A Silica Bilayer Supported on Ru(0001): Following the Crystalline-to Vitreous Transformation in Real Time with Spectro-microscopy.

The crystalline-to-vitreous phase transformation of a SiO2 bilayer supported on Ru(0001) was studied by time-dependent LEED, local XPS, and DFT calculations. The silica bilayer system has parallels to 3D silica glass and can be used to understand the mechanism of the disorder transition. DFT simulations show that the formation of a Stone-Wales-type of defect follows a complex mechanism, where the two layers show decoupled behavior in terms of chemical bond rearrangements. The calculated activation energy of the rate-determining step for the formation of a Stone-Wales-type of defect (4.3 eV) agrees with the experimental value. Charge transfer between SiO2 bilayer and Ru(0001) support lowers the activation energy for breaking the Si-O bond compared to the unsupported film. Pre-exponential factors obtained in UHV and in O2 atmospheres differ significantly, suggesting that the interfacial ORu underneath the SiO2 bilayer plays a role on how the disordering propagates within the film.

A Two-Dimensional 'Zigzag' Silica Polymorph on a Metal Support.

We present a new polymorph of the two-dimensional (2D) silica film with a characteristic 'zigzag' line structure and a rectangular unit cell which forms on a Ru(0001) metal substrate. This new silica polymorph may allow for important insights into growth modes and transformations of 2D silica films as a model system for the study of glass transitions. Based on scanning tunneling microscopy, low energy electron diffraction, infrared reflection absorption spectroscopy, and X-ray photoelectron spectroscopy measurements on the one hand, and density functional theory calculations on the other, a structural model for the 'zigzag' polymorph is proposed. In comparison to established monolayer and bilayer silica, this 'zigzag' structure system has intermediate characteristics in terms of coupling to the substrate and stoichiometry. The silica 'zigzag' phase is transformed upon reoxidation at higher annealing temperature into a SiO2 silica bilayer film which is chemically decoupled from the substrate.

Water Formation under Silica Thin Films: Real-Time Observation of a Chemical Reaction in a Physically Confined Space.

Using low-energy electron microscopy and local photoelectron spectroscopy, water formation from adsorbed O and H2 on a Ru(0001) surface covered with a vitreous SiO2 bilayer (BL) was investigated and compared to the same reaction on bare Ru(0001). In both cases the reaction is characterized by moving reaction fronts. The reason for this might be related to the requirement of site release by O adatoms for further H2 -dissociative adsorption. Apparent activation energies (Eaapp ) are found for the front motion of 0.59 eV without cover and 0.27 eV under cover. We suggest that the smaller activation energy but higher reaction temperature for the reaction on the SiO2 BL covered Ru(0001) surface is due to a change of the rate-determining step. Other possible effects of the cover are discussed. Our results give the first values for Eaapp in confined space.

An environmental assessment of the parasite fauna of the reef-associated grouper Epinephelus areolatus from Indonesian waters.

Sixty Epinephelus areolatus were examined for metazoan fish parasites in Indonesia, off Segara Anakan lagoon, Java and in Balinese waters. The study revealed 21 different parasite species, and 14 new host and locality records. The anisakid nematodes Anisakis typica and, for the first time in Indonesia, Anisakis sp. HC-2005 were identified by using molecular methods. Ecological parameters were calculated for both sites off the anthropogenically influenced Segara Anakan lagoon and the relatively undisturbed reference site at the southern Balinese coast. The fish from Segara Anakan demonstrated a significantly higher enzymatic activity (Hepatosomatic index) and a significantly reduced number of heteroxenous gut helminths (e.g. the digenean Didymodiclinus sp., the nematode Raphidascaris sp. and the acanthocephalan Serrasentis sagittifer). Other regional differences for E. areolatus included ecto-/endoparasite ratio, endoparasite diversity, the parasite species composition and prevalence of infection of the respective parasite species. We applied the stargraph method to visualize observed regional differences using grouper parasites as biological indicators for the sampled coastal ecosystems at both sampling sites.

