Intermediate Spectra Research Articles

The Reaction of Synechocystis Catalase–Peroxidase (KatG) with Isoniazid Investigated by Multifrequency (9–285 GHz) EPR Spectroscopy

Three distinct electron paramagnetic resonance (EPR) spectra of radical intermediates formed as reactive intermediates in the catalytic cycle of Synechocystis catalase–peroxidase were identified. Multifrequency EPR spectroscopy, combined with site-directed mutagenesis and selective deuterium labeling of Trp and Tyr residues, allowed us to unequivocally assign such intermediates to an [Fe(IV) = O Por·+] species, the first committed intermediate in monofunctional peroxidases and two protein-based radicals, identified as Trp106· and a Tyr·, formed subsequently to the [Fe(IV) = O Por·+] species by intramolecular electron transfer. Our recent characterization of the Mycobacterium tuberculosis catalase–peroxidase showed that the Trp· sites differ among these enzymes, and that the [Fe(IV) = O Trp·] species was the reactive intermediate with the prodrug isoniazid. Accordingly, the question to address was whether the dissimilarity in the sites for the formation of the Trp· intermediates and in the geometry of the distal side was reflected by differences in the peroxidase-like reaction of Synechocystis and Mycobacterium tuberculosis catalase–peroxidases with the prodrug isoniazid. Our findings show that in the Synechocystis enzyme, the isoniazid substrate can get closer to the heme distal side and can react readily with the [Fe(IV) = O Por·+] species, at variance to the situation in the M. tuberculosis catalase–peroxidase. These results indicate that, as in the case of monofunctional peroxidases, the difference in the sites for the formation of the Trp· as alternative reactive intermediates to the [Fe(IV) = O Por·+] species is correlated to differences in substrate binding sites.

M22: AN [Fe/H] ABUNDANCE RANGE REVEALED

Intermediate resolution spectra at the Ca II triplet have been obtained for 55 candidate red giants in the field of the globular cluster M22 with the VLT/FORS instrument. Spectra were also obtained for a number of red giants in standard globular clusters to provide a calibration of the observed line strengths with overall abundance [Fe/H]. For the 41 M22 member stars that lie within the V-V_HB bounds of the calibration, we find an abundance distribution that is substantially broader than that expected from the observed errors alone. We argue that this broad distribution cannot be the result of differential reddening. Instead we conclude that, as has long been suspected, M22 is similar to omega Cen in having an intrinsic dispersion in heavy element abundance. The observed M22 abundance distribution rises sharply to a peak at [Fe/H] = -1.9 with a broad tail to higher abundances: the highest abundance star in our sample has [Fe/H] = -1.45 dex. If the unusual properties of omega Cen have their origin in a scenario in which the cluster is the remnant nucleus of a disrupted dwarf galaxy, then such a scenario likely applies also to M22.

Bidirectional Introgressive Hybridization between a Cattle and Human Schistosome Species

Schistosomiasis is a disease of great medical and veterinary importance in tropical and subtropical regions, caused by parasitic flatworms of the genus Schistosoma (subclass Digenea). Following major water development schemes in the 1980s, schistosomiasis has become an important parasitic disease of children living in the Senegal River Basin (SRB). During molecular parasitological surveys, nuclear and mitochondrial markers revealed unexpected natural interactions between a bovine and human Schistosoma species: S. bovis and S. haematobium, respectively. Hybrid schistosomes recovered from the urine and faeces of children and the intermediate snail hosts of both parental species, Bulinus truncatus and B. globosus, presented a nuclear ITS rRNA sequence identical to S. haematobium, while the partial mitochondrial cox1 sequence was identified as S. bovis. Molecular data suggest that the hybrids are not 1st generation and are a result of parental and/or hybrid backcrosses, indicating a stable hybrid zone. Larval stages with the reverse genetic profile were also found and are suggested to be F1 progeny. The data provide indisputable evidence for the occurrence of bidirectional introgressive hybridization between a bovine and a human Schistosoma species. Hybrid species have been found infecting B. truncatus, a snail species that is now very abundant throughout the SRB. The recent increase in urinary schistosomiasis in the villages along the SRB could therefore be a direct effect of the increased transmission through B. truncatus. Hybridization between schistosomes under laboratory conditions has been shown to result in heterosis (higher fecundity, faster maturation time, wider intermediate host spectrum), having important implications on disease prevalence, pathology and treatment. If this new hybrid exhibits the same hybrid vigour, it could develop into an emerging pathogen, necessitating further control strategies in zones where both parental species overlap.

