Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans.

Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear "ferroxidase centre" in individual subunits. We have measured X-ray absorption spectra of four mutants (K86Q, K86Q/E27D, K86Q/E107D, and K86Q/E27D/E107D, involving variations of Glu to Asp on either or both sides of the dinuclear ferroxidase site) of recombinant human H-chain ferritin (rHuHF) in their complexes with reactive Fe(II) and redox-inactive Zn(II). The results for Fe-rHuHf are compared with those for recombinant Desulfovibrio desulfuricans bacterioferritin (DdBfr) in three states: oxidised, reduced, and oxidised/Chelex-treated. The X-ray absorption near-edge region of the spectrum allows the oxidation state of the iron ions to be assessed. Extended X-ray absorption fine structure simulations have yielded accurate geometric information that represents an important refinement of the crystal structure of DdBfr; most metal-ligand bonds are shortened and there is a decrease in ionic radius going from the Fe(II) to the Fe(III) state. The Chelex-treated sample is found to be partly mineralised, giving an indication of the state of iron in the cycled-oxidised (reduced, then oxidised) form of DdBfr, where the crystal structure shows the dinuclear site to be only half occupied. In the case of rHuHF the complexes with Zn(II) reveal a surprising similarity between the variants, indicating that the rHuHf dinuclear site is rigid. In spite of this, the rHuHf complexes with Fe(II) show a variation in reactivity that is reflected in the iron oxidation states and coordination geometries.

Bridging-type changes facilitate successive oxidation steps at about 1 V in two binuclear manganese complexes--implications for photosynthetic water-oxidation.

The redox behavior of two synthetic manganese complexes illustrates a mechanistic aspect of importance for light-driven water oxidation in Photosystem II (PSII) and design of biomimetic systems (artificial photosynthesis). The coupling between changes in oxidation state and structural changes was investigated for two binuclear manganese complexes (1 and 2), which differ in the set of first sphere ligands to Mn (N(3)O(3) in 1, N(2)O(4) in 2). Both complexes were studied by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) in three oxidation states which had been previously prepared either electro- or photochemically. The following bridging-type changes are suggested. In 1: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(II)<-->Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(III). In 2: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)<-->Mn(III)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(IV). In both complexes, the first one-electron oxidation proceeds without bridging-type change, but involves a redox-potential increase by 0.5-1V. The second one-electron oxidation likely is coupled to mu-oxo-bridge (or mu-OH) formation which seems to counteract a further potential increase. In both complexes, mu-O(H) bridge formation is associated with a redox transition proceeding at approximately 1V, but the mu-O(H) bridge is observed at the Mn(2)(III,III) level in 1 and at the Mn(III,IV) level in 2, demonstrating modulation of the redox behavior by the terminal ligands. It is proposed that also in PSII bridging-type changes facilitate successive oxidation steps at approximately the same potential.

Solution structure of the partially folded high-risk human papilloma virus 45 oncoprotein E7.

The oncoprotein E7 of human papilloma viruses (HPV) is involved in the pathogenesis and maintenance of human cervical cancers. The most prevalent HPV types found in cervix carcinomas are HPV16, 18 and 45. The structure of the E7 dimer from HPV45 (PDB 2F8B) was determined by nuclear magnetic resonance spectroscopy. Each monomer comprises an unfolded N-terminus and a well-structured C-terminal domain with a beta1beta2alpha1beta3alpha2 topology representing a unique zinc-binding fold found only for E7. Dimerization occurs through the alpha1/alpha1' helices and intermolecular beta-sheet formation but excludes the zinc-binding sites. E7 is reported to interact with a number of cellular proteins (e.g. pRb, p21(CIP1)). Binding of a peptide derived from the C-terminus of p21(CIP1) to the C-terminal domain of E7 was characterized by monitoring chemical shift perturbations of the amide groups of E7. This provides direct evidence that a shallow groove situated between alpha1 and beta1 of the E7 C-terminal domain is interacting with the C-terminus of p21(CIP1). Intriguingly, this binding site overlaps with the low-affinity binding site on E7 for the C-domain of pRb.