In Situ Patterning of Ultrasharp Dopant Profiles in Silicon.

We develop a method for patterning a buried two-dimensional electron gas (2DEG) in silicon using low kinetic energy electron stimulated desorption (LEESD) of a monohydride resist mask. A buried 2DEG forms as a result of placing a dense and narrow profile of phosphorus dopants beneath the silicon surface; a so-called δ-layer. Such 2D dopant profiles have previously been studied theoretically, and by angle-resolved photoemission spectroscopy, and have been shown to host a 2DEG with properties desirable for atomic-scale devices and quantum computation applications. Here we outline a patterning method based on low kinetic energy electron beam lithography, combined with in situ characterization, and demonstrate the formation of patterned features with dopant concentrations sufficient to create localized 2DEG states.

Analysis of strain-induced EPR-line shapes and anisotropic spin-lattice relaxation in a [2Fe-2S] ferredoxin.

The electron paramagnetic resonance spectrum of the [2Fe-2S]1+(2+;1+) cluster in spinach-leaf ferredoxin has been measured at four microwave frequencies from 1 to 35 GHz. Using a modified g-strain formula, the asymmetrical spectrum has been simulated in detail without the assumption of signal multiplicity. In all but the lowest frequency bands the line width is dominated by an extremely anisotropic g-shift distribution, caused by a statistical distribution in dislocation strains. The crossover point of domination by unresolved proton splittings is around 2 GHz. The angle-dependent elasticity of the cluster can be related to an anisotropy in the spin-lattice relaxation rate. Intensity behaviour under continuous saturation, at temperatures in the two-phonon region, is in qualitative agreement with elementary theory. On the basis of these results it is argued that biochemists should be aware of the questionable nature of some ad hoc assumptions commonly made to interpret EPR of metalloproteins. Specifically, a physically meaningful determination of the number and stoicheiometry of distinguishable compounds, represented in a complex spectrum, may well require more advanced theoretical tools than the frequently employed deconvolution in symmetrical Gaussians with associated unique relaxation times.

On the reduction potentials of Fe and Cu-Zn containing superoxide dismutases.

The reduction potentials of bovine erythrocyte copper-zinc superoxide dismutase and Escherichia coli iron superoxide dismutase were determined in EPR-monitored redox titrations in homogeneous solution. The copper-zinc enzyme is reduced and reoxidized with a midpoint potential of +120 mV versus standard hydrogen electrode (SHE) at pH 7.5. The iron enzyme can be reduced with an apparent midpoint potential of -67 mV versus SHE at pH 7.5. However, reaction with ferricyanide affords only slow, partial re-oxidation. Cyclic voltammetry of the copper-zinc enzyme in the presence of 50 mM Sc3+ at pH 4.0 using a glassy carbon electrode results in asymmetric voltammograms. The midpoint potential of the enzyme at this pH value, calculated as the average of the anodic and cathodic peak potentials, is +400 mV versus SHE. The physiological relevance of this value is limited, since EPR experiments indicated that reduction of the copper-zinc enzyme at pH 4.0 is not reversible. Consequences of the irreversible behavior of the two dismutases for the previously reported studies on their redox properties are discussed.

Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin. Possible new fingerprints for three iron clusters.

We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus. First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers. This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2. Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the [4Fe-4S]+ (S = 1/2) and the reduced 3Fe center. A theoretical explanation is given for the observation of interaction signals with constant effective g values.

Dual-mode EPR spectrometry of O2-pulsed cytochrome c oxidase.

O2-activated bovine heart cytochrome c oxidase has been examined by dual-mode EPR spectrometry. Resonances have been observed at g = 10 and 4.5 in the parallel mode and at g = 10, 5, 1.8 and 1.7 in the normal mode. The bulk of these signals are interpreted to come from a stoichiometric S = 2 system with magnitude of a = 0.17 cm-1, D = +2.1 cm-1, magnitude of E = 0.026 cm-1, g = 2. Exchange coupling between cytochrome a3 and CuB is not indicated.

