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The structures of two types of guanidine-quinoline copper complexes have been investigated by single-crystal X-ray crystallography, K-edge X-ray absorption spectroscopy (XAS), resonance Raman and UV/Vis spectroscopy, cyclic voltammetry, and density functional theory (DFT). Independ- ent of the oxidation state, the two structures, which are virtually identical for solids and complexes in solution, resemble each other strongly and are connected by a reversible electron transfer at 0.33 V. By resonant excitation of the two entatic copper complexes, the transition state of the electron transfer is accessible through vibrational modes, which are coupled to metal-ligand charge transfer (MLCT) and ligand-metal charge transfer (LMCT) states. Copper is one of the most important redox-active metals and plays a central role in many biological processes. (1) Blue copper electron-transfer proteins are natures workhorses for electron transfer (ET). They use copper as a one-electron relay that shuttles between the cuprous and cupric oxidation states. Their Cu II/I reduction potentials span a large window (E8' = 0.18 to > 1 V versus NHE (normal hydrogen elec- trode)), (2) which tailors these proteins to interact with a wide variety of ET partners. The inner coordination sphere most directly affects the redox properties of metal ions. (3) Hence, tuning the Cu II/I redox couple is central for electron transfer in nature and also important in synthetic complexes for catalytic applications. (4-6) There are multiple factors governing the redox potential, including the effects of the first coordination

The zinc(II) coordination chemistry of the bioinspired bis(tetradentate) ligand 3,5-bis{bis[2-(1-methylimidazolyl)methyl]aminomethyl}pyrazole (L) was studied in detail both in solution and in the solid state. Six different species were determined in aqueous solution by means of potentiometry within the pH range 211, namely mononuclear [ZnH2L]4+, dinuclear [Zn2L]4+, [Zn2H1L]3+ and [Zn2H2L]2+, as well as tetranuclear [Zn4H5L2]3+ and [Zn4H6L2]2+. Complexes representing the protonation state of four of these species could be isolated and structurally characterized by X-ray diffraction, namely [ZnH2L(MeCN)](ClO4)4 (1), [Zn2H1L](SO3CF3)3 (4), [Zn2H1L(MeOHOMe)](ClO4)2 (5a), [Zn2H1L(OMe)](ClO4)2 (5b), and [(Zn2H1L)2(mu-O)(mu-OH)](ClO4)3 (6). In addition, a bis(pyrazolato)-bridged complex, [(ZnH1L)2](ClO4)2 (2), was obtained and structurally characterized, and it was found to exhibit ligand dynamics in solution. Both 5a and 5b are likely present in solution, which requires significant plasticity of the Zn...Zn distance (4.21 angstrom in 5a vs. 3.38 angstrom in 5b). Interconversion of 5a and 5b is proposed to be a facile process, supporting the idea that the ZnO2H3Zn moiety is a functionally relevant hydrolytic zincaqua structure in oligozinc metalloenzymes. The tetranuclear complex 6 that features two {Zn2H1L} subunits connected by a central mu-oxido-mu-hydroxido moiety is unprecedented in zinc(II) chemistry. Combining X-ray crystallography with potentiometry, ESI mass spectrometry, as well as NMR and EXAFS spectroscopy has provided a comprehensive picture of the multitude of species present in this rather complex system.

Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls, which replicate key structural features of the active site of the hydrogenase Hmd. The reaction of Ph(2)PC(6)H(4)C(O)SPh and sources of Fe(0) generates both Fe(SPh)(Ph(2)PC(6)H(4)CO)(CO)(3) (1) and the diferrous diacyl Fe(2)(SPh)(2)(CO)(3)(Ph(2)PC(6)H(4)CO)(2), which carbonylates to give 1. For the extremely bulky arylthioester Ph(2)PC(6)H(4)C(O)SC(6)H(3)-2,6-(2,4,6-trimethylphenyl)(2), oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained. Complex 1 reacts with cyanide to give Et(4)N[Fe(SPh)(Ph(2)PC(6)H(4)CO)(CN)(CO)(2)] (Et(4)N[2]). (13)C and (31)P NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2](-) in ν(CN) and ν(CO) regions very closely matches that for Hmd(CN). XANES and EXAFS measurements also indicate close structural and electronic similarity of Et(4)N[2] to the active site of wild-type Hmd. Complex 1 also stereospecifically forms a derivative with TsCH(2)NC, but the adduct is more labile than Et(4)N[2]. Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd.

