X-ray absorption study of Rhus laccase: evidence for a copper-copper interaction, which disappears on type 2 copper removal

X-ray absorption spectra are reported for the multi-Cu oxidase Rhus vernicifera laccase in oxidized and fully reduced forms and for laccase from which the type 2 Cu has been depleted (T2D). The structure of the Cu K edge for both preparations shows the presence of CuII and CuI in the oxidized and reduced states, respectively. As previously reported by LuBien et al. (1981), removal of the type 2 Cu leads to reduction of the type 3 center, which can be reoxidized with H2O2. Fourier transforms of the extended X-ray absorption fine structure (EXAFS) give well-defined first and outer shell scattering peaks. Analysis of the first shell peak is complicated by the heterogeneity of the Cu sites. When (imidazole)4CuIISO4 is used as a model of the average Cu-ligand interactions, it is shown that all of the first shell peaks contain 2.7-3.5 near neighbors per Cu, at an average distance of 1.97-1.98 A. For T2D laccase, the fit is improved by inclusion of one-third of a sulfur atom at 2.19 A, corresponding to the presumptive cysteine ligand of the type 1 Cu, which remains in the preparation containing three Cu atoms per molecule. The outer shell region shows two peaks characteristic of scattering from distant imidazole atoms. For T2D laccase the filtered outer shell contribution can be satisfactorily fit by scattering from an average of 2.1-2.4 imidazole groups. For native laccase, however, imidazole alone cannot satisfactorily model the outer shell contribution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural study of the copper and zinc sites in metallothioneins by using extended X-ray-absorption fine structure.

Zn-metallothionein 1 from rabbit liver was investigated by means of Zn K-edge extended X-ray-absorption fine structure (e.x.a.f.s.). Also, the Cu and Zn K-edge e.x.a.f.s. were measured for two samples of mixed Cu Zn-metallothionein 2, with Cu/Zn ratios of 5:2 and 6:3, from pig liver. Detailed simulation of the Cu sites shows a primary co-ordination with three sulphur atoms, presumably from cysteine residues at 0.225 nm +/- 0.001 nm (2.25 +/- 0.01 A). The data for the Zn sites are best reproduced by four Zn-S separations at 0.233 +/- 0.001 nm (2.33 +/- 0.01 A). The Zn K-edge e.x.a.f.s. recorded for rabbit metallothionein 1 at 77 K shows, in addition to the primary co-ordination shell, evidence for two Zn-Zn separations at approx. 0.50 nm (5.0 A). This latter result provides the first information concerning the internal arrangement of zinc atoms in Zn7-metallothionein.     

Effect of cyanide binding on the copper sites of cytochrome c oxidase: an X-ray absorption spectroscopic study

Cu x-ray absorption spectroscopy (XAS) has been used to investigate the effect of cyanide treatment on the structures of the copper sites in beef heart cytochrome c oxidase. The Cu K-edge spectrum changes significantly upon cyanide binding to resting state enzyme, as does the Cu extended x-ray absorption fine structure (EXAFS) spectrum. The Cu EXAFS Fourier transfer (FT) exhibits an enhanced peak for the cyanide-treated enzyme in the region containing the Cu...Fe peak in the resting state FT (at R' approximately equal to 2.6-2.7 A). This peak in the cyanide-treated sample is hypothesized to arise from "outer shell" scattering from a linear Cu-cyanide moiety, suggesting cyanide binding to CuB only (CuB 2+-CN-) or cyanide bridging between the Fe of heme a3 and CuB (Fe3+-(CN-)-CuB 2+).     

Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy.

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approximately 2.5 A. The Cu K-edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 A, two sulfur atoms at 2.24 A and a Cu atom at 2. 43 A, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 A. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.     

Direct structural information for the copper site of dopamine beta-mono-oxygenase obtained by using extended X-ray-absorption fine structure.

