Solid-state NMR study of the charge-transfer complex between ubiquinone-8 and disulfide bond generating membrane protein DsbB

Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB, and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ~500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge-transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (~20 ppm), methoxys (~60 ppm), olefin carbons (120-140 ppm), and carbonyls (150-160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge-transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.     

Sub-Doppler infrared spectroscopy of CH2D radical in a slit supersonic jet: isotopic symmetry breaking in the CH stretching manifold

First high-resolution infrared absorption spectra in the fundamental symmetric/asymmetric CH stretching region of isotopically substituted methyl radical, CH(2)D, are reported and analyzed. These studies become feasible in the difference frequency spectrometer due to (i) high density radical generation via dissociative electron attachment to CH(2)DI in a discharge, (ii) low rotational temperatures (23 K) from supersonic cooling in a slit expansion, (iii) long absorption path length (64 cm) along the slit axes, and (iv) near shot noise limited absorption sensitivity (5 × 10(-7)/√(Hz)). The spectra are fully rovibrationally resolved and fit to an asymmetric top rotational Hamiltonian to yield rotational/centrifugal constants and vibrational band origins. In addition, the slit expansion collisionally quenches the transverse velocity distribution along the laser probe direction, yielding sub-Doppler resolution of spin-rotation structure and even partial resolution of nuclear hyperfine structure for each rovibrational line. Global least-squares fits to the line shapes provide additional information on spin-rotation and nuclear hyperfine constants, which complement and clarify previous FTIR studies [K. Kawaguchi, Can. J. Phys. 79, 449 (2001)] of CH(2)D in the out-of-plane bending region. Finally, analysis of the spectral data from the full isotopomeric CH(m)D(3-m) series based on harmonically coupled Morse oscillators establishes a predictive framework for describing the manifold of planar stretching vibrations in this fundamental combustion radical.     

Cu adsorption on pyrite (1 0 0): Ab initio and spectroscopic studies

Surface optimised S 2p photoelectron spectra show that both surface S²⁻ monomers and (S-S)²⁻ dimers are present at pyrite (1 0 0) fracture surfaces. In order to determine which sulfur species are involved in Cu adsorption, fresh pyrite surfaces were exposed to Cu²⁺ in solution. The S 2p spectra suggest that both types of S surface species are involved in the mechanism of Cu adsorption (activation). Ab initio density functional theory was used to model Cu adsorbed onto pyrite (1 0 0) to support the interpretation of the spectroscopy. Mulliken population analysis confirms the charge distribution suggested by the core line shifts as observed in the photoelectron spectra. The ab initio calculations were consistent with a two-coordinate bond between Cu(I), a surface S monomer and a surface S dimer.     

Large amplitude quantum mechanics in polyatomic hydrides. I. A particles-on-a-sphere model for XH(n)

A framework is presented for converged quantum mechanical calculations on large amplitude dynamics in polyatomic hydrides (XH(n)) based on a relatively simple, but computationally tractable, "particles-on-a-sphere" (POS) model for the intramolecular motion of the light atoms. The model assumes independent two-dimensional (2D) angular motion of H atoms imbedded on the surface of a sphere with an arbitrary interatomic angular potential. This assumption permits systematic evolution from "free rotor" to "tunneling" to "quasi-rigid" polyatomic molecule behavior for small, but finite, values of total angular momentum J. This work focuses on simple triatom (n=2) and tetratom (n=3) systems as a function of interatomic potential stiffness, with explicit consideration of H2O, NH3, and H3O+ as limiting test cases. The POS model also establishes the necessary mathematical groundwork for calculations on dynamically much more challenging XH(n) species with n>3 (e.g., models of CH5+) where such a reduced dimensionality approach offers prospects for being quantum mechanically tractable at low J values (i.e., J=0, 1, 2) characteristic of supersonic jet expansion conditions.     

Quantum state-resolved energy transfer dynamics at gas-liquid interfaces: IR laser studies of CO2 scattering from perfluorinated liquids

An apparatus for detailed study of quantum state-resolved inelastic energy transfer dynamics at the gas-liquid interface is described. The approach relies on supersonic jet-cooled molecular beams impinging on a continuously renewable liquid surface in a vacuum and exploits sub-Doppler high-resolution laser absorption methods to probe rotational, vibrational, and translational distributions in the scattered flux. First results are presented for skimmed beams of jet-cooled CO(2) (T(beam) approximately 15 K) colliding at normal incidence with a liquid perfluoropolyether (PFPE) surface at E(inc) = 10.6(8) kcal/mol. The experiment uses a tunable Pb-salt diode laser for direct absorption on the CO(2) nu(3) asymmetric stretch. Measured rotational distributions in both 00(0)0 and 01(1)0 vibrational manifolds indicate CO(2) inelastically scatters from the liquid surface into a clearly non-Boltzmann distribution, revealing nonequilibrium dynamics with average rotational energies in excess of the liquid (T(s) = 300 K). Furthermore, high-resolution analysis of the absorption profiles reveals that Doppler widths correspond to temperatures significantly warmer than T(s) and increase systematically with the J rotational state. These rotational and translational distributions are consistent with two distinct gas-liquid collision pathways: (i) a T approximately 300 K component due to trapping-desorption (TD) and (ii) a much hotter distribution (T approximately 750 K) due to "prompt" impulsive scattering (IS) from the gas-liquid interface. By way of contrast, vibrational populations in the CO(2) bending mode are inefficiently excited by scattering from the liquid, presumably reflecting much slower T-V collisional energy transfer rates.     

