Calibration of electron spectra

Energy calibration procedures, which have been used in ESCA, are reviewed. Binding energies of calibration lines suitable for solid and for gaseous samples have been determined. Charging effects of nonconducting samples have been investigated for different X-radiation, sample thickness and material. A calibration relative to lines from some insulating standard compound mixed into the sample was shown to be unreliable, while experiments indicated that in calibrating relative to a surface layer, the carbon line from the hydrocarbon contamination layer can be used as well as a line from a thick layer of a noble metal on a part of the sample.     

High covalence in CuSO4 and the radicalization of sulfate: an X-ray absorption and density functional study

Sulfur K-edge X-ray absorption spectroscopy (XAS) of anhydrous CuSO(4) reveals a well-resolved preedge transition feature at 2478.8 eV that has no counterpart in the XAS spectra of anhydrous ZnSO(4) or copper sulfate pentahydrate. Similar but weaker preedge features occur in the sulfur K-edge XAS spectra of [Cu(itao)SO(4)] (2478.4 eV) and [Cu[(CH(3))(6)tren]SO(4)] (2477.7 eV). Preedge features in the XAS spectra of transition metal ligands are generally attributed to covalent delocalization of a metal d-orbital hole into a ligand-based orbital. Copper L-edge XAS of CuSO(4) revealed that 56% of the Cu(II) 3d hole is delocalized onto the sulfate ligand. Hybrid density functional calculations on the two most realistic models of the covalent delocalization pathways in CuSO(4) indicate about 50% electron delocalization onto the sulfate oxygen-based 2p orbitals; however, at most 14% of that can be found on sulfate sulfur. Both experimental and computational results indicated that the high covalence of anhydrous CuSO(4) has made sulfate more like the radical monoanion, inducing an extensive mixing and redistribution of sulfur 3p-based unoccupied orbitals to lower energy in comparison to sulfate in ZnSO(4). It is this redistribution, rather than a direct covalent interaction between Cu(II) and sulfur, that is the origin of the observed sulfur XAS preedge feature. From pseudo-Voigt fits to the CuSO(4) sulfur K-edge XAS spectrum, a ground-state 3p character of 6% was quantified for the orbital contributing to the preedge transition, in reasonable agreement with the DFT calculation. Similar XAS fits indicated 2% sulfur 3p character for the preedge transition orbitals in [Cu(itao)SO(4)] and [Cu[(CH(3))(6)tren]SO(4)]. The covalent radicalization of ligands similar to sulfate, with consequent energy redistribution of the virtual orbitals, represents a new mechanism for the induction of ligand preedge XAS features. The high covalence of the Cu sites in CuSO(4) was found to be similar to that of Cu sites in oxidized cupredoxins, including its anistropic nature, and can serve as the simplest inorganic examples of intramolecular electron-transfer processes.     

Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent, nonheme iron enzyme that catalyzes the hydroxylation of L-Phe to L-Tyr in the rate-limiting step of phenylalanine catabolism. This reaction is tightly coupled in the wild-type enzyme to oxidation of the tetrahydropterin cofactor. Dysfunction of PAH activity in humans leads to the disease phenylketonuria (PKU). We have investigated two PKU-inducing mutants, Arg158Gln and Glu280Lys, using kinetic methods, magnetic circular dichrosim (MCD) spectroscopy, and X-ray absorption spectroscopy (XAS). Analysis of the products produced by the mutant enzymes shows that although both oxidize pterin at more than twice the rate of wild-type enzyme, these reactions are only approximately 20% coupled to production of L-Tyr. Previous MCD and XAS studies had demonstrated that the resting Fe(II) site is six-coordinate in the wild-type enzyme and converts to a five-coordinate site when both L-Phe and reduced pterin are present in the active site. Although the Arg158Gln mutant forms the five-coordinate site when both cosubstrates are bound, the Fe(II) site of the Glu280Lys mutant remains six-coordinate. These results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species, which subsequently reacts with the Fe(II) site if the pterin is properly oriented for formation of an Fe-OO-pterin bridge and an open coordination position is available on the Fe(II).     

Sulfur K-edge X-ray absorption spectroscopy of 2Fe-2S ferredoxin: covalency of the oxidized and reduced 2Fe forms and comparison to model complexes

Ligand K-edge X-ray absorption spectroscopy (XAS) provides a direct experimental probe of ligand-metal bonding. In previous studies, this method has been applied to mononuclear Fe-S and binuclear 2Fe-2S model compounds as well as to rubredoxins and the Rieske protein. These studies are now extended to the oxidized and reduced forms of ferredoxin I from spinach. Because of its high instability, the mixed-valence state was generated electrochemically in the protein matrix, and ligand K-edge absorption spectra were recorded using an XAS spectroelectrochemical cell. The experimental setup is described. The XAS edge data are analyzed to independently determine the covalencies of the iron-sulfide and -thiolate bonds. The results are compared with those obtained previously for the Rieske protein and for 2Fe-2S model compounds. It is found that the sulfide covalency is significantly lower in oxidized FdI compared to that of the oxidized model complex. This decrease is interpreted in terms of H bonding present in the protein, and its contribution to the reduction potential E degrees is estimated. Further, a significant increase in covalency for the Fe(III)-sulfide bond and a decrease of the Fe(II)-sulfide bond are observed in the reduced Fe(III)Fe(II) mixed-valence species compared to those of the Fe(III)Fe(III) homovalent site. This demonstrates that, upon reduction, the sulfide interactions with the ferrous site decrease, allowing greater charge donation to the remaining ferric center. That is the dominant change in electronic structure of the Fe(2)S(2)RS(4) center upon reduction and can contribute to the redox properties of this active site.     