An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions.

The high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum contains a cubane prosthetic group that shuttles between the [4Fe-4S]3+,2+ states. We find that the EPR spectra from this protein can be explained as a sum of two components, a major one with g = 2.02; 2.04; 2.12, and a minor one with g = 2.04; 2.07; approximately 2.13. In the presence of 0.1-2.0 M NaCl, freezing induces polymerization of the protein (presumably dimers), which is detected as intercluster spin-spin interaction in the EPR. The observed spin-spin interactions are interpreted as being due to two very similar dimeric structures in an approx. 1:2 ratio. Computer simulation of the X- and Q-band EPR spectra shows that the z-components of the g-tensors in each dimer pair must be co-linear, with center-to-center distances between the clusters of approximately 13 A and approximately 16 A. Inspection of possible dimeric structures of C. vinosum HiPIP by standard molecular graphics procedures revealed that the Fe/S cluster is exposed toward a flattened surface and is accessible to solvent. Moreover, the Fe/S clusters in two HiPIP molecules can easily achieve a center-to-center distance of approximately 14 A when approaching along a common 3-fold axis that extends through the S4 sulfur atom of the cubane; the z-component of the EPR g-tensor is co-linear with this symmetry axis.

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy.

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.

Overproduction of the prismane protein from Desulfovibrio desulfuricans ATCC 27774 in Desulfovibrio vulgaris (Hildenborough) and EPR spectroscopy of the [6Fe-6S] cluster in different redox states.

The Desulfovibrio desulfuricans ATCC 27774 prismane protein was isolated from a Desulfovibrio vulgaris (Hildenborough) strain that contained the gene for this protein in expression vector pSUP104. A redox titration demonstrated that the [Fe-S] cluster in this protein may attain four different redox states, indicated as +3, +4, +5 and +6, with midpoint potentials for the transitions of approx. -220, +50/-25 and +370 mV, respectively. EPR spectra of the protein in the various redox states are reminiscent of those of the D. vulgaris prismane protein (Pierik et al. (1992) Eur. J. Biochem. 206, 705-719), but differ in details. In the +5-state, virtually all the iron is in a S = 9/2 spin state, indicative for a cluster that is more complex than common [4Fe-4S] or [2Fe-2S] clusters. Similarity of the EPR spectrum of the protein in the +3-state with those of inorganic [6Fe-6S] model compounds suggests that the cluster in the protein is also [6Fe-6S]. In the +4-state of the protein a broad signal due to an integer-spin system can be detected with normal-mode EPR. A dramatic sharpening-up and increase of intensity of this band (g = 14.7) is observed with parallel-mode EPR. In accordance with the chemically determined iron content of the protein (6.0 +/- 0.45 moles of iron/mole of protein), the spectroscopic data indicate one [6Fe-6S] cluster in this protein. We did not find evidence for a previous claim (Moura et al. (1992) J. Biol. Chem. 267, 4489-4496) that the D. desulfuricans protein contains two [6Fe-6S] clusters.

VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties.

Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe-2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the [2Fe-2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements.

A novel S = 3/2 EPR signal associated with native Fe-proteins of nitrogenase.

In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g approximately equal to 5. Temperature dependence of the signal was consistent with an S = 3/2 system with negative zero-field splitting, D = -5 +/- 0.7 cm-1. The ms = +/- 3/2 ground state doublet gives rise to a transition with geff = 5.90 and the transition within the excited ms = +/- 1/2 doublet has a split geff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin . mol-1 which summed with the spin intensity of the S = 1/2 g = 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S = 3/2 signal with no concomitant changes in intensity of the S = 1/2 signal.

Pyrococcus furiosus ferredoxin is a functional dimer.

Pyrococcus furiosus ferredoxin is subject to a monomer/dimer equilibrium as a function of ionic strength. At physiological ionic strength, approximately 0.35 M NaCl, the protein is very predominantly homodimer. The monomeric form exhibits impaired electron transfer on glassy carbon; it also has a decreased S=3/2 over S=1/2 ratio as shown by electron paramagnetic resonance spectroscopy. Even following sterilization at 121 degrees C the dimer is stable in denaturing gel electrophoresis.