The influence of sulfur versus selenium coordination to molybdenum on the oxo transfer reaction mechanisms of functional models for oxidoreductases has been studied. The solution structure of the dimeric molybdenum compound with tridentate bis-anionic ligands containing a thioether function (−O(CH2)3S(CH2)3O−) has been determined using EXAFS spectroscopy to be able to compare a feature of its solution structure to that of its selenoether analogue. A significant difference is found for the solution structures of the two compounds. The thioether group remains coordinated in solution, whereas the selenoether does not. The influence of this difference on the catalytic oxo transfer has been investigated in detail by following the catalytic transition of PPh3 to OPPh3 with DMSO as oxygen donor with variation of both substrate concentrations.

Zinc serves regulatory functions in cells and thus, several mechanisms exist for tight control of its homeostasis. One mechanism is storage in and retrieval from vesicles, so-called zincosomes, but the chemical speciation of zincosomal zinc has remained enigmatic. Here, we determine the intravesicular zinc-coordination in isolated zincosomes in comparison to intact RAW264.7 murine macrophage cells. In elemental maps of a cell monolayer, generated by microbeam X-ray fluorescence, zincosomes were identified as spots of high zinc accumulation. A fingerprint for the binding motif obtained by μXANES (X-ray absorption near edge structure) matches the XANES from isolated vesicles; zinc is not free, but present as a complexed form (average coordination; 1.0 sulfur, 2,5 histidines 30 and 1.0 oxygen), resembling regulatory or catalytic zinc sites in proteins. Such coordination enables reversible binding, acting as a ‘zinc sink’, facilitating the accumulation of high amounts of zinc against a concentration gradient.

The Dps-like peroxide resistance protein (Dpr) is a dodecameric protein that protects the human and swine pathogen Streptococcus suis from hydrogen peroxide by removing free Fe2+ from the cytosol. Subsequent oxidation of iron by Dpr results in the deposition of Fe3+ inside the protein's central cavity. Structural changes that occur in the ferroxidase center were studied by X-ray crystallography after soaking Dpr crystals with Fe2+ in the presence of sodium dithionite. Twelve iron-binding sites were identified with each site formed by residues Asp74 and Glu78 from one subunit, and Asp63, His47 and His59 from a 2-fold symmetry-related subunit. Compared to the iron-free Dpr, Asp74 and Glu78 were found to be the most flexible amino acid residues and able to adopt a variety of conformations in different subunits. The crystal structure of an Asp74Ala Dpr mutant soaked with a Fe2+ -solution revealed variations in the Asp63 position and no iron bound to the ferroxidase center. These results indicate an intrinsic flexibility in the active site that may be important for the catalytic reaction and subsequent nucleation events. Two iron cores with remarkably different features were identified in Dpr using X-ray absorption spectroscopy. Purified Dpr was found to have a small-size iron core with only approximately 16 iron atoms/dodecamer forming a ferritin-like ferrihydrite structure. Because of its size, this core represents the smallest iron core identified so far in ferritins and other Dps-like proteins. A large-size core (approximately 180 iron atoms/dodecamer) formed after incubating the protein with a ferrous solution shows differences in iron coordination compared to the small size core. Characterization of the two iron cores in Dpr could provide insights into nucleation events and the mechanism of iron core growth in the Dps family of proteins.

Peptide nucleic acid (PNA) oligomers with linear topology are synthesised with histidines at the central positions in order to provide metal-ion coordination sites within the water shielded and non-polar environment of a nucleobase stack that emerges upon duplex formation. Variation of the linker length used for attachment of the nucleobases to the regular peptide backbone allows for fine tuning of the distances and coordination environment at the metal binding site. The effect of zinc and copper incorporation into the modified alanyl-PNA scaffolds on duplex stability was probed by temperature-dependent UV spectroscopy, and additional insight was gained from EPR spectroscopy and EXAFS. Whenever four histidines (two per oligomer strand) are available, addition of Zn2+ significantly enhances double-strand stability, thus supporting coordination of Zn2+ by the histidine-N and incorporation of the metal ion within the base stack. In contrast to Zn2+, however, Cu2+ ions do not induce double-strand formation of an alanyl/norvalyl-PNA oligomer, as confirmed by EPR and EXAFS, which reveal a mixed {N2Ox} donor environment. While metal-ion binding to histidine-modified alanyl-PNA oligomers (and homologues) is apparently feasible, a proper matching of the binding-site geometry and the stereoelectronic requirements of the specific metal ion are essential in order to mediate double-strand formation. This might offer new options for alternative pairing schemes in alanyl-PNA chemistry in which metal coordination substitutes the common base-pair H-bonding pattern, and for metal-dependent regulation of alanyl-PNA duplex formation. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The regulatory Ni–Fe hydrogenase (RH) from Ralstonia eutropha which forms a [HoxBC] 2 complex functions as a hydrogen sensor under aerobic conditions. We have studied a novel Strep-tag isolate of the RH large subunit, HoxC ST , which lacks the Fe–S clusters of HoxB, allowing for structure determination of the catalytic site by X-ray absorption spectroscopy both at the Ni and, for the first time, also at the Fe K-edge. This technique, together with Fourier-transform infrared spectroscopy, revealed a Ni–Fe site with [O 1 (CysS) 2 Ni II (μ-SCys) 2 Fe II (CN) 2 (CO)] structure in about 50% of HoxC ST and a [(CysS) 2 Fe II (CN) 2 (CO)] site lacking Ni in the remainder protein. Possibly both sites may be intermediates in the maturation process of the RH.

Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.

Cu(II) binding to the α prion protein (αPrP) can be both intramolecular and intermolecular. X-ray absorption spectroscopy at the copper K-edge has been used to explore the site geometry under each binding mode using both insoluble polymeric Cu(II)·αBoPrP-(24-242) (bovine PrP) complexes and soluble Cu(II) complexes of peptides containing one, two, and four copies of the octarepeat. Analysis of the extended region of the spectra using a multiple scattering approach revealed two types of sites differing in the number of His residues in the first coordination shell of Cu(II). Peptides containing one and two-octarepeat copies in sub-stoichiometric Cu(II) complexes showed the direct binding of a single His in accord with crystallographic intra-repeat geometry. Alternatively, the polymeric Cu(II)·αBoPrP-(24-242) complex and Cu(II) in its soluble complex with a four-octarepeat peptide at half-site-occupancy showed Cu(II) directly bound to two His residues, consistent with an inter-repeat binding mode. Increasing the Cu(II) site occupancy from 0.5 to 0.75 in the peptide containing four octarepeats resulted in spectral features that are intermediate to those of the inter- and intra-repeat modes. The transition from His-Cu-His (inter-repeat) to Cu-His (intra-repeat) on increasing Cu(II) saturation offers a structural basis for the positive cooperativity of the cation binding process and explains the capacity of αPrP to participate in Cu(II)-mediated intermolecular interactions.

The analysis of XANES patterns is very indicative for screening samples. Powerful X-ray absorption spectroscopy data-analysis programs can simulate these patterns. Here, a case study on two structural motifs is presented: a non-heme Fe site (2-His-1-carboxylate motif) and the metallo β-lactamase dinuclear Zn site. Simulations of the edge shapes for different structural models will be compared with experimental results, pointing out limitations and challenges. The influence of single neighbouring atoms in the first and second shell on the resulting XANES pattern is discussed. Insights into catalytic mechanisms and the requirements for future theory development are addressed.

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.

The cellular prion protein (PrP(C)) is a Cu(2+) binding protein connected to the outer cell membrane. The molecular features of the Cu(2+) binding sites have been investigated and characterized by spectroscopic experiments on PrP(C)-derived peptides and the recombinant human full-length PrP(C )(hPrP-[23-231]). The hPrP-[23-231] was loaded with (63)Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP(C)/Cu(2+)-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu(2+) structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu(2+) coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP(C).

Guanidinium nitroprusside GNP, (CN3H6)2[Fe(CN)5NO] has been investigated in single‐crystalline form by nuclear resonant forward scattering (NFS) using synchrotron radiation (Mossbauer spectroscopy in the time domain). This method provides a direct measure of effective absorber thickness and therefore also of the Lamb–Mossbauer factor fLM. GNP has the advantage that all [Fe(CN5)NO]2- anions are practically aligned within the crystal. For the two different crystal orientations, with the crystallographic a- and c-direction parallel to the synchrotron beam, respectively, we have obtained fLM(a)= 0.122(10) and fLM(c)= 0.206(10), i.e., GNP exhibits significant anisotropic vibrational behavior. The quantum beat pattern of the NFS spectra obtained for the two different crystal orientations is discussed on the basis of radiation characteristics of the polarized synchrotron beam and the multipole transitions of oriented 57Fe nuclei.

X-ray absorption spectroscopy at the Mn K-edge has been performed on multilayers of photosystem II-enriched fragments of the native thylakoid membrane prepared from a higher plant (spinach) and a unicellular green alga (Scenedesmus obliquus). Spectra collected for various angles between the prevailing orientation of the thylakoid membrane normal and the X-ray electric field vector contain information on the atomic structure of the tetranuclear manganese complex of photosystem II (PS II) and its orientation with respect to the membrane normal. The previously used approach for evaluation of the dichroism of extended X-ray absorption fine structure (EXAFS) spectra [George, G. N., et al. (1989) Science 243, 789-791] is modified, and the following results are obtained for PS II in its dark-stable state (S1-state): (1) structure and orientation of the PS II manganese complexes of green algae and higher plants are highly similiar or fully identical; (2) two 2.7-A vectors, which, most likely, connect the Mn nuclei of a planar Mn2(mu-O2) structure, are at an average angle of 80 degrees +/- 10 degrees with respect to the thylakoid normal; (3) the plane of the Mn2(mu-O2) structures is rather in parallel with the thylakoid plane than perpendicular. Structural models for the oxygen-evolving manganese complex and its orientation in the thylakoid membrane are discussed within the context of the presented results.

