Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to an apo target protein. The intermediate [2Fe-2S] ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor has thus far not been established. In this paper we demonstrate human frataxin to bind from six to seven iron ions. Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements [K(D)(Fe(3+)) approximately 11.7 microM; (K(D)(Fe(2+)) approximately 55.0 microM] and isothermal titration calorimetry (ITC) [K(D)(Fe(3+)) approximately 10.2 microM]. Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements. Both fluorescence (K(D) 0.45 microM) and ITC measurements (K(D) 0.15 microM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities. Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU. Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for [2Fe-2S] cluster formation on ISU. We have demonstrated elsewhere [J. Am. Chem. Soc. 2002, 124, 8774-8775] that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery. Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield. With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained. Both UV-vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins. The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the [2Fe-2S] cluster assembly reaction. A kinetic rate constant k(obs) approximately 0.075 min(-)(1) was determined with 100 microM ISU, 2.4 mM Na(2)S, and 40 microM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT. Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.     

Characterization of [2Fe–2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

Many iron–sulfur proteins involved in cluster trafficking form [2Fe–2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe–2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA-like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe–2S] species, such as (FDX2:GLRX5:[2Fe–2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe–2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron–sulfur-cluster-bridged protein complexes and transient iron–sulfur-cluster transfer intermediates.     

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.     

Hexahydroxybenzene–2,2′-bipyridine (1/2)

2,2′-Bi­pyridine (2BPY) and hexa­hydroxy­benzene (HHB) crystallize in a 2:1 ratio as a neutral molecular adduct, C₆H₆O₆·2C₁₀H₈N₂, in space group P with Z = 1 and with the HHB molecule lying on an inversion centre. HHB, of which this is the first single-crystal X-ray structure determination, forms O—H⃛O hydrogen-bonded chains parallel to the a axis, with O⃛O distances of 2.761 (1) and 2.782 (1) Å. O—H⃛N hydrogen bonds to the 2BPY molecules crosslink these chains, with O⃛N distances of 2.707 (1) and 2.735 (1) Å.     

Development and single laboratory validation of an optical biosensor assay for tetrodotoxin detection as a tool to combat emerging risks in European seafood

Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 μg/kg. The decision limit (CCα) was 100 μg/kg, with the detection capability (CCβ) found to be ≤200 μg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 μg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4 % and 7.8, 8.3, and 3.7 % for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99 %.     

ESR Study of the Plasma Polymerizations of Trimethylsilane and Methane

Electron Spin Resonance (ESR) was used to study, at the molecular level, the plasma polymerization of trimethylsilane (TMS) and methane. Direct ESR analysis of the plasma coated Al substrate required the use of a novel ESR technique. TMS plasma deposit on Al showed a single broad resonance line near g = 2.003. The signal was stable in vacuum and decayed on exposure to air, with a significant fraction persisting for days. Results show that this signal arises from silicon dangling bonds. Identical TMS signals were observed from films prepared by the DC cathodic or the AF glow discharge method but their decay rates were different. In contrast, the deposition of methane produced two distinct types of carbon-based signals depending upon the method of deposition. TMS or CH₄ films deposited by the DC cathodic method showed slow signals decay and high refractive indices value. While the use of Al as the substrate showed plasma-coating radicals, only substrate radicals were observed when PE was used as the substrate. The nature of radicals formed depends not only on the deposition method used but also on the substrate type.     

Dynamics of mass transport and magnetic fields in low-wire-number-array Z pinches

The dynamics of mass transport were observed in a wire array implosion with multiframe laser probing. Plasma bubbles arise at breaks in the wires. Interferometry shows that the leading edge of the bubbles brings material to the axis of the array. The speed of this material was measured to be > or =3 x 10(7) cm/s during the wire array implosion. A shock was observed during the collision of the bubbles with the precursor. The Faraday effect indicates current flowing in breaks on the wires. The current switches from the imploding mass to the on-axis plasma column at the beginning of the x-ray pulse.     

Reversible switching of a cobalt complex by thermal, pressure, and electrochemical stimuli: abrupt, complete, hysteretic spin crossover

Triply switchable [Co(II)(dpzca)(2)] shows an abrupt, reversible, and hysteretic spin crossover (T(1/2)↓ = 168 K, T(1/2)↑ = 179 K, and ΔT(1/2) = 11 K) between the high-spin (HS) and low-spin (LS) states of cobalt(II), both of which have been structurally characterized. The spin transition is also reversibly triggered by pressure changes. Moreover, in a third reversible switching mechanism for this complex, the magnetic properties can be switched between HS cobalt(II) and LS cobalt(III) by redox.     

Direct observation of adatom-substitutional atom interaction on dilute metal alloy surfaces

The field ion microscope is used to study interactions of a migrating tungsten adatom with substitutional rhenium atoms in the (110) plane of a W-3%Re alloy. By observing about 300 migration periods on a single (110) surface plane of a small field ion emittier, each position of the adatom with respect to deduced Re atom locations can be identified. The interaction is found to be attractive with a strength of 90 ± 7 meV at the closest equilibrium separation, and repulsive with a strength of at least 80 meV at the second closest separation. No interaction could be observed for larger separations indicating a strength of less than 10 meV. Results of a control experiment, diffusion of single W adatoms on the (110) plane of pure W, are also presented for comparison.