Copper K-edge e.x.a.f.s (extended X-ray-absorption fine structure) was measured for dopamine beta-mono-oxygenase in aqueous solution. Comparison with the Cu K-edge e.x.a.f.s. of bovine erythrocyte superoxide dismutase shows a close resemblance. Detailed analysis of the e.x.a.f.s. indicates that the copper atom is bound to four imidazole groups at 0.201 nm with one or two oxygen atoms at 0.23 nm.     

X-ray absorption spectroscopic study of the active copper sites in dopamine beta-hydroxylase

X-ray absorption spectroscopy has been used to investigate the local environment of the copper sites in bovine dopamine beta-hydroylase, the enzyme that catalyzes the conversion of dopamine to norepinephrine in the adrenal medulla and noradrenergic nerve cells. The marked similarity of the x-ray absorption edge features of the oxidized and ascorbate-reduced forms of the enzyme with those of the corresponding Cu(imidazole)4 complexes suggests that the ligation in both cases is very similar. Furthermore, this similarity is found for the extended x-ray absorption fine structure data, and analysis shows only nitrogen (or oxygen) ligation for both enzyme forms. Thus, four nitrogen atoms provide the best fit to the data at an average distance of 1.97 +/- 0.02 A for the oxidized enzyme and four nitrogen atoms at 2.05 +/- 0.02 A for the ascorbate-reduced form. The present data analysis also indicates that there is little change in the average copper ligand environment upon reduction of the enzyme-bound copper from Cu(II) to the Cu(I). The data for the oxidized form of the enzyme are in agreement with previous spin-echo EPR experiments that show three to four imidazole nitrogen ligands for each copper (McCracken, J., Desai, P. R., Papadopoulos, N. J., Villafranca, J. J., and Peisach, J. (1988) Biochemistry 27, 4133-4137). In addition, the data do not indicate the presence of any heavy atom (sulfur or chlorine) ligation to the ascorbate-reduced form of the enzyme as reported by Scott et al. (Scott, R. A., Sullivan, R. J., DeWolf, W. E., Jr., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417).     

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.     

X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (Mo^VI) protein has been investigated in solution at 77?K; the Mo K-edge position (20006.4?eV) is consistent with the presence of Mo^VI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71?Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32?Å and two at 2.47?Å, and two O atoms, one at 1.92?Å (considered to be H-bonded to Trp 116) and one at 2.27?Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (Mo^VI) DMSO reductase at 77?K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (Mo^VI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4?eV and 20004.5?eV, respectively; these values, together with the lack of a significant presence of Mo^V in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of Mo^IV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05?Å and one at 2.51?Å, and four S atoms at 2.36?Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69?Å, one at 1.91?Å and one at 2.11?Å, plus four S atoms, two at 2.28?Å and two at 2.37?Å. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to Mo^IV with an Mo-O bond of length 1.92?Å.     

Studies on the active site of deacetoxycephalosporin C synthase

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.     

X-ray absorption spectroscopy of xanthine oxidase. The molybdenum centres of the functional and the desulpho forms.

X-ray absorption spectra have been recorded for the molybdenum K-edge region of xanthine oxidase. Both the absorption edge and the extended fine structure (e.x.a.f.s.) regions were investigated. Spectra were obtained for samples of the desulpho enzyme as well as for mixtures of this with the active enzyme. The spectrum of the pure active form was then obtained by difference. The desulpho enzyme shows a pronounced step in the absorption edge, of a type previously associated terminal oxygen ligands. In the active enzyme this step has decreased markedly. Satisfactory simulations of the e.x.a.f.s. spectrum of the desulpho enzyme could be obtained by assuming the molybdenum to be bonded to two terminal oxygen atoms (Mo = O about .175 nm), two sulphur atoms (presumably from cysteine residues, Mo-S about .0250 nm) and one sulphur atom (presumably from a methionine residue, Mo-S about 0.290 nm). E.x.a.f.s. of the active enzyme differed appreciably from this. In keeping with earlier proposals [Gutteridge, Tanner & Bray (1978) Biochem. J. 175, 887-897], the spectrum of the active enzyme could be simulated if a sulphur atom at about 0.225 nm (i.e. presumably a terminal sulphur atom) replaced one of the terminal oxygen atoms of the desulpho from, with small changes in the other bond distances. Validity of the interpretative procedures, which involved phase shift and amplitude calculations ab initio, was demonstrated by using low molecular weight compounds of known structure.     

