High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast

High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y*)-diiron cofactor in the Y2-containing R2 subunit of ribonucleotide reductase (RNR) from yeast. The present work represents the first use of 140-GHz time domain EPR and ENDOR to examine this system and demonstrates the capabilities of the method to elucidate the electronic structure and the chemical environment of protein radicals. Low-temperature spin-echo-detected EPR spectra of yeast Y* reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y* from E. coli RNR, a substantial upfield shift of the g(1)-value is observed. The origin of the shift in g(1) was investigated by 140-GHz (1)H and (2)H pulsed ENDOR experiments of the Y2-containing subunit in protonated and D(2)O-exchanged buffer. (2)H ENDOR spectra and simulations provide unambiguous evidence for one strongly coupled (2)H arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule. Orientation-selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bond with respect to the molecular symmetry axes and the bond length (1.81 A). Finally, we have performed saturation recovery experiments and observed enhanced spin lattice relaxation rates of the Y* above 10 K. At temperatures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction between Y* and the first excited paramagnetic state of the diiron cluster adjacent to it. From the activation energy of the rates, we determine the exchange interaction between the two irons of the cluster, J(exc) = -85 cm(-)(1). The relaxation mechanism and the presence of the hydrogen bond are discussed in terms of the differences in the structure of the Y*-diiron cofactor in yeast Y2 and other class I R2s.     

Elongated conformers of charge states +11 to +15 of bovine ubiquitin studied using ESI-FAIMS-MS

Recent advancements in high-field asymmetric waveform ion mobility spectrometry (FAIMS) have led to significant improvements in the application of this technology to the study of protein conformers. Compared with previous work, the maximum value of the separation voltage (i.e., the dispersion voltage) has increased, thereby enabling multiple, elongated conformers of individual charge states of bovine ubiquitin to be separated in the gas phase (e.g., four conformers of each of the +11 and +12 charge states were separated). The use of a carrier gas mixture of 40% nitrogen and 60% helium changed the separation selectivity compared with pure nitrogen and enhanced the signal intensity, especially for the +14 and +15 charge states (the latter was not detected in a nitrogen carrier gas). Conformer cross sections were determined using the FAIMS/energy-loss method and found to be similar within a given charge state. The cross sections for conformers of charge states +13, + 14, and +15 plateau at about 2000 A2 suggesting that the structure of bovine ubiquitin is essentially unfolded after the addition of the 13th proton.     

Palladium-catalyzed synthesis of arylamines from aryl halides and lithium bis(trimethylsilyl)amide as an ammonia equivalent

[reaction: see text]. A simple, palladium-catalyzed method to convert aryl halides to the parent anilines using lithium bis(trimethylsilyl)amide (LiN(SiMe3)2) is reported. The reaction is catalyzed by Pd(dba)2 and P(t-Bu)3 and can be run with as little as 0.2 mol % of catalyst. The reaction is faster than competing generation of benzyne intermediates and, therefore, provides the aniline products regiospecifically.     

Analysis for metallothioneins using coupled techniques

Analytical chemistry of metallothioneins based on the coupling of a high resolution separation technique with an element or species selective detection technique is discussed. The role of size-exclusion chromatography (SEC) with on-line atomic spectrometric detection for the quantification of metallothionein fraction in cell cytosols is evaluated. Particular attention is given to the conditions for the separation of metallated metallothionein isoforms (MT-1, MT-2, MT-3) and sub-isoforms within these classes by anion-exchange and reversed-phase HPLC. Techniques for interfacing chromatography with atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP AES) and ICP mass spectrometry (MS) are assessed. The potential of electrospray (tandem) mass spectrometry for the characterization of metallothionein isoforms with respect to molecular mass and aminoacid sequence is highlighted. Perspectives for capillary zone electrophoresis (CZE), microbore and capillary HPLC with ICP MS and electrospray MS(/MS) detection for the probing of metallothioneins are discussed. Applications of hyphenated techniques to the analysis of real-world samples are reviewed.     

