxy-Mb), direct comparison of EXAFS spectra indicates that structurally MbCO is much closer to MbCO than to deoxy-Mb.(ABSTRACT TRUNCATED AT 250 WORDS)     

The local structure of solid diorganotin(IV) complexes formed with carbohydrates by X-ray absorption spectroscopy

Diethyltin(IV) complexes formed with carbohydrate ligands (aldoses, polyalcohols, sugar acids and disaccharides) containing the diethyltin(IV) moiety and the carbohydrate ligand in a 1:1 ratio were prepared. Their local structures were determined by extended X-ray absorption fine structure (EXAFS) in the solid state. The results showed that the dioxastannolane units are associated into an infinite chain polymer, in which tin(IV) is bound by two carbon atoms and three or four oxygen atoms either in highly distorted octahedral and trigonal bipyramidal arrangements or in a purely trigonal bipyramidal arrangement. The present structure models are consistent with the results of previous Mössbauer studies, proving the advantages of the use of the partial quadrupole splitting concept for the determination of the symmetry of the coordination sphere in tin(IV) organic complexes.     

The Fe-heme structure of met-indoleamine 2,3-dioxygenase-2 determined by X-ray absorption fine structure

Multiple-scattering (MS) analysis of EXAFS data on met-indoleamine 2,3-dioxygenase-2 (IDO2) and analysis of XANES have provided the first direct structural information about the axial donor ligands of the iron center for this recently discovered protein. At 10 K, it exists in a low-spin bis(His) form with Fe–N_p(av) = 1.97 Å, the Fe–N_Im bond lengths of 2.11 Å and 2.05 Å, which is in equilibrium with a high-spin form at room temperature. The bond distances in the low-spin form are consistent with other low-spin hemeproteins, as is the XANES spectrum, which is closer to that of the low-spin met-Lb than that of the high-spin met-Mb. The potential physiological role of this spin equilibrium is discussed.     

The active site of hemerythrin as determined by X-ray absorption fine structure

Extensive X-ray absorption fine structure measurements and analysis have been made on azidomet- and methemerythrin and on the native forms of oxy- and deoxyhemerythrin. Due to the availability of models that have been synthesized to mimic the active site of hemerythrin, it was possible to make a thorough assessment of the various errors in the structural parameters determined by the analysis. It is found that the largest source of error is the lack of complete transferability of amplitude and phase between the standards and hemerythrin. This is of particular importance in distinguishing the contributions of the second-shell low-Z atoms and, thus, has a substantial influence on the determination of the iron-iron distance. The internal consistencies of the various checks and a new formulation of error analysis for the structural parameters give us confidence in the structure determined for the active site. The main result is that as O2 is released from oxyhemerythrin, the mu-oxo bridge between the two iron atoms in the active site with an Fe-O distance of 1.8 A converts to a mu-hydroxo bridge in deoxyhemerythrin, expanding the Fe-O distance to 2.0 A. The Fe-Fe distance expands proportionally from 3.24 A in oxyhemerythrin to 3.57 A in deoxyhemerythrin so as to keep the Fe-O-Fe bridging angle approximately constant. These conclusions provide experimental support for the structures of oxy- and deoxyhemerythrin proposed previously on the basis of spectroscopic and preliminary X-ray crystallographic data.     

Similarity of the structure of ferritin and iron . dextran (imferon) determined by extended X-ray absorption fine structure analysis.

Ferritin, a natural complex of iron oxide encased in protein, and iron . dextran, a synthetic complex of iron oxide coated with dextran, have the similar properties of maintaining high concentrations of iron in solution at physiological pH and releasing iron relatively slowly in vivo. Extended x-ray absorption fine structure (EX-AFS) analysis was performed on each complex and compared to see if the structures of the iron cores were similar. The results obtained from the extended x-ray absorption fine structure technique show that the near-neighbor environment around the average iron atom in ferritin and iron . dextran is identical, within experimental uncertainty, for the first three shells. The similarity of the iron cores in both complexes may explain the similarity of iron release in vivo. Ferritin has a protein coat which is composed of 24 subunits arranged in a hollow sphere with six channels through which the iron may move during deposition and release. However, little is known about the requirements of the protein structure in ferritin for the maintenance of high concentrations of iron in a soluble, nontoxic form or about the role of the protein in the release of iron from ferritin. The results suggest that iron . dextran will be a useful model compound in studies of the relation of the iron core and protein in ferritin to function.     

Role of aliphatic and phenolic hydroxyl groups in uranyl complexation by humic substances

Relativistic density functional calculations of uranyl complexes with alcohols were carried out to study how phenolic and aliphatic hydroxyl groups of humic substances may contribute to uranyl complexation by humic substances. According to recent experimental work, blocking of phenolic OH groups decreases the loading capacity, but has no effect on the key geometric parameters of uranyl humate complexes. This can be understood on the basis of our calculations which showed uranium-oxygen distances to be very similar for complexes with rather different types of O-donor ligands, with average U-O_eq ∼ 237 pm for fivefold coordinated uranyl (VI) complexes, both for O⁻ and OH functional groups. Uranyl complexation by alcohol moieties seems unlikely at environmental conditions as a high pH is required for the deprotonation of these groups; we confirm an alternative complexation mechanism that overcomes the ligand deprotonation problem. Similarities in structures and energetic suggest that complexes of both aliphatic and phenolic alcoholates may well contribute in comparable fashion to the complexation of uranyl by humic acids.     