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM

It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST elastic scattering cross section database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy-metal clusters used to label intracellular proteins. In sectioned cells that have been stained only with osmium tetroxide, we find an average of 1.2+/-0.1 Os atom per nm(3), corresponding to an atomic ratio of Os:C atoms of approximately 0.02, which indicates that small heavy atom clusters of Undecagold and Nanogold can be detected in lightly stained specimens.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

Redox-dependent structural changes in the Azotobacter vinelandii bacterioferritin: new insights into the ferroxidase and iron transport mechanism

In this work, we report the X-ray crystal structure of the aerobically isolated (oxidized) and the anaerobic dithionite-reduced (at pH 8.0) forms of the native Azotobacter vinelandii bacterioferritin to 2.7 and 2.0 A resolution, respectively. Iron K-edge multiple anomalous dispersion (MAD) experiments unequivocally identified the presence of three independent iron-containing sites within the protein structure. Specifically, a dinuclear (ferroxidase) site, a b-type heme site, and the binding of a single iron atom at the four-fold molecular axis of the protein shell were observed. In addition to the novel observation of iron at the four-fold pore, these data also reveal that the oxidized form of the protein has a symmetrical ferroxidase site containing two five-coordinate iron atoms. Each iron atom is ligated by four carboxylate oxygen atoms and a single histidyl nitrogen atom. A single water molecule is found within hydrogen bonding distance of the ferroxidase site that bridges the two iron atoms on the side opposite the histidine ligands. Chemical reduction of the protein under anaerobic conditions results in an increase in the average Fe-Fe distance in the ferroxidase site from approximately 3.5 to approximately 4.0 A and the loss of one of the ligands, H130. In addition, there is significant movement of the bridging water molecule and several other amino acid side chains in the vicinity of the ferroxidase site and along the D helix to the three-fold symmetry axis. In contrast to previous work, the higher-resolution data for the dithionite-reduced structure suggest that the heme may be bound in multiple conformations. Taken together, these data allow a molecular movie of the ferroxidase gating mechanism to be developed and provide further insight into the iron uptake and/or release and mineralization mechanism of bacterioferritins in general.     

Iron K-edge absorption spectroscopic investigations of the cores of ferritin and haemosiderins.

The extended X-ray absorption fine structure (EXAFS) associated with the iron K-edge has been measured and interpreted for ferritin and haemosiderin extracted from horse spleen, and haemosiderin extracted from the livers of humans with treated primary haemochromatosis, and from the spleens of humans with treated secondary haemochromatosis. For ferritin, the data are consistent with, on average, each iron atom being in an environment comprised of approx. six oxygen atoms at 1.93 +/- 0.02 A, approx. 1.5 iron atoms at 2.95 +/- 0.02 A and approx. 1.1 iron atoms at 3.39 +/- 0.02 A, with a further shell of oxygens at approx. 3.6 A. Iron in horse spleen haemosiderin is in an essentially identical local environment to that in horse spleen ferritin. In contrast, the EXAFS data for primary haemochromatosis haemosiderin indicate that the iron-oxide core is amorphous; only a single shell of approx. six oxygen atoms at approx. 1.94 +/- 0.02 A being apparent. Secondary haemochromatosis haemosiderin shows an ordered structure with approx. 1.4 iron atoms at both 2.97 +/- 0.02 and 3.34 +/- 0.02 A. This arrangement of iron atoms is similar to that in horse spleen haemosiderin, but the first oxygen shell is split with approx. 2.9 atoms at 1.90 +/- 0.02 A and approx. 2.7 at 2.03 +/- 0.02 A, indicative of substantial structural differences between secondary haemochromatosis haemosiderin and horse spleen haemosiderin.     