EPR spectroscopic characterization of an 'iron only' nitrogenase. S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus.

The alternative nitrogenase of Rhodobacter capsulatus, isolated from a nifHDK deletion mutant, has been purified to near homogeneity and identified as an 'iron only' nitrogenase. The dithionite-reduced component 1 ('FeFe protein') of this enzyme showed an EPR spectrum consisting of two components: a minor S = 1/2 signal at g = 1.93 and a very characteristic S = 3/2 signal of near-stoichiometric intensity at g = 5.44. This resonance is very close to the highest possible g value (g = 5.46) for the coinciding two intradoublet subspectra of an S = 3/2 system of maximal rhombicity (E/D = 0.33). The deviation from axial symmetry (increasing E/D) correlates with the stability, activity and substrate selectivity of the different (Mo, V, Fe) nitrogenases.

On the prosthetic group(s) of component II from nitrogenase. EPR of the Fe-protein from Azotobacter vinelandii.

The EPR spectrum of the reduced Fe-protein from nitrogenase has been reinvestigated. The dependences on temperature, microwave power, and microwave frequency all suggest that the observed signal represents a magnetically isolated [4Fe-4S]1+(2+;1+) cluster. Also, the signal can be simulated assuming a simple, g-strained S = 1/2 system. However, the integrated intensity amounts to no more than 0.2 spins per protein molecule. It is, therefore, impossible that Fe-protein preparations contain a single type of [4Fe-4S] cluster.

Electrochemical study of the redox properties of [2Fe-2S] ferredoxins. Evidence for superreduction of the Rieske [2Fe-2S] cluster.

Direct, unmediated electrochemistry has been used to compare the redox properties of [2Fe-2S] clusters in spinach ferredoxin, Spirulina platensis ferredoxin and the water soluble fragment of the Rieske protein. The use of electrochemistry enabled, for the first time, the observation of the second reduction step, [Fe(III), Fe(II)] to [Fe(II), Fe(II)], in a biological [2Fe-2S] system. A water-soluble fragment of the Rieske protein from bovine heart bc1 complex exhibits two subsequent quasi-reversible responses in cyclic voltammetry on activated glassy carbon. In contrast the ferredoxins from spinach and Spirulina platensis only show one single reduction potential. These results support a seniority scheme for biological iron-sulfur clusters related cluster size to electron transfer versatility. Electrochemical reduction of spinach ferredoxin in the presence of NADP+ and ferredoxin: NADP+ oxidoreductase results in the generation of NADPH. The second order rate constant for the reaction between the ferredoxin and the reductase was estimated from cyclic voltammetry experiments to be > 3.10(5) M-1.s-1.

Redox properties of the sulfhydrogenase from Pyrococcus furiosus.

The sulfhydrogenase from the extreme thermophile Pyrococcus furiosus has been re-investigated. The alpha beta gamma delta heterotetrameric enzyme of 153.3 kDa was found to contain 17 Fe, 17 S2-, and 0.74 Ni. The specific activity of the purified protein was 80 U/mg. Three EPR signals were found. A rhombic S = 1/2 signal (g = 2.07, 1.93, 1.89) was observed reminiscent in its shape and temperature dependence of spectra from [4Fe-4S](2+; 1+) clusters. However, in reductive titrations the spectrum appeared at the unusually high potential Em,7.5 = -90 mV. Moreover, the signal disappeared again at Em7.5 = -328 mV. Also, two other signals appear upon reduction: a near-axial (g = 2.02, 1.95, 1.92) S = 1/2 spectrum (Em,7.5 = -303 mV) indicative for the presence of a [2Fe-2S](2+; 1+) cluster, and a broad spectrum of unknown origin with effective g-values 2.25, 1.89 (Em,7.5 = -310 mV). We hypothesize that the latter signal is caused by magnetic interaction of the rhombic signal and a third cluster.