Structural and oxidation state changes of the photosystem II manganese complex in four transitions of the water oxidation cycle (S0 --> S1, S1 --> S2, S2 --> S3, and S3,4 --> S0) characterized by X-ray absorption spectroscopy at 20 K and room temperature.

Structural and electronic changes (oxidation states) of the Mn(4)Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), and S(3),(4) --> S(0)), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and RT data, S(0)-S(3), XANES and EXAFS) obtained by the three approaches leads to the following conclusions. (i) In all S-states, the gross structural and electronic features of the Mn complex are similar at 20 K and room temperature. There are no indications for significant temperature-dependent variations in structure, protonation state, or charge localization. (ii) Mn-centered oxidation likely occurs on each of the three S-state transitions, leading to the S(3) state. (iii) Significant structural changes are coupled to the S(0) --> S(1) and the S(2) --> S(3) transitions which are identified as changes in the Mn-Mn bridging mode. We propose that in the S(2) --> S(3) transition a third Mn-(mu-O)(2)-Mn unit is formed, whereas the S(0) --> S(1) transition involves deprotonation of a mu-hydroxo bridge. In light of these results, the mechanism of accumulation of four oxidation equivalents by the Mn complex and possible implications for formation of the O-O bond are considered.

Bone tissue incorporates in vitro gallium with a local structure similar to gallium-doped brushite.

During mineral growth in rat bone-marrow stromal cell cultures, gallium follows calcium pathways. The dominant phase of the cell culture mineral constitutes the poorly crystalline hydroxyapatite (HAP). This model system mimics bone mineralization in vivo. The structural characterization of the Ga environment was performed by X-ray absorption spectroscopy at the Ga K-edge. These data were compared with Ga-doped synthetic compounds (poorly crystalline hydroxyapatite, amorphous calcium phosphate and brushite) and with strontium-treated bone tissue, obtained from the same culture model. It was found that Sr(2+) substitutes for Ca(2+) in the HAP crystal lattice. In contrast, the replacement by Ga(3+) yielded a much more disordered local environment of the probe atom in all investigated cell culture samples. The coordination of Ga ions in the cell culture minerals was similar to that of Ga(3+), substituted for Ca(2+), in the Ga-doped synthetic brushite (Ga-DCPD). The Ga atoms in the Ga-DCPD were coordinated by four oxygen atoms (1.90 A) of the four phosphate groups and two oxygen atoms at 2.02 A. Interestingly, the local environment of Ga in the cell culture minerals was not dependent on the onset of Ga treatment, the Ga concentration in the medium or the age of the mineral. Thus, it was concluded that Ga ions were incorporated into the precursor phase to the HAP mineral. Substitution for Ca(2+ )with Ga(3+) distorted locally this brushite-like environment, which prevented the transformation of the initially deposited phase into the poorly crystalline HAP.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

The X-ray absorption spectroscopy Debye-Waller factors of an iron compound and of met-myoglobin as a function of temperature.

Protein dynamics can be characterized by the mean square displacements of the individual atoms of a molecule. This concept is extended to X-ray absorption spectroscopy (XAS) of proteins where the physical information in the Debye-Waller factor is in general neglected. In a first step, a procedure for the investigation of the temperature dependence of XAS spectra has been developed for a small iron compound. Subsequently, experiments have been performed on met-myoglobin. It is shown that the mean square displacements of XAS are smaller than those obtained by Mössbauer spectroscopy and far smaller than crystallographic mean square displacements. This behavior is explained by the different sensitivity of the methods. XAS measures a relative mean square displacement between the absorbing and backscattering atoms only. A comparison with mean square displacements calculated from normal modes shows that static displacements contribute significantly. It becomes obvious that the atoms of the active center show a high correlation of their motions.

Molecular mechanism of hydrogen peroxide conversion and activation by Cu(II)-amikacin complexes.

The interactions between Cu(II)-amikacin complexes [Cu(II)-Ami] and hydrogen peroxide were studied by spectroscopy (EPR, UV-vis, CD, XAS) and cyclic voltammetry. A monomer-dimer equilibrium was detected at complex concentrations above 5 mM (log K(dim) = 1.84 +/- 0.03). The dimeric complex undergoes easy, although irreversible oxidation (ca. 0.5-0.6 V) to a Cu(III) species on platinum electrode. However, the monomeric complexes are able to catalyze hydrogen peroxide disproportionation reaction at pH 7.4 in a multistep process, mediated by hydroxyl radicals and involving both Cu(I)/Cu(II) and Cu(II)/Cu(III) redox pairs.