Two different zinc sites in bovine 5-aminolevulinate dehydratase distinguished by extended X-ray absorption fine structure

The zinc coordination in 5-aminolevulinate dehydratase was investigated by extended X-ray absorption fine structure (EXAFS) associated with the zinc K-edge. The enzyme binds 8 mol of zinc/mol of octameric protein, but only four zinc ions seem sufficient for full activity. We have undertaken a study on four forms of the enzyme: (a) the eight-zinc native enzyme; (b) the enzyme with only the four zinc sites necessary for full activation occupied; (c) the enzyme with the vacant sites of (b) occupied by four lead ions; (d) the product complex between (b) and porphobilinogen. We have shown that two structurally distinct types of zinc sites are available in the enzyme. The site necessary for activity has an average zinc environment best described by two/three histidines and one/zero oxygen from a group such as tyrosine or a solvent molecule at 2.06 +/- 0.02 A, one tyrosine or aspartate at 1.91 +/- 0.03 A, and one cysteine sulfur at 2.32 +/- 0.03 A with a total coordination of five ligands. The unoccupied site in (b), obtained by taking the difference spectrum between the spectra from samples (a) and (b), is dominated by a single contribution of four cysteinyl sulfur atoms at 2.28 +/- 0.02 A. Spectra from samples (c) and (d) show only small changes from that of (b), reflecting a slight rearrangement of the ligands around the zinc atom.     

X-ray absorption studies of the Cu-dependent phenylalanine hydroxylase from Chromobacterium violaceum. Comparison of the copper coordination in oxidized and dithionite-reduced enzymes.

The coordination chemistry of the Cu sites of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum has been studied by X-ray absorption spectroscopy (XAS). The EXAFS of the Cu(II) form of the enzyme resembles that of other non-blue copper proteins such as plasma amine oxidases and dopamine-beta-hydroxylase and is characteristic of a mixed N/O coordination shell containing histidine ligation. Detailed simulations of the raw EXAFS data have been carried out using full curved-wave restrained refinement methodologies which allow imidazole ligands to be treated as structural units. The results suggest a Cu(II) coordination of two histidines and two additional O/N-donor groups. A reasonable fit to both data sets can be obtained by assuming that the non-imidazole first-shell donor atoms are derived from solvent (H2O or OH-). The EXAFS of the reduced enzyme shows major differences. The amplitude of the first shell in the Fourier transform is only 50% of that of the oxidized enzyme, indicative of a substantial reduction in coordination number. In addition, the first shell of the transform is split into two components. Simulations of the reduced data can be obtained by either two histidines at a long distance of 2.08 A and an O ligand at a short distance of 1.88 A or two histidines at a short distance of 1.90 A and one second-row scatterer such as S or Cl at 2.20 A. Comparison of absorption edge data on the reduced enzyme with data from Cu(I) bis- and tris(1,2-dimethylimidazole) complexes suggests a pseudo-three-coordinate structure.     