Cu(II) binding to the alpha prion protein (alphaPrP) can be both intramolecular and intermolecular. X-ray absorption spectroscopy at the copper K-edge has been used to explore the site geometry under each binding mode using both insoluble polymeric Cu(II).alphaBoPrP-(24-242) (bovine PrP) complexes and soluble Cu(II) complexes of peptides containing one, two, and four copies of the octarepeat. Analysis of the extended region of the spectra using a multiple scattering approach revealed two types of sites differing in the number of His residues in the first coordination shell of Cu(II). Peptides containing one and two-octarepeat copies in sub-stoichiometric Cu(II) complexes showed the direct binding of a single His in accord with crystallographic intra-repeat geometry. Alternatively, the polymeric Cu(II).alphaBoPrP-(24-242) complex and Cu(II) in its soluble complex with a four-octarepeat peptide at half-site-occupancy showed Cu(II) directly bound to two His residues, consistent with an inter-repeat binding mode. Increasing the Cu(II) site occupancy from 0.5 to 0.75 in the peptide containing four octarepeats resulted in spectral features that are intermediate to those of the inter- and intra-repeat modes. The transition from His-Cu-His (inter-repeat) to Cu-His (intra-repeat) on increasing Cu(II) saturation offers a structural basis for the positive cooperativity of the cation binding process and explains the capacity of alphaPrP to participate in Cu(II)-mediated intermolecular interactions.

The guanidine–quinoline ligand dimethylethyleneguanidinoquinoline (DMEGqu) is able to stabilise bis(chelate) copper complexes in an intermediate geometry between tetrahedral and square-planar environments. The structures of the obtained complexes model the entatic state and have been investigated in solid state by single-crystal X-ray diffraction and in the solid state and in solution by X-ray absorption spectroscopy. The dimethylethyleneguanidine (DMEG) unit of the DMEGqu ligand displays a smaller steric encumbrance than the tetramethylguanidine (TMG) counterpart; this allows slightly larger structural changes upon oxidation than those for the TMG counterparts. Moreover, triflate coordination was possible for the CuII DMEG complexes. DFT analyses revealed that good structural and optical descriptions are possible through the use of the hybrid functionals B3LYP and TPSSh in combination with the triple-zeta basis set def2-TZVP and the inclusion of empirical dispersion with Becke–Johnson damping and a suitable solvent model. The orbital analysis gives insights into the electronic structure of the complexes and their charge-transfer behaviour.

Streptococcus suis Dpr belongs to the Dps family of bacterial and archaeal proteins that oxidize Fe(2+) to Fe(3+) to protect microorganisms from oxidative damage. The oxidized iron is subsequently deposited as ferrihydrite inside a protein cavity, resulting in the formation of an iron core. The size and the magnetic properties of the iron core have attracted considerable attention for nanotechnological applications in recent years. Here, the magnetic and structural properties of the iron core in wild-type Dpr and four cavity mutants were studied. All samples clearly demonstrated a superparamagnetic behavior in superconducting quantum interference device magnetometry and Mössbauer spectroscopy compatible with that of superparamagnetic ferrihydrite nanoparticles. However, all the mutants exhibited higher magnetic moments than the wild-type protein. Furthermore, measurement of the iron content with inductively coupled plasma mass spectrometry revealed a smaller amount of iron in the iron cores of the mutants, suggesting that the mutations affect nucleation and iron deposition inside the cavity. The X-ray crystal structures of the mutants revealed no changes compared with the wild-type crystal structure; thus, the differences in the magnetic moments could not be attributed to structural changes in the protein. Extended X-ray absorption fine structure measurements showed that the coordination geometry of the iron cores of the mutants was similar to that of the wild-type protein. Taken together, these results suggest that mutation of the residues that surround the iron storage cavity could be exploited to selectively modify the magnetic properties of the iron core without affecting the structure of the protein and the geometry of the iron core.

Local structure of Y1–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 Θ E = 350 K, while a minimum value of Θ E = 300 K is reached for the compound with x = 0.025, which has the highest critical temperature T c = 12.5 K of the superconductive transition. This correlates with the further suppressing of superconductivity and with the appearance of the local magnetic moments in the investigated Y1–x Yb x Ni2B2C series for x ≥ 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 temperaturre were explained in the frame of the band filling effect.