Metal ion binding and coordination geometry for wild type and mutants of metallo-beta -lactamase from Bacillus cereus 569/H/9 (BcII): a combined thermodynamic, kinetic, and spectroscopic approach.

One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.

Determination of metal-metal distances: significance and accuracy.

Nature utilises a variety of metal clusters as catalytic centers. Some of them make use of two or more metals in the catalytic site. The variation of metal-metal distances plays an important role in several processes like charge transfer and weakening of bonds. X-ray absorption spectroscopy can determine these metal-metal distances in several states (crystal, solution or amorphous). However sometimes backscattering from light elements hides the metal-metal contribution to the fine structure. Here we point out significance and accuracy of metal-metal distances in a model system. Therefore a number of different refinement protocols are applied to the data. These protocols will be discussed focusing on the significance and accuracy of the metal-metal distances extracted from the data.

Structural properties of Y1-xYbxNi2B2C synthesized at high pressure: EXAFS data analysis.

Local structure of Y(1-x)Yb(x)Ni2B2C series synthesized at high pressure 8 GPa has been studied using EXAFS. Measurements were performed at the Ni K-edge in temperature range 5-300 K. The results show that the Debye-Waller factor for Ni-Ni bond in the parent YNi2B2C compound is characterized by the Einstein temperature O(E) = 350 K, while a minimum value O(E) = 300 K is reached for the compound with = 0.025, which has the highest critical temperature T(c) = 12.5K of the superconductive transition. This correlates with the further suppressing of superconductivity and with the appearance of the local magnetic moments in the investigated Y(1-x)Yb(x)Ni2B2C series for x > or = 0.05 compounds. Observed changes in the local electronic and the local crystal structure of this system as a function of Yb concentration and of temperature were explained in the frame of the band filling effect.

The role of Se, Mo and Fe in the structure and function of carbon monoxide dehydrogenase.

CO dehydrogenase (EC 1.2.99.2) catalyzes the oxidation of CO according to the following equation: CO + H2O-->CO2 + 2 e- + 2 H+. It is a selenium-containing molybdo-iron-sulfur-flavoenzyme, which has been crystallized and structurally characterized in its oxidized state from the aerobic CO utilizing bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudoflava. Both CO dehydrogenase structures show only minor differences, and the enzymes are dimers of two heterotrimers. Each heterotrimer is composed of a molybdoprotein, a flavoprotein, and an iron-sulfur protein. CO oxidation takes place at the molybdoprotein which contains a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and a Mo-ion, along with a catalytically essential S-selanylcysteine. The latter is appropriately positioned in the SeMo-active site by a unique VAYRCSFR active site loop. In H. pseudoflava the arginine preceeding the cysteine in the active site loop is modified to a Cgamma-hydroxy arginine residue which has no obvious function. The substituents in the first coordination sphere of the Mo-ion are the enedithiolate sulfur atoms of the molybdopterin-cytosine dinucleotide, two oxo- and a sulfido-group. Extended X-ray absorption fine structure spectroscopy (EXAFS), along with the crystal structure of CO dehydrogenase (23.2 U mg(-1)) at 1.85 A resolution, have identified a sulfur atom at 2.3 A from the Mo-ion. The sulfur reacts with cyanide yielding thiocyanate. The corresponding inactive desulfo-CO dehydrogenase shows a typical desulfo inhibited-type of Mo-electron paramagnetic resonance (EPR) spectrum. Structural changes at the SeMo-site during catalysis are suggested by the Mo to Se distance of 3.7 A and the Mo-S-Se angle of 113 degrees in the oxidized enzyme which increase to 4.1 A, and 121 degrees, respectively, in the reduced enzyme. The intramolecular electron transport chain in CO dehydrogenase involves the following prosthetic groups and minimal distances: CO-->[Mo of the molybdenum cofactor] - 14.6 A - [2Fe-2S]I - 12.4 A - [2Fe-2S]II - 8.7 A - [FAD].