Electronic property and reactivity of (hydroperoxo)metal compounds

DFT calculations were done for the (hydroperoxo)metal complexes with eta1-coordination mode, where metal ions are Fe(III), Al(III), Cu(II) and Zn(II). Results shows that 1) the electron density at the two oxygen atoms of the hydroperoxide ion is highly dependent on the angle O-O-H in M-OOH species and the difference in electron density between the two oxygen atoms reaches a maximum at the angle O-O-H = 180 degrees, 2) total electron density at the two oxygen atoms of the peroxide ion increases by approach of methane to the (hydroperoxo)metal species in the cases of Fe(III) and Cu(II); on the other hand, significant decrease of the electron density on peroxide oxygen atoms was observed for the cases of Al(III) and Zn(II) compounds. These findings suggest that the (hydroperoxo)metal species acts as an electrophile in the former cases (M = Fe(III), Cu(II)) and as a nucleophile for the latter two compounds (M = Zn(II), Al(III)). The electrophilicity observed for the Fe(III) and Cu(II) complexes is attributed to the presence of unoccupied- or half-filled d-orbitals interacting with the hydroperoxide ion. 3) Two oxygen atoms of the (hydroperoxo)-compounds of Fe(III) and Cu(II) complexes exhibit quite different reactivity toward the substrate, such as methane. When methane approaches the oxygen atom which is coordinated to a metal ion, a strong decrease of electron density at the methane carbon atom occurs with concomitant increase of electron density at the peroxide oxygen atoms inducing its heterolytic O-O cleavage. When methane approaches the terminal oxygen atom, an oxidative coupling reaction occurs between peroxide ion and methane; at first a nucleophilic attack by the terminal electron-rich oxygen atom occurs at the carbon atom to induce C-O bond formation, and a subsequent oxidative electron transfer proceeds from substrate to the metal-peroxide species yielding CH3-OOH, CH3OH, or other oxidized products. These results clearly demonstrate that the (hydroperoxo)-metal compound itself is a rather stable compound, and activation of the peroxide ion is induced by interaction with the substrate, and the products obtained by the oxygenation reaction are dependent on the chemical property of the substrate, redox property of a metal ion, and stability of the compounds formed in the intermediate process.     

Spectroscopic studies for identifying the chemical states of the periostracum of the Corbicula species in Lake Biwa

Corbicula clam shells consist of thin periostracum and calcareous layers made of calcium carbonate (CaCO₃). Depending on habitat conditions, the shell exhibits various colorations, such as yellow, brown, and black. The chemical state of the periostracum of the Corbicula species in Lake Biwa was studied by X-ray absorption fine structure (XAFS) and Raman scattering spectroscopies. Fe K-edge X-ray absorption near edge structure (XANES) revealed that the Fe³⁺ intensity increases as the color of the shell changes from yellow to black. Raman spectra suggested that quinone-based polymers cover the yellow shell, and the black shell is further covered by dihydroxyphenylalanine (DOPA) rings of amino acid derivatives. From Fe K-edge extended X-ray absorption fine structure (EXAFS), it was found that Fe³⁺ in the periostracum was surrounded by five to six oxygen atoms with an average Fe-O ligand distance of 2.0 Å. Accordingly, a tris-DOPA-Fe³⁺ complex is formed, which is responsible for the periostracum’s black color.     

Multiple scattering x-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q(B). On the basis of x-ray diffraction at 2.5 angstroms resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+). Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn(2+) binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.     

Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa strain P2 were studied by x-ray and electron paramagnetic resonance (EPR) spectroscopy. The enzyme is dimeric and contains four Cu atoms and about seven cysteine residues/subunit of Mr = 73,000. The extended x-ray absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as isolated (oxidized or slightly reduced), enzyme exposed briefly to air, reduced enzyme, and enzyme at pH 7 after having been activated by standing at pH 10. The average Cu ligand environment in the first shell was best modeled for all forms of the enzyme by a combination of N/O and S atoms at a total coordination number between 3 and 4 and bond distances ranging from 1.96-2.03 A for Cu-N/O and 2.20-2.25 A for Cu-S. The data could be fit without using Cu-Cu interactions. Overall the results are similar to those reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086). The first derivative EPR spectra of the Cu(II) centers at 15 and 45 K were qualitatively similar among enzyme as isolated and enzyme exposed to N2O or air. These three nominally oxidized samples showed an axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16. Hyperfine structure was observed in both the g parallel and g perpendicular regions with splittings of 43 and 25 gauss, respectively. These hyperfine components are attributed to exchange coupled Cu(I)-Cu(II) S = 1/2 (half-met) centers. In the enzyme as isolated and after exposure to N2O, about 3/4 of the Cu was EPR silent, whereas after exposure to air the signal integrated to about half the Cu concentration. The EPR spectrum of enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra from activated and inactive species. The activated species presented a complex set of narrow hyperfine components which may arise from contributions from more than one species of half-met center.