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including Zn(II), Cd(II), and Cu(I). MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT E(c)-1 from Triticum aestivum, common bread wheat, is a Zn(II)-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding β(E) domain was recently described. Here we present the structure of the N-terminal second domain, γ-E(c)-1, determined by NMR spectroscopy. The γ-E(c)-1 domain enfolds an M (2) (II) Cys(6) cluster and was characterized as part of the full-length Zn(6)E(c)-1 protein as well as in the form of the separately expressed domain, both in the Zn(II)-containing isoform and the Cd(II)-containing isoform. Extended X-ray absorption fine structure analysis of Zn(2)γ-E(c)-1 clearly shows the presence of a ZnS(4) coordination sphere with average Zn-S distances of 2.33 Å. (113)Cd NMR experiments were used to identify the M(II)-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-E(c)-1 were probed with Cd(II) binding experiments as well as by pH titrations of the Zn(II) and Cd(II) forms, the latter suggesting an interaction of the γ domain and the β(E) domain within the full-length protein.

The [Fe]-hydrogenase is an ideal system for studying the electronic properties of the low spin iron site that is common to the catalytic centres of all hydrogenases. Because they have no auxiliary iron-sulfur clusters and possess a cofactor containing a single iron centre, the [Fe]-hydrogenases are well suited for spectroscopic analysis of those factors required for the activation of molecular hydrogen. Specifically, in this study we shed light on the electronic and molecular structure of the iron centre by XAS analysis of [Fe]-hydrogenase from Methanocaldococcus jannashii and five model complexes (Fe(ethanedithiolate)(CO)(2)(PMe(3))(2), [K(18-crown-6)](2)[Fe(CN)(2)(CO)(3)], K[Fe(CN)(CO)(4)], K(3)[Fe(III)(CN)(6)], K(4)[Fe(II)(CN)(6)]). The different electron donors have a strong influence on the iron absorption K-edge energy position, which is frequently used to determine the metal oxidation state. Our results demonstrate that the K-edges of Fe(II) complexes, achieved with low-spin ferrous thiolates, are consistent with a ferrous centre in the [Fe]-hydrogenase from Methanocaldococcus jannashii. The metal geometry also strongly influences the XANES and thus the electronic structure. Using in silico simulation, we were able to reproduce the main features of the XANES spectra and describe the effects of individual donor contributions on the spectra. Thereby, we reveal the essential role of an unusual carbon donor coming from an acyl group of the cofactor in the determination of the electronic structure required for the activity of the enzyme.

Herein we present an innovative combination of EXAFSspectroscopy and resonant Raman scattering for the charac-terisation of the ground state and structural dynamics of athermally stable binuclear bisACHTUNGRE(m-oxo) dicopperACHTUNGRE(III) species.Peralkylated bis(guanidine)-based ligands are used in thesynthesis of this compound.

We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn(II) and Cu(I). Recently, we could show that wheat E(c)-1 is coordinating its six Zn(II) ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn(II) ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn(II) ion of the N-terminal domain is relative resistant to complete replacement with Cd(II) indicating the presence of a Zn(II)-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE(c)-1 with an excess of Zn(II) shows a certain amount of sub-stoichiometrically metal-loaded species. The possible relevance of these finding for the proposed biological functions of wheat E(c)-1 will be discussed. In addition, extended X-ray absorption fine structure (EXAFS) measurements on both, the full-length and the truncated protein, provide final evidence for His participation in metal ion binding.

Journal of biological inorganic chemistry 14(5), 663 - 672 (2009). doi:10.1007/s00775-009-0479-7, Bacteria and archaea import molybdenum andtungsten from the environment in the form of theoxyanions molybdate (MoO42-) and tungstate (WO42-).These substrates are captured by an external, high-affinitybinding protein, and delivered to ATP binding cassettetransporters, which move them across the cell membrane.We have recently reported a crystal structure of themolybdate/tungstate binding protein ModA/WtpA fromArchaeoglobus fulgidus, which revealed an octahedrallycoordinated central metal atom. By contrast, the previouslydetermined structures of three bacterial homologs showedtetracoordinate molybdenum and tungsten atoms in theirbinding pockets. Until then, coordination numbers abovefour had only been found for molybdenum/tungsten inmetalloenzymes where these metal atoms are part of thecatalytic cofactors and coordinated by mostly non-oxygenligands. We now report a high-resolution structure ofA. fulgidus ModA/WtpA, as well as crystal structures offour additional homologs, all bound to tungstate. Thesecrystal structures match X-ray absorption spectroscopymeasurements from soluble, tungstate-bound protein, andreveal the details of the distorted octahedral coordination.Our results demonstrate that the distorted octahedralgeometry is not an exclusive feature of the A. fulgidusprotein, and suggest distinct binding modes of the bindingproteins from archaea and bacteria., Published by Springer, Berlin