Decomposition of Urea in the SCR Process: Combination of DFT Calculations and Experimental Results on the Catalytic Hydrolysis of Isocyanic Acid on TiO2 and Al2O3

In selective catalytic reduction (SCR) systems for diesel vehicles the injected urea solution decomposes to ammonia and isocyanic acid (HNCO), which reacts with water to another ammonia molecule and carbon dioxide over the SCR catalyst or a special urea decomposition catalyst. The second reaction step, i.e. the catalytic hydrolysis of HNCO was studied on the anatase TiO2(101) surface and Al2O3(100) surface with ab initio density functional theory (DFT) calculations using a cluster model as well as with in situ diffuse reflectance infrared Fourier transform spectra (DRIFTS) investigations and kinetic experiments. The following mechanistic pathway has been identified to be most feasible: HNCO dissociatively adsorbs on the metal oxide surface as isocyanates, which are attacked by water, thereby forming carbamic acid at the surface. In a further step this intermediate is transformed to a carbamate complex, which leads to CO2 desorption and consequently NH3 formation. The comparison between the sum of the theoretical vibrational spectra of the reaction intermediates with the in situ DRIFT spectra also strongly supports the accuracy of the second reaction pathway. This mechanism holds also for the HNCO hydrolysis over γ-Al2O3 and the reactivity compared to TiO2 was found to be consistent with the heights of the barriers in the energy diagrams. Based on these promising preliminary results a computational screening has been started in order to predict the most active metal oxides and surfaces for this reaction.

Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization–enhanced solid-state NMR

Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximately 90. Multidimensional spectroscopy of U-(13)C,(15)N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.

Possible evidence of asymmetry in SN 2007rt, a type IIn supernova

An optical photometric and spectroscopic analysis of the slowly-evolving Type IIn SN2007rt is presented, covering a duration of 481 days after discovery. Its earliest spectrum, taken approximately 100 days after the explosion epoch, indicates the presence of a dense circumstellar medium, with which the supernova ejecta is interacting. This is supported by the slowly-evolving light curve. A notable feature in the spectrum of SN 2007rt is the presence of a broad He I 5875 line, not usually detected in Type IIn supernovae. This may imply that the progenitor star has a high He/H ratio, having shed a significant portion of its hydrogen shell via mass-loss. An intermediate resolution spectrum reveals a narrow Halpha P-Cygni profile, the absorption component of which has a width of 128 km/s. This slow velocity suggests that the progenitor of SN 2007rt recently underwent mass-loss with wind speeds comparable to the lower limits of those detected in luminous blue variables. Asymmetries in the line profiles of H and He at early phases bears some resemblance to double-peaked features observed in a number of Ib/c spectra. These asymmetries may be indicative of an asymmetric or bipolar outflow or alternatively dust formation in the fast expanding ejecta. In addition, the late time spectrum, at over 240 days post-explosion, shows clear evidence for the presence of newly formed dust.

Conformational Changes of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a member of the new class of light-gated ion channels which serve as phototaxis receptors in the green alga Chlamydomonas reinhardtii. The protein is employed in optogenetics where neural circuits are optically stimulated under high spatiotemporal control. Despite its rapidly growing use in physiological experiments, the reaction mechanism of ChR2 is poorly understood. Here, we applied vibrational spectroscopy to trace structural changes of ChR2 after light-excitation of the retinal chromophore. FT-IR difference spectra of the various photocycle intermediates revealed that stages of the photoreaction preceding (P(1) state) and succeeding (P(4)) the conductive state of the channel (P(3)) are associated with large conformational changes of the protein backbone as indicate by strong differences in the amide I bands. Critical hydrogen-bonding changes of protonated carboxylic amino acid side chains (D156, E90) were detected and discussed with regard to the functional mechanism. We used the C128T mutant where the lifetime of P(3) is prolonged and applied FT-IR and resonance Raman spectroscopy to study the conductive P(3) state of ChR2. Finally, a mechanistic model is proposed that links the observed structural changes of ChR2 to the changes in the channel's conductance.

Real‐time molecular assessment on oxidative injury of single cells using Raman spectroscopy

AbstractOxidative stress is encountered in many biological systems; the resultant oxidative injury plays a significant role in the pathogenesis of diverse diseases. Conventional measurements on oxidative injury are employed almost exclusively on a large population of cells either by counting the fraction of cell death or by observing the fluorometric change resulting from exogenous reagents, thereby lacking in molecular detail and temporal specificity. In this work we combine laser tweezers and Raman spectroscopy to observe the response of single cells to oxidative stress. By measuring the temporal changes of vibrational spectra of single optically trapped cells, we demonstrate a molecular‐level assessment of cellular oxidative injury in real time, both qualitatively and quantitatively, without the introduction of exogenous reagents. The main experimental findings are supported by the observation of Raman spectra of intermediates and downstream products. The abrogation of the above changes by ascorbic acid further illustrates the therapeutic effect of antioxidants against cellular oxidative injury. This approach is extensible to studies exploring the biochemical transformation of single cells or intracellular organelles in response to various chemical or physical stimuli. With the aid of ‘molecular fingerprints’, single‐cell Raman spectroscopy exhibits a great potential for accessing the chemical aspects of cellular bioactivity, yielding insight into pathophysiological processes and assisting the development of novel therapeutic interventions against diseases. Copyright © 2009 John Wiley & Sons, Ltd.

Applications of X-ray absorption spectroscopy to biologically relevant metal-based chemistry

Recent developments in the understanding of the biosynthesis of the active site of the nitrogenase enzyme, the structure of the iron centre of [Fe]-hydrogenase and the structure and biomimetic chemistry of the [FeFe] hydrogenase H-cluster as deduced by application of X-ray spectroscopy are reviewed. The techniques central to this work include X-ray absorption spectroscopy either in the form of extended X-ray absorption fine structure (EXAFS), X-ray absorption near-edge structure (XANES) and nuclear resonant vibrational spectroscopy (NRVS). Examples of the advances in the understanding of the chemistry of the system through integration of a range of spectroscopic and computational techniques with X-ray spectroscopy are highlighted. The critical role played by ab initio calculation of structural and spectroscopic properties of transition-metal compounds using density functional theory (DFT) is illustrated both by the calculation of nuclear resonance vibrational spectroscopy (NRVS) spectra and the structures and spectra of intermediates through the catalytic reactions of hydrogenase model compounds.

SPECTROSCOPIC CONFIRMATION OF THE PLANETARY NEBULA NATURE OF PM 1-242, PM 1-318, AND PM 1-333 AND MORPHOLOGICAL ANALYSIS OF THE NEBULAE

We present intermediate resolution long-slit spectra and narrowband H{alpha}, [N II], and [O III] images of PM 1-242, PM 318, and PM 1-333, three IRAS sources classified as possible planetary nebulae. The spectra show that the three objects are true planetary nebulae and allow us to study their physical properties; the images provide a detailed view of their morphology. PM 1-242 is a medium- to high-excitation (e.g., He II{lambda}4686/H{beta} {approx} 0.4; [N II]{lambda}6584/H{alpha} {approx} 0.3) planetary nebula with an elliptical shape containing [N II] enhanced point-symmetric arcs. An electron temperature [T {sub e}([S III])] of {approx} 10250 K and an electron density [N {sub e}([S II])] of {approx} 2300 cm{sup -3} are derived for PM 1-242. Abundance calculations suggest a large helium abundance (He/H {approx} 0.29) in PM 1-242. PM 1-318 is a high-excitation (He II{lambda}4686/H{beta} {approx} 1) planetary nebula with a ring-like inner shell containing two enhanced opposite regions, surrounded by a fainter round attached shell brighter in the light of [O III]. PM 1-333 is an extended planetary nebula with a high-excitation (He II{lambda}4686/H{beta} up to {approx} 0.9) patchy circular main body containing two low-excitation knotty arcs. A low N {sub e}([S II]) of {approx} 450 cm{sup -3}more » and T {sub e}([O III]) of {approx} 15000 K are derived for this nebula. Abundance calculations suggest that PM 1-333 is a type I planetary nebula. The lack of a sharp shell morphology, low electron density, and high excitation strongly suggest that PM 1-333 is an evolved planetary nebula. PM 1-333 also shows two low-ionization polar structures whose morphology and emission properties are reminiscent of collimated outflows. We compare PM 1-333 with other evolved planetary nebulae with collimated outflows and find that outflows among evolved planetary nebulae exhibit a large variety of properties, in accordance with these observed in younger planetary nebula.« less

Identification of reaction intermediates during hydrogenation of CD 3CN on Raney-Co

Reaction intermediates formed on the surface of Raney-Co after co-adsorption of acetonitrile- d 3 (CD 3CN) and hydrogen were investigated by inelastic neutron scattering (INS). Detailed information on the structure of the adsorbed molecules was obtained by comparison of the spectra of potential reaction intermediates with reference spectra of H 2, CD 3CN, and CD 3CH 2NH 2 as well as INS spectra calculated by ab initio methods. We concluded that CD 3CN reacted preferentially with hydrogen bound to η 3 sites on the Co-001 plane. On the surface, a mixture of a nitrene-like intermediate and co-adsorbed CD 3CH 2NH 2 was formed, as indicated by a strong CH 2 twisting vibrational mode, which decreased in intensity relative to the other CH 2 vibrational modes, when the amount of hydrogen was increased. The surface study is complemented with detailed catalyst characterisation as well as kinetic data on the hydrogenation of acetonitrile- d 3 .

Approaches to alkane functionalization with Tp′Pt and (nacnac)Pt reagents

In this perspective article we give an overview of our studies of C–H activation with platinum over the past decade. The scorpionate ligand Tp′ (Tp′ = hydrotris(3,5-dimethylpyrazolyl)borate) has exhibited the ability to stabilize both Pt(IV) and Pt(II) complexes due to flexibility between κ3 and κ2 binding modes. Control of the Tp′ coordination mode and Pt oxidation state is tightly coupled with acid/base chemistry. This ability allowed us to model a spectrum of intermediates in C–H activation reactions, including Pt(IV) alkyl hydrides, five-coordinate Pt(IV) silyl hydrides, and Pt(II) benzene complexes. We have been able to observe sturdy Pt(II) benzene complexes and probe their equilibria with the respective C–H activated Pt(IV) species in the presence of an added ligand. We have also taken steps toward functionalizing hydrocarbons with Tp′Pt by dehydrogenation of alkanes and by arylation of olefins. We have recently turned to bidentate nacnac (nacnac = N-phenyl-β-enamineimine) ligands in the hopes of catalytically functionalizing hydrocarbons.Key words: C–H activation, reductive elimination, Tp′Pt, five-coordinate, β-diiminate.

Alternative data reduction procedures for UVES: Wavelength calibration and spectrum addition

This paper addresses alternative procedures to the ESO supplied pipeline procedures for the reduction of UVES spectra of two quasar spectra to determine the value of the fundamental constant μ=Mp/Me at early times in the universe. The procedures utilize intermediate product images and spectra produced by the pipeline with alternative wavelength calibration and spectrum addition methods. Spectroscopic studies that require extreme wavelength precision need customized wavelength calibration procedures beyond that usually supplied by the standard data reduction pipelines. An example of such studies is the measurement of the values of the fundamental constants at early times in the universe. This article describes a wavelength calibration procedure for the UV–visual Echelle spectrometer on the very large telescope, however, it can be extended to other spectrometers as well. The procedure described here provides relative wavelength precision of better than 3×10-7 for the long-slit Thorium–Argon calibration lamp exposures. The gain in precision over the pipeline wavelength calibration is almost entirely due to a more exclusive selection of Th/Ar calibration lines.

Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.

Conformation and Membrane Orientation of Amphiphilic Helical Peptides by Oriented Circular Dichroism

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic α-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of ∼1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L ∼1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L ∼1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with β-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.

Competition of coalescence and “fireball” processes in nonequilibrium emission of light charged particles fromp+Aucollisions

The energy and angular dependence of the double differential cross sections ${d}^{2}\ensuremath{\sigma}/d\ensuremath{\Omega}\mathit{dE}$ were measured for $p,d,t,$$^{3,4,6}\mathrm{He}$, $^{6,7,8,9}\mathrm{Li}$, $^{7,9,10}\mathrm{Be}$, and $^{10,11,12}\mathrm{B}$ isotopes produced in reactions of 1.2- and 1.9-GeV protons on a Au target. The beam energy dependence of the data, supplemented by the cross sections from a previous experiment at 2.5 GeV, is very smooth. The shape of the spectra and angular distributions do not change significantly in the beam energy range from 1.2 to 2.5 GeV. However, the absolute value of the cross sections increases for all ejectiles. The intermediate mass fragment spectra and their angular distributions are very well reproduced by a phenomenological model of two emitting, moving sources, with parameters smoothly varying with energy. The double differential cross sections for light charged particles were analyzed in the framework of the microscopic model calculations of intranuclear cascade, including the coalescence of nucleons and a statistical model for evaporation of particles from excited residual nuclei. However, the energy and angular dependencies of the data agree with neither predictions of the microscopic intranuclear cascade calculations for protons nor the coalescence calculations for other light charged particles. A very good description of the data is achieved by the phenomenological inclusion of the emission of light charged particles from a ``fireball'' (i.e., a fast and hot moving source). It was found that the nonequilibrium processes are very important for the production of light charged particles. They exhaust 40%--80% of the total cross section---depending on the emitted particles. Coalescence and fireball emission yield comparable contributions to the cross sections with the exception of the $^{3}\mathrm{He}$ data where coalescence clearly dominates. For all light charged particles, the ratio of nonequilibrium processes to processes proceeding through a phase of statistical equilibrium does not change significantly between the beam energies of 1.2 and 2.5 GeV.

Assigning Vibrational Spectra of Ferryl-Oxo Intermediates of Cytochrome c Oxidase by Periodic Orbits and Molecular Dynamics

Complexity is inherent in biological molecules not only because of the large number of atoms but also because of their nonlinear interactions responsible for chaotic behaviours, localized motions, and bifurcation phenomena. Thus, versatile spectroscopic techniques have been invented to achieve temporal and spatial resolution to minimize the uncertainties in assigning the spectra of complex molecules. Can we associate spectral lines to specific chemical bonds or species in a large molecule? Can energy stay localized in a bond for a substantial period of time to leave its spectroscopic signature? These longstanding problems are investigated by studying the resonance Raman spectra of ferryl-oxo intermediates of cytochrome c oxidase. The difference spectra of isotopically substituted ferryl oxygen ((16)O minus (18)O) in the cytochrome c oxidase recorded in several laboratories show one or two prominent positive peaks which have not been completely elucidated yet. By applying the hierarchical methods of nonlinear mechanics, and particularly the study of periodic orbits in the active site of the enzyme, in conjunction with molecular dynamics calculations of larger systems which include the embraced active site by the protein and selected protonated/deprotonated conformations of amino acids, we translate the spectral lines to molecular motions. It is demonstrated that for the active site stable periodic orbits exist for a substantial energy range. Families of periodic orbits which are associated with the vibrations of Fe(IV)=O bond mark the regions of phase space where nearby trajectories remain localized, as well as assign the spectral bands of the active site in the protein matrix. We demonstrate that proton movement adjacent to active site, which occurs during the P --> F transition, can lead to significant perturbations of the Fe(IV)=O isotopic difference vibrational spectra in cytochrome c oxidase, without a change in oxidation state of the metal sites. This finding links spectroscopic characteristics to protonation events occurring during enzymatic turnover.

Electron and proton transfer in the ba 3 oxidase from Thermus thermophilus

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O(2) to water is catalysed at a haem a(3)-Cu(B) catalytic site. The three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba( 3 )-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 10(4) s(-1)) and formation of the peroxy (P(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 10(4) s(-1), associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s(-1), accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the P(R) and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa(3)-type oxidases.

Nα Resonating-Group Calculation with a Quark-Model G-Matrix NN Interaction

We calculate nα phase-shifts and scattering observables in the resonating-group method, using the nuclear-matter G-matrix of an SU6 quark-model NN interaction. The G-matrix is generated in the recent energy-independent procedure of the quark-model NN interaction with the continuous prescription for intermediate spectra, by assuming an appropriate Fermi momentum kF = 1.2 fm −1. The nα RGM interaction kernels are evaluated with explicit treatments of the nonlocality and momentum dependence of partial-wave G-matrix components. The momentum dependence of the G-matrix components is different for each of the nucleon-exchange and interaction types. Without introducing any artificial parameters except for kF , the central and spin-orbit components of the nα Born kernel are found to have reasonable strengths under the assumption of a rigid translationally invariant shell-model wave function of the α-cluster. The characteristic behaviors of three different exchange terms, corresponding to knockout, heavy-particle pickup and nucleon-rearrangement processes, are essentially the same between the case of previous local effective NN forces and the case of nonlocal G-matrix NN interactions.

THE UNUSUAL DISTRIBUTIONS OF IONIZED MATERIAL AND MOLECULAR HYDROGEN IN NGC 6881: SIGNPOSTS OF MULTIPLE EVENTS OF BIPOLAR EJECTION IN A PLANETARY NEBULA

The planetary nebula NGC 6881 displays in the optical a quadrupolar morphology consisting of two pairs of highly collimated bipolar lobes aligned along different directions. An additional bipolar ejection is revealed by the hydrogen molecular emission, but its wide hourglass morphology is very different from that of the ionized material. To investigate in detail the spatial distribution of molecular hydrogen and ionized material within NGC 6881, and to determine the prevalent excitation mechanism of the H2 emission, we have obtained new near-IR Br-gamma and H2 and optical H-alpha and [N II] images, as well as intermediate resolution JHK spectra. These observations confirm the association of the H2 bipolar lobes to NGC 6881 and find that the prevalent excitation mechanism is collisional. The detailed morphology and very different collimation degree of the H2 and ionized bipolar lobes of NGC 6881 not only imply that multiple bipolar ejections have occurred in this nebula, but also that the dominant shaping agent is different for each bipolar ejection: a bipolar stellar wind most likely produced the H2 lobes, while highly collimated outflows are carving out the ionized lobes into the thick circumstellar envelope. The asymmetry between the southeast and northwest H2 bipolar lobes suggests the interaction of the nebula with an inhomogeneous interstellar medium. We find evidence that places NGC 6881 in the H II region Sh 2-109 along the Orion local spiral arm.