Approaching non-destructive surface chemical analysis of CMSX-4 superalloy with double-pulsed laser induced breakdown spectroscopy

Laser induced breakdown spectroscopy (LIBS) was performed on the CMSX-4 Ni-based superalloy using a femtosecond pulsed laser. An orthogonal double-pulse technique was used to minimize surface damage associated with LIBS. With this technique, the depth of ablation craters was reduced from 200 nm for single-pulse LIBS down to less than 60 nm using orthogonal double-pulse LIBS. The technique also allowed the average velocity of the ablated material to be determined, which ranged from 4720 ± 560 m/s at a pump laser fluence of 3.1 J/cm² to 8150 ±1800 m/s at 10.1 J/cm².     

Large-amplitude quantum mechanics in polyatomic hydrides. II. A particle-on-a-sphere model for XH(n) (n=4,5)

This paper describes the application of a relatively simple, but computationally tractable, "particle-on-a-sphere" (POS) model for quantum-mechanical calculation of large-amplitude, H atom dynamics in polyatomic hydrides (XH(n)), based on radially relaxed, two-dimensional angular motion of H atoms on the surface of a sphere. This work focuses on systems with many degrees of freedom, i.e., XH(4) (eight dimensional) and XH(5) (ten dimensional), with corresponding molecular analogs of CH(4) and CH(5) (+) and is applicable to rovibrationally excited states with J> or =0. A pairwise-additive potential fit for CH(5) (+), which yields remarkable agreement with geometries, energies, and barrier heights on the full-dimensional surface of Brown et al. [J. Chem. Phys. 121, 4105 (2004)] is presented. Comparisons with experimental data and diffusion quantum Monte Carlo (DMC) methods test convergence for the POS model and provide insight into multidimensional quantum rovibrational dynamics. In particular, POS energy-level patterns for a series of scaled CH(5) (+) potentials indicate an absence of strong tunneling behavior, consistent with the highly delocalized wave functions, large zero-point energies, and small interconversion barriers noted in previous DMC studies of Brown et al.     

Vibrational spectra of melamine diborate,C3N6H62H3BO3

IR and Raman spectra of melamine diborate have been recorded and analysed. Band assignments are given based on the vibrations of melamine and boric acid molecules. Three sets of frequencies observed for the N-H stretching mode region is ascribed to different types of hydrogen bonds in the amino groups of the triazine ring. Due to the lower symmetry of the melamine and boric acid molecules in the crystal, activation of inactive modes and lifting of the degeneracy of certain vibrational modes are observed. Lower symmetry of the melamine molecule in the crystal has resulted in the decrease of intensity of the Raman active melamine band around 1490 cm(-1). All the internal modes of boric acid molecule were identified. All the ring breathing modes of melamine molecule show frequency shift towards the high wavenumber side. In other words, hydrogen bonding affects the ring breathing modes of melamine.     

High-resolution infrared spectroscopy of jet-cooled vinyl radical: symmetric CH2 stretch excitation and tunneling dynamics

First high-resolution IR spectra of jet-cooled vinyl radical in the C-H stretch region are reported. Detailed spectral assignments and least squares fits to an A-reduction Watson asymmetric top Hamiltonian yield rotational constants and vibrational origins for three A-type bands, assigned to single quantum excitation of the symmetric CH(2) stretch. Two of the observed bands arise definitively from ground state vinyl radical, as rigorously confirmed by combination differences predicted from previous midinfrared CH(2) wagging studies of Kanamori et al. [J. Chem. Phys. 92, 197 (1990)] as well as millimeter wave rotation-tunneling studies of Tanaka et al. [J. Chem. Phys. 120, 3604 (2004)]. The two bands reflect transitions out of symmetric (0(+)) and antisymmetric (0(-)) tunneling levels of vinyl radical populated at 14 K slit-jet expansion temperatures. The band origins for the lower-lower (0(+)<--0(+)) and upper-upper (0(-)<--0(-)) transitions occur at 2901.8603(7) and 2901.9319(4) cm(-1), respectively, which indicates an increase in the tunneling splitting and therefore a decrease in the effective tunneling barrier upon CH(2) symmetric stretch excitation. The third A-type band with origin at 2897.2264(3) cm(-1) exhibits rotational constants quite close to (but at high-resolution distinguishable from) the vinyl radical ground state, consistent with a CH(2) symmetric stretch hot band built on one or more quanta of excitation in a low frequency vibration. The observed CH(2) symmetric stretch bands are in excellent agreement with anharmonically scaled high level density functional theory (DFT) calculations and redshifted considerably from previous low resolution assignments. Of particular dynamical interest, Boltzmann analysis indicates that the pair of 0(+) and 0(-) tunneling bands exhibits 1:1 nuclear spin statistics for K(a)=even:odd states. This differs from the expected 3:1 ratio for feasible exchange of the two methylenic H atoms but is consistent with a 4:4 ratio predicted for interchange between all three H atoms. This suggests the novel dynamical possibility of large amplitude "roaming" of all three H atoms in vinyl radical, promoted by high internal vibrational excitation arising from dissociative electron attachment in the discharge.     

Vibrational spectroscopic studies and ab initio calculations of Goniothalamin, a natural product

5,6-Dihydro-6-styryl-2-pyrone (Goniothalamin), is isolated from the leaves of Goniothalamus wightii and identified by spectral analysis and X-ray diffraction studies. FT-IR spectroscopy has also been used to characterize the vibrational bands. The vibrational wavenumbers and corresponding vibrational assignments are examined theoretically using the Gaussian03 set of quantum chemistry codes. Predicted IR and Raman intensities are reported.