Local and global chirality at surfaces: succinic acid versus tartaric acid on Cu110

A detailed comparison of tartaric acid (HOOC-CHOH-CHOH-COOH) and succinic acid (HOOC-CH(2)-CH(2)-COOH) molecules on a Cu(110) surface is presented with a view to elucidate how the two-dimensional chirality exhibited by such robust, chemisorbed systems is affected when both OH groups of the former molecule are replaced with H groups, a stereochemical change that leaves the metal-bonding functionalities of the molecule untouched but destroys both chiral centers. It is found that this change does not significantly affect the thermodynamically preferred chemical forms that are adopted, namely the doubly deprotonated bicarboxylate at low coverages (theta 相似文献   

Myoglobin-CO substate structures and dynamics: multidimensional vibrational echoes and molecular dynamics simulations

Spectrally resolved infrared stimulated vibrational echo data were obtained for sperm whale carbonmonoxymyoglobin (MbCO) at 300 K. The measured dephasing dynamics of the CO ligand are in agreement with dephasing dynamics calculated with molecular dynamics (MD) simulations for MbCO with the residue histidine-64 (His64) having its imidazole epsilon nitrogen protonated (N(epsilon)-H). The two conformational substate structures B(epsilon) and R(epsilon) observed in the MD simulations are assigned to the spectroscopic A(1) and A(3) conformational substates of MbCO, respectively, based on the agreement between the experimentally measured and calculated dephasing dynamics for these substates. In the A(1) substate, the N(epsilon)-H proton and N(delta) of His64 are approximately equidistant from the CO ligand, while in the A(3) substate, the N(epsilon)-H of His64 is oriented toward the CO, and the N(delta) is on the surface of the protein. The MD simulations show that dynamics of His64 represent the major source of vibrational dephasing of the CO ligand in the A(3) state on both femtosecond and picosecond time scales. Dephasing in the A(1) state is controlled by His64 on femtosecond time scales, and by the rest of the protein and the water solvent on longer time scales.     

The standard redox potential of the phenyl radical/anion couple

The voltammogram of aryldiazonium tetrafluoroborates in acetonitrile (ACN), at low concentration, shows a first one-electron wave followed at a more negative potential by a small second wave; this last one corresponds to the reduction of the radical formed at the level of the first wave. Simulation of the voltammogram permits one to determine the standard redox potential of the radical/anion couple Eo(Ph./Ph-) = 0.05 V/SCE and the reduction mechanism of the diazonium cation. An electron transfer concerted with the cleavage of the C-N bond furnishes the aryl radical; this radical undergoes two competitive reactions: reduction at the electrode and H-atom transfer.     

Determination of uranium after its selective extraction as phenylacetate

The method described is based on the extraction of uranium with a chloroform solution of phenylacetic acid from slightly acidic solution containing nitrilotriacetic acid, which masks all interfering metals. After stripping into very dilute hydrochloric acid, uranium is reduced with ascorbic acid and determined complexometrically. The method permits reliable determination of uranium in the presence of all quadri-, ter- and bivalent metals investigated, molybdenum(VI), tungsten(VI), and vanadium(V).     

Differential pulse adsorptive stripping voltammetry of osmium-modified peptides

Complexes of osmium tetroxide with nitrogen ligands were developed and used in our laboratory as probes of the DNA structure. Here, we show that the complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) can be used for modification and electrochemical detection of proteins at neutral pH. Salmon luteinizing hormone (SLH) containing two tryptophan (Trp) residues and human luteinizing hormone (HLH) containing one Trp were modified by Os,bipy and measured by differential pulse adsorptive stripping voltammetry (DPAdSV) at a hanging mercury drop electrode (HMDE). The intensity of the DPAdSV catalytic signals corresponded to the number of Trp residues in the peptide molecule. Decreasing pH of the background electrolyte from 6.6 to 3.8 led to the increase of DPAdSV signals, suggesting that at pH 3.8, the DPAdSV detection limit might be well below 1 ng/ml. Our results suggest that Os,bipy is potentially useful for chemical modification of proteins.