X-ray absorption spectroscopy (XAS) of toxic metal mineral transformations by fungi

Fungi can be highly efficient biogeochemical agents and accumulators of soluble and particulate forms of metals. This work aims to understand some of the physico-chemical mechanisms involved in toxic metal transformations focusing on the speciation of metals accumulated by fungi and mycorrhizal associations. The amorphous state or poor crystallinity of metal complexes within biomass and relatively low metal concentrations make the determination of metal speciation in biological systems a challenging problem but this can be overcome by using synchrotron-based element-specific X-ray absorption spectroscopy (XAS) techniques. In this research, we have exposed fungi and ectomycorrhizas to a variety of copper-, zinc- and lead-containing minerals. X-ray absorption spectroscopy studies revealed that oxygen ligands (phosphate, carboxylate) played a major role in toxic metal coordination within the fungal and ectomycorrhizal biomass during the accumulation of mobilized toxic metals. Coordination of toxic metals within biomass depended on the fungal species, initial mineral composition, the nitrogen source, and the physiological state/age of the fungal mycelium.     

Structural investigations by extended X-ray absorption fine structure spectroscopy of the iron center of mitochondrial aconitase in higher plant cells.

We have obtained iron K-edge extended x-ray absorption fine structure spectra of the plant mitochondrial aconitase in its active state, in the presence (aconitase (+)) and absence (aconitase (-)) of the substrate citrate. Analysis of the data indicates that oxygens are present in the first coordination shell, at an average Fe-O distance of 1.96/1.98 A (aconitase (+)/aconitase(-)). Part of these oxygens is provided by the citrate, which binds at 1.99 A from the iron in aconitase (+). The second shell (sulfur) contribution is split and is consistent with Fe-S distances of 2.30/2.29 and 2.56/2.59 A, and the third shell (iron) is consistent with an Fe-Fe distance of 2.83/2.84 A. Both Fe-S and Fe-Fe distances are longer than similar distances found in most Fe-S centers. A strong scattering at approximately 5 A has been identified as originating from an iron atom which is near to, but not part of, the Fe-S cluster. These data indicate that active plant mitochondrial aconitase contains a novel type of iron center.     

Effects of humic substances on the oxidation of pentachlorophenol by peroxosulfate catalyzed by iron(III)-phthalocyanine-tetrasulfonic acid

In an attempt to enhance the oxidation of pentachlorophenol (PCP) in the Fe(III)-PcTS/KHSO5 system, the presence of added humic substances (HSs) was studied, investigating the chemical properties of HSs related to the enhancement in PCP oxidation by correlations with the degree of enhancement in PCP oxidation (%delta(PCP)60). The %delta(PCP)60 value increased with a decrease in the content of oxygen-containing functional groups, such as carboxylic acids. This indicated that HSs with a lower content of oxygen-containing functional groups would be useful for enhancing the oxidation of PCP. A negative correlation between %delta(PCP)60 and the kinetic constants of Fe(III)-PcTS self-oxidation indicated that the enhancement by added HSs could be attributed to the suppression of Fe(III)-PcTS deactivation by self-oxidation. Such a stabilization of Fe(III)-PcTS could be attributed to hydrophobic interactions between the catalyst and HSs.     

Nitrogen chemical structure in DNA and related molecules by X-ray absorption spectroscopy.

The electronic environment of nitrogen in nucleic acid bases, nucleotides, polynucleotides and DNA has been studied, for the first time using X-Ray Absorption Near-Edge Spectroscopy (XANES). Generally, the spectra of these complex molecules consist of low energy bands corresponding to 1s-->pi* transitions and high energy bands corresponding to 1s-->sigma* transition, as illustrated using several nitrogen model compounds. The 1s-->pi* transitions show particular sensitivity to the chemical environment of the nitrogen. Oxygen substitution on ring carbon atoms generally results in a significant blue shift of the lowest 1s-->pi* bands while halogen substitution results in a small blue shift. These observations illustrate the significance of the disturbance of the aromatic ring system produced by exocyclic carbonyl groups. Direct substitution on the nitrogen frequently results in significant spectral perturbations. Differences between the spectra of the polynucleotides and the sums of spectra of the individual nucleotides point to the effects of hydrogen-bonding in complementary double-helix structures. The XANES spectrum of a DNA sample with a known ratio of the polynucleotides is equivalent to the weighted sum of the spectra of individual polynucleotides, indicating that the difference in base stacking interactions produces negligible spectral effects. The variability of nitrogen K-edge spectra in these samples and in protein may be useful for chemically specific imaging using X-ray microscopes.