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin.

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and (15)N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His-->Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS*L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)(3) donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS*N donor set deriving from His46, Cys112 and Met121. The Cu-S(delta)(Met) distance is reduced from about 3.2 to 2.3 A. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the beta-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.

Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036.

Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Generation of oxoiron (IV) tetramesitylporphyrin pi-cation radical complexes by m-CPBA oxidation of ferric tetramesitylporphyrin derivatives in butyronitrile at - 78 degrees C. Evidence for the formation of six-coordinate oxoiron (IV) tetramesitylporphyrin pi-cation radical complexes FeIV = O(tmp*)X (X = Cl-, Br-), by Mössbauer and X-ray absorption spectroscopy.

The generation of six-coordinate oxoiron (IV) tetramesitylporphyrin pi-caption radical complexes by m-CPBA (meta-chloroperbenzoic acid) oxidation of ferric tetramesitylporphyrin derivatives in butyronitrile at - 78 degrees C was investigated. UV-Vis and EPR spectroscopies indicate that the axial ligand present in the ferric starting derivatives is retained in the high-valent iron complexes. Indirect evidence for the formation of six-coordinate oxoiron (IV) tetramesitylporphyrin complexes FeIV = O(tmp*)X (X=Cl-, Br-) by m-CPBA oxidation of FeX(tmp) (X=Cl-, Br-) in butyronitrile at - 78 degrees C was also obtained by Mössbauer spectroscopy. Direct confirmation of the presence of a halide ion as second axial ligand of iron in these high-valent iron species was obtained by X-ray absorption spectroscopy. The EXAFS spectra of the samples obtained by m-CPBA oxidation of FeX(tmp) (X=Cl-, Br-) were refined using two different coordination models including both four porphyrinato-nitrogens and the axial oxo group. The two models include (model I) or exclude (model II) the axial halogen. The statistical tests indicate the presence of a halide ion as second axial ligand of iron in both derivatives. The refinements led to the following bond distances: FeIV=O(tmp*)Cl(3):Fe-O=1.66(1),Fe-Cl=2.39(2) and Fe-Np=1.99(1) A;FeIV=O(tmp*)Br(4):Fe-O=1.65(1),Fe-Br=2.93(2), Fe-Np=2.02(1) A. The lengthening of the Fe-X(X=Cl-, Br-) distances relative to those occurring in the ferric precursor porphyrins is, most probably, related to the strong trans influence of the oxoiron(IV) fragment present in 3 or 4.

Extended X-ray absorption fine structure studies on periplasmic and intracellular molybdenum-binding proteins.

We have studied the molybdenum K-edge X-ray absorption spectra of Mo bound in the Mo-binding proteins Mop from Haemophilus influenzae, ModG from Azotobacter vinelandii and the Escherichia coli ModE transcriptional regulatory protein, and compared them with the absorption spectra of A. vinelandii ModA and monomeric molybdate. Pre-edge and extended fine structure data indicate that the Mo-binding proteins with molbindin-like domains bind tetrahedral molybdate with a Mo-O distance of 1.76 A. The molbindin subunits or sub-domains represent a novel protein fold that is used by proteins with distinct functions to bind molybdate in the cytoplasm. The high specificity of the proteins for molybdenum does not depend on a change of coordination number or geometry.

Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy.

Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6 x histidine) N-terminal tags. After reconstitution with 57Fe(II), the Mössbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization. Before dehydration, > 90% of the iron in hTH1 had Mössbauer parameters typical for high-spin Fe(II) in a six-coordinate environment [isomer shift delta (1.8-77 K) = 1.26-1.24 mm s-1 and quadrupole splitting delta EQ = 2.68 mm s-1]. After dehydration, the Mössbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (delta = 1.07 mm s-1 and delta EQ = 2.89 mm s-1 at 77 K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (delta = 1.24 mm s-1 and delta EQ = 2.87 mm s-1 at 77 K). Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag. After rehydration of hTH1 the spectroscopic changes were completely reversed. The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mössbauer spectra. The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five-coordinate iron center. Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron. The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center.

Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism.

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.