YtfE was recently shown to be a newly discovered protein required for the recovery of the activity of iron-sulfur-containing enzymes damaged by oxidative and nitrosative stress conditions. The Escherichia coli YtfE purified protein is a dimer with two iron atoms per monomer and the type and properties of the iron center were investigated by using a combination of resonance Raman and extended X-ray absorption fine structure spectroscopies. The results demonstrate that YtfE contains a non-heme dinuclear iron center having μ-oxo and μ-carboxylate bridging ligands and six histidine residues coordinating the iron ions. This is the first example of a protein from this important class of di-iron proteins to be shown to be involved in the repair of iron-sulfur centers. Fil: Todorovic, Smilja. Universidade Nova de Lisboa; Portugal Fil: Justino, Marta C.. Universidade Nova de Lisboa; Portugal Fil: Wellenreuther, Gerd. The European Molecular Biology Laboratory; Alemania Fil: Hildebrandt, Peter. Technishe Universitat Berlin; Alemania Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Technishe Universitat Berlin; Alemania Fil: Meyer Klaucke, Wolfram. The European Molecular Biology Laboratory; Alemania Fil: Saraiva, Lígia M.. Universidade Nova de Lisboa; Portugal

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-beta (Abeta) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Abeta peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Abeta(1-40), Abeta(1-16), Abeta(1-28), Abeta(5-23), and Abeta(17-40)). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His(6), His(13), and His(14)). In zinc-Abeta complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Abeta peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.

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.

EXAFS allows the determination of metal binding motifs in proteins. Here we present an algorithm for the identification of one subgroup, the Zinc finger proteins, by an automated refinement of biological EXAFS data. In combination with scoring criteria inspired by high resolution crystal structures these refinements led to the identification of these motifs, that playing an important role in protein‐DNA and protein‐RNA interactions. Robust criteria were identified investigating several EXAFS data sets from different zinc binding proteins. For all structural zinc motifs the automated EXAFS refinement led to results consistent with published ones.

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.

The elaC gene product from Escherichia coli , ZiPD, is a 3′ tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis , E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3′ end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 Å. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima , but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.

X-ray absorption spectroscopy data show different metal binding site structures in beta-amyloid peptides according to whether they are complexed with Cu(2+) or Zn(2+) ions. While the geometry around copper is stably consistent with an intra-peptide binding with three metal-coordinated Histidine residues, the zinc coordination mode depends on specific solution conditions. In particular, different sample preparations are seen to lead to different geometries around the absorber that are compatible with either an intra- or an inter-peptide coordination mode. This result reinforces the hypothesis that assigns different physiological roles to the two metals, with zinc favoring peptide aggregation and, as a consequence, plaque formation.

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

Azotobacter vinelandii is a diazotrophic bacterium characterized by the outstanding capability of storing Mo in a special storage protein, which guarantees Mo-dependent nitrogen fixation even,under growth conditions of extreme Mo starvation. The Mo storage protein is constitutively synthesized with respect to the nitrogen source and is regulated by molybdenum at an extremely low concentration level (0-50 nm). This protein was isolated as an alpha(4)beta(4) octamer with a total molecular mass of about 240 kg mol(-1) and its shape was determined by small-angle X-ray scattering. The genes of the alpha and beta subunits were unequivocally identified; the amino acid sequences thereby determined reveal that the Mo storage protein is not related to any other known molybdoprotein. Each protein molecule can store at least 90 Mo atoms. Extended X-ray absorption fine-structure spectroscopy identified a metal-oxygen cluster bound to the Mo storage protein. The binding of Mo (biosynthesis and incorporation of the duster) is de pendent on adenosine triphosphate (ATP); Mo release is ATP-independent but pH-regulated, occurring only above pH 7.1. This Mo storage protein is the only known noniron metal storage system in the biosphere containing a metal-oxygen cluster.

On the basis of EXAFS and MS/MS experimental results and a reinterpretation of the electron density map obtained by X-ray crystallography, we describe a new post-translational modification, that is, a 3,5-dibromotyrosine residue that is incorporated in the polypeptide chain of a vanadium haloperoxidase.

Since high-intensity synchrotron radiation is available, "extended X-ray absorption fine structure" spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu(2+) complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.

The structural and spectroscopic properties of the biologically active [Fe—4S] site of three different mutants of the wild-type rubredoxin from the archaeon Pyrococcus abyssi were investigated and compared with each other and additionally with those of the rubredoxin from the bacterium Clostridium pasteurianum.