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

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

Electronic structure description of the mu(4)-sulfide bridged tetranuclear Cu(Z) center in N(2)O reductase

Spectroscopy coupled with density functional calculations has been used to define the spin state, oxidation states, spin distribution, and ground state wave function of the mu4-sulfide bridged tetranuclear CuZ cluster of nitrous oxide reductase. Initial insight into the electronic contribution to N2O reduction is developed, which involves a sigma superexchange pathway through the bridging sulfide.     

S K-edge X-ray absorption spectroscopic investigation of the Ni-containing superoxide dismutase active site: new structural insight into the mechanism

Superoxide dismutases protect cells from the toxic effects of reactive oxygen species derived from superoxide. Nickel-containing superoxide dismutases (NiSOD), found in Streptomyces species and in cyanobacteria, are distinct from Mn-, Fe-, or Cu/Zn-containing SODs in amino acid sequence and metal ligand environment. Sulfur K-edge X-ray absorption spectroscopic investigations were carried out for a series of mono- and binuclear Ni model compounds with varying sulfur ligation, and for oxidized and reduced NiSOD to elucidate the types of Ni-S interactions found in the two oxidation states. The S K-edge XAS spectra clearly indicate the presence of Ni(III)-bound terminal thiolate in the oxidized enzyme and the absence of such coordination to Ni(II) in the peroxide-reduced enzyme. This striking change in the S ligation for Ni with redox suggests that, upon peroxide reduction, an electron is transferred to the Ni(III) site and the terminal thiolate becomes protonated, providing an efficient mechanism for proton-coupled electron transfer.     

Metallocyclopeptide complexes with MII(S.Cys)4 chromophores

The tetracysteinyl peptide cyclo[Lys1,12](Gln-Cys-Gly-Val-Cys-Gly-Lys-Cys-Ile-Ala-Cys-Lys) ([symbol: see text] L(Cys.SH)4) was synthesized by solid-phase methods using an Fmoc/t-Bu/allyl strategy on a PAL-PEG-PS support. The formation of the 1:1 complexes with M = Fe2+, Co2+, and Ni2+ was observed by spectrophotometric monitoring of reactions in aqueous solution at pH 7.5. Size exclusion chromatography indicated that the peptide is a monomer and the complexes are dimers [M2([symbol: see text]L(Cys.S)4)2] in aqueous buffer at pH 7.5. Cobalt and nickel K-edge X-ray absorption data and EXAFS analysis of [Co2([symbol: see text] L(Cys.S)4)2] and [Ni2([symbol: see text] L(Cys.S)4)2] as lyophilized solids are reported. Derived bond distances are Co-S = 2.30 A and Ni-S = 2.21 A. From the collective results provided by absorption spectra, K-edges, EXAFS, and bond length comparisons with known structures, it is shown that [Fe2([symbol: see text] L(Cys.S)4)2] and [Co2([symbol: see text] L(Cys.S)4)2] possess distorted tetrahedral structures and [Ni2([symbol: see text] L(Cys.S)4)2] has distorted square planar stereochemistry. The Co(II) chromophore is particularly distinctive of the assigned structure, displaying three components of the parent tetrahedral ligand field transition 4A2-->4T1(P) (610, 685, 740 nm). The observed structures conform to the intrinsic stereochemical preferences of the metal ions. Structures for the binuclear complexes are suggested. These are the first characterized metal complexes of a cysteinyl cyclopeptide and among the few well-documented complexes of synthetic cyclopeptides. This study is a desirable first step in the design of cyclic peptides for the binding of mononuclear and polynuclear metal centers.     

Spectroscopic and density functional theory studies of the blue-copper site in M121SeM and C112SeC azurin: Cu-Se versus Cu-S bonding

S K-edge X-ray absorption, UV-vis absorption, magnetic circular dichroism (MCD), and resonance Raman spectroscopies are used to investigate the electronic structure differences among WT, M121SeM, and C112SeC Pseudomonas aeruginosa (P.a) azurin. A comparison of S K-edge XAS of WT and M121SeM azurin and a CuII-thioether model complex shows that the 38% S character in the ground state wave function of the blue-copper (BC) sites solely reflects the Cu-SCys bond. Resonance Raman (rR) data on WT and C112SeC azurin give direct evidence for the kinematic coupling between the Cu-SCys stretch and the cysteine deformation modes in WT azurin, which leads to multiple features in the rR spectrum of the BC site. The UV-vis absorption and MCD data on WT, M121SeM, and C112SeC give very similar C0/D0 ratios, indicating that the C-term MCD intensity mechanism involves Cu-centered spin-orbit coupling (SOC). The spectroscopic data combined with density functional theory (DFT) calculations indicate that SCys and SeCys have similar covalent interactions with Cu at their respective bond lengths of 2.1 and 2.3 A. This reflects the similar electronegativites of S and Se in the thiolate/selenolate ligand fragment and explains the strong spectroscopic similarities between WT and C112SeC azurin.