Global Fitting Analysis Research Articles

Determining electronic spectra at interfaces by electronic sum frequency generation: One- and two-photon double resonant oxazine 750 at the air/water interface

The second-order nonlinear electronic spectra were measured for a dye oxazine 750 (OX750) adsorbed at the air/water interface using the multiplex electronic sum frequency generation (ESFG) spectroscopy recently developed by our group. The excitation-wavelength dependence of the ESFG spectrum was investigated, and a global fitting analysis was performed to separate contributions of one- and two-photon resonances. The analysis yielded linear interface electronic spectra in the one- and two-photon resonance regions, which can be directly compared to bulk absorption spectra. A two-dimensional plot of the linear interface electronic spectra is newly proposed to graphically represent all the essential information on the electronic structure of interfacial molecules. On this new analytical basis of the ESFG spectroscopy, the spectroscopic properties of OX750 at the interface are discussed.

Multiple Steps Determine the Overall Rate of the Reduction of 5α-Dihydrotestosterone Catalyzed by Human Type 3 3α-Hydroxysteroid Dehydrogenase: Implications for the Elimination of Androgens

Human type 3 3alpha-hydroxysteroid dehydrogenase, or aldo-keto reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5alpha-dihydrotestosterone (DHT, potent androgen) to form 3alpha-androstanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The k(cat) for the NADPH-dependent reduction of DHT catalyzed by AKR1C2 is 0.033 s(-1). We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low k(cat) value. Stopped-flow experiments to measure the formation of the AKR1C2.NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on k(cat) (1.5) and a slightly larger effect on k(cat)/K(m) (2.1) were observed in the steady state. In the transient state, the maximum rate constant for the single turnover of DHT (k(trans)) was determined to be 0.11 s(-1) for the NADPH-dependent reaction, which was approximately 4-fold greater than the corresponding k(cat) x k(trans) was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent (D)k(trans) of 3.5. Thus, the effects of isotopic substitution on the hydride transfer step were masked by slow events that follow or precede the chemical transformation. Transient multiple-turnover reactions generated curvilinear reaction traces, consistent with the product formation and release occurring at comparable rates. Global fitting analysis of the transient kinetic data enabled the estimate of the rate constants for the three-step cofactor binding/release model and for the minimal ordered bi-bi turnover mechanism. Results were consistent with a kinetic mechanism in which a series of slow events, including the chemical step (0.12 s(-1)), the release of the steroid product (0.081 s(-1)), and the release of the cofactor product (0.21 s(-1)), combine to yield the overall observed low turnover number.

Conformational Transitions as Determinants of Specificity for the DNA Methyltransferase EcoRI

Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M.EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. Chem. 274, 19269-19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M.EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500- and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by an approximately 2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.

Joint ro-vibrational analysis of the HDS high resolution infrared data

High resolution Fourier transform spectra of the HDS molecule were recorded and analyzed for the first time in the region of the bands ν 1 + 2 ν 2 (3938.6 cm −1), ν 1 + ν 3 (4522.6 cm −1), 2 ν 2 + ν 3 (4638.8 cm −1), 2 ν 1 + ν 2 (4767.7 cm −1), ν 1 + ν 2 + ν 3 (5525.2 cm −1), 3 ν 1 (5560.6 cm −1), ν 1 + 2 ν 3 (7047.2 cm −1), and 2 ν 2 + 2 ν 3 (7123.9 cm −1). The ro-vibrational energies of the upper vibrational states of these bands together with the ro-vibrational energies of 12 other bands already studied at high resolution were used as inputs in a Global Fit analysis firstly described in [O.N. Ulenikov, G.A. Onopenko, H. Lin, J.-H. Zhang, Z.-Y. Zhou, Q.-S. Zhu, R.N. Tolchenov, J. Mol. Spectrosc. 189 (1998) 29–39]. In this case, the resonance interactions between the states ( v 1 v 2 v 3) and ( v 1 ± 2 v 2 ∓ 1 v 3 ∓ 1) were taken into account. The resulting set of 143 parameters reproduces all the experimental data (2984 vibration–rotation energies of 20 vibrational states which correspond to about 9700 ro-vibrational transitions with J max = 23) with accuracies comparable with the experimental uncertainties.

Global fit analysis including the ν9 + ν4 − ν4 hot band of ethane: Evidence of an interaction with the ν12 fundamental

The ν 9 fundamental band of ethane occurs in the 12 μm region. It is the strongest band of ethane in a terrestrial window and is commonly used for the identification of ethane in the Jovian planets. The ν 9 + ν 4 − ν 4 band occurs in the same region; neither can be analysed as an isolated band, since both are embedded in the torsional bath of the ground vibrational state. We report here two global fit models including data from both of these bands as well as the ν 3 fundamental and the ν 4, 2 ν 4 − ν 4, and 3 ν 4 torsional transitions. The first is restricted to −5 ⩽ KΔ K ⩽ 15 in the hot band and gives an excellent fit to the included data. Three resonant interactions are identified in this fit—a Coriolis interaction with two resonant cases between the ν 9 torsional stack and that of the ground vibrational state (gs) and a resonant Fermi interaction between the ν 3 fundamental and the gs. Hot band lines with KΔ K < −5 are influenced by a fourth perturbation, with a crossing at −11 < KΔ K < −10, which has been attributed to an interaction with the ν 12 fundamental. A second fit, demonstrating a promising treatment of this interaction, is also presented.

The Cytoplasmic Heme-binding Protein (PhuS) from the Heme Uptake System of Pseudomonas aeruginosa Is an Intracellular Heme-trafficking Protein to the δ-Regioselective Heme Oxygenase

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.

Temperature dependence of chaperone-like activity and oligomeric state of αB-crystallin

The chaperone-like activity and the oligomeric state of αB-crystallin were studied at different temperatures and in the presence of urea and thiocyanate. The activity, assessed measuring the ability of αB-crystallin to prevent the aggregation of denatured insulin, strongly depends on