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 symmetrically ligated complexes 1, 2, and 3 with a (mu-oxo)bis(mu-acetato)diferric core can be one-electron oxidized electrochemically or chemically with aminyl radical cations [*NR3][SbCl6] in acetonitrile yielding complexes which contain the mixed-valent [(mu-oxo)bis(mu-acetato)iron(IV)iron(III)]3+ core: [([9]aneN3)(2FeIII2)(mu-O)(mu-CH3CO2)2](ClO4)2 (1(ClO4)2), [(Me3[9]aneN3)(2FeIII2)(mu-O)(mu-CH3CO2)2](PF6)2 (2(PF6)(2)), and [(tpb)(2FeIII2)(mu-O)(mu-CH3CO2)2] (3) where ([9]aneN3) is the neutral triamine 1,4,7-triazacyclononane and (Me3[9]aneN3) is its tris-N-methylated derivative, and (tpb)(-) is the monoanion trispyrazolylborate. The asymmetrically ligated complex [(Me3[9]aneN3)FeIII(mu-O)(mu-CH3CO2)2FeIII(tpb)](PF6) (4(PF6)) and its one-electron oxidized form [4ox]2+ have also been prepared. Finally, the known heterodinuclear species [(Me3[9]aneN3)CrIII(mu-O)(mu-CH3CO2)2Fe([9]aneN3)](PF6)2 (5(PF6)(2)) can also be one-electron oxidized yielding [5ox]3+ containing an iron(IV) ion. The structure of 4(PF6).0.5CH3CN.0.25(C2H5)2O has been determined by X-ray crystallography and that of [5ox]2+ by Fe K-edge EXAFS-spectroscopy (Fe(IV)-O(oxo): 1.69(1) A; Fe(IV)-O(carboxylato) 1.93(3) A, Fe(IV)-N 2.00(2) A) contrasting the data for 5 (Fe(III)-O(oxo) 1.80 A; Fe(III)-O(carboxylato) 2.05 A, Fe-N 2.20 A). [5ox]2+ has an St = 1/2 ground state whereas all complexes containing the mixed-valent [FeIV(mu-O)(mu-CH3CO2)2FeIII]3+ core have an St = 3/2 ground state. Mossbauer spectra of the oxidized forms of complexes clearly show the presence of low spin FeIV ions (isomer shift approximately 0.02 mm s(-1), quadrupole splitting approximately 1.4 mm s(-1) at 80 K), whereas the high spin FeIII ion exhibits delta approximately 0.46 mm s(-1) and DeltaE(Q) approximately 0.5 mm s(-1). Mossbauer, EPR spectral and structural parameters have been calculated by density functional theoretical methods at the BP86 and B3LYP levels. The exchange coupling constant, J, for diiron complexes with the mixed-valent FeIV-FeIII core (H = -2J S1.S2; S(1) = 5/2; S2 = 1) has been calculated to be -88 cm(-1) (intramolecular antiferromagnetic coupling) and for the reduced diferric form of -75 cm(-1) in reasonable agreement with experiment (J = -120 cm(-1)).

Cytosolic glyoxalase 2 (GLX2-2) from Arabidopsis thaliana is a metalloenzyme that has been shown to bind a mixture of Zn, Fe, or Mn when produced in cells grown in rich media. In an effort to prepare metal-enriched samples, GLX2-2 was over-expressed in minimal media containing either Zn, Fe, or Mn. The resulting enzymes bound an average of 1 equivalent of metal ion and were partially enriched with a specific metal ion. The enzymes produced in minimal media were active towards the substrate S-D-lactoylglutathione, yielding kcat/ Km values similar to those of rich media GLX2-2. EPR studies on minimal media GLX2-2 samples revealed spectra which were identical to those over-expressed in rich media that contained nearly 2 equivalents of metal. The EPR spectra showed the presence of antiferromagnetically and ferromagnetically coupled, dinuclear metal centers. EXAFS spectra on the minimal media GLX2-2 samples over-expressed in the presence of Fe or Zn were also very similar to those of the rich media GLX2-2 samples, indicating the presence of dinuclear metal centers. The EXAFS studies also demonstrate that Zn(II) and Fe (in the Fe-enriched sample) are distributed in the dinuclear site. These data indicate that the minimal media GLX2-2 samples are a mixture of fully loaded, dinuclear metal-containing enzyme and metal-free enzyme. This characteristic of A. thaliana GLX2-2 makes it unique among the other members of the metallo-beta-lactamase family in that it does not ever appear to exist as a mononuclear metal ion containing enzyme and that it exhibits positive cooperativity in metal binding.

Glyoxalase II belongs to the metallo-beta-lactamase superfamily of proteins, possessing the characteristic dinuclear active site. Within this protein family, glyoxalase II from Arabidopsis thaliana is the first member to be isolated with significant amounts of iron, manganese, and zinc when being recombinantly produced in Escherichia coli. Enzyme preparations with different ratios of these three metals all yield k(cat)/K(M) values in the range of 1.5-1.9 s(-1) microM(-1) with the substrate S-d-lactoylglutathione. X-ray absorption spectroscopy reveals binding of all three metals to the dinuclear active site with 5-6-fold coordination consisting of 2.5 +/- 0.5 histidine and 2.5 +/- 0.5 oxygen ligands. This model does not distinguish site-specific or distributed binding. The metal-metal distance is determined to be 3.18 +/- 0.06 A. Electron paramagnetic resonance spectroscopy gives evidence for several different types of dimetal sites, including spin-coupled Fe(III)Fe(II), Fe(III)Zn(II), and Mn(II)Mn(II) centers. The metal-ligand distances measured by X-ray absorption spectroscopy vary depending on the metal type and comply with their element-specific, characteristic values. This reflects a high degree of structural flexibility within the glyoxalase II dinuclear active site, which is considered as the structural basis for its broad metal selectivity.

The reaction of mononuclear [LFe(III)] where L represents the trianionic ligand 1,4,7-tris(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane with NiCl(2). 6H(2)O and subsequent oxidations with [Ni(III)(tacn)(2)](ClO(4))(3) (tacn = 1,4,7-triazacyclononane) and PbO(2)/methanesulfonic acid produced an isostructural series of complexes [LFeNiFeL](n+) (n = 2 (1), n = 3 (2), n = 4 (3)), which were isolated as PF(6)(-) (1, 3) or ClO(4)(-) salts (2). The molecular structures were established by X-ray crystallography for [LFeNiFeL](ClO(4))(2). 5CH(3)CN (1*), C(88)H(123)Cl(2)Fe(2)N(11)NiO(8)S(6), and [LFeNiFeL](ClO(4))(3). 8acetone (2*), C(102)H(156)Cl(3)Fe(2)N(6)NiO(20)S(6). Both compounds crystallize in the triclinic space group P (1) over bar with a = 13.065(2) Angstrom (13.155(2) Angstrom), b = 13.626(3) Angstrom (13.747(3) Angstrom), c = 14.043(3) Angstrom (16.237(3) Angstrom), alpha = 114.47(3)degrees (114.20(2)degrees), beta = 97.67(3)degrees (96.57(2)degrees), gamma = 90.34(3)degrees (98.86(2)degrees), Z = 1(1) (values in parentheses refer to 2*). The cations in 1, 2, and 3 have been determined to be isostructural by Fe and Ni K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. All compounds contain linear trinuclear cations (face-sharing octahedral) with an N(3)Fe(mu-SR)(3)Ni(mu-SR)(3)FeN(3) core structure. The electronic structures of 1, 2, and 3 have been studied by Fe and Ni K-edge X-ray absorption near edge (XANES), UV-vis, EPR, and Mossbauer spectroscopy as well as by temperature-dependent magnetic susceptibility measurements. Complexes 1 and 3 possess an S(t) = 0 whereas 2 has an S(t) = 1/2 ground state. It is shown that the electronic structures cannot be described by using localized valences (oxidation states). Delocalized models invoking the double-exchange mechanism are appropriate; i.e., spin-dependent delocalization via a double-exchange mechanism yields the correct ground state in each case, 1, 2, and 3 represent the first examples where double exchange stabilizes a ground state of minimum spin multiplicity.

The temperature dependence of the mean square displacement of the (57)Fe nuclei due to motion faster than 100 ns are measured by temperature-dependent Mossbauer spectroscopy for oxidized and reduced HiPIPs from Ectothiorhodospira halophila, Chromatium vinosum WT and a Cys77Ser mutant. The behaviour is interpretable in the frame of the general model of protein dynamics distinguishing two temperature intervals. The character of harmonic and quasi-diffusional modes in HiPIPs is discussed. Dynamic information obtained from Mossbauer spectroscopy and Fe K-edge EXAFS are compared. Structure dynamics of the iron-sulfur cluster in the partially unfolded reduced HiPIP from C. vinosum was investigated by Mossbauer spectroscopy and EXAFS, indicating an intact metal centre and a protein backbone with a largely collapsed secondary structure. The role of the cofactor during protein folding is discussed. Differences in the dynamics between the native protein and the molten globule are found at physiological temperatures only. The structure and dynamic behaviour of the [Fe(4)S(4)]Cys(3)Ser cluster in the Cys77Ser mutant of the HiPIP from C. vinosum are analysed. The temperature dependence of electron relaxation in oxidized HiPIPs is investigated by Mossbauer spectroscopy and analysed theoretically, considering spin-spin and spin-lattice relaxation. The latter consists of contributions from direct phonon bottleneck and Orbach mechanisms. The data agree with former pulsed EPR results. Orbach relaxation is interpreted as due to transitions between electronic isomers of oxidized HiPIPs. With this interpretation, the energetic difference between both isomers equals the energy gap estimated from the temperature dependence of the Orbach relaxation.

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.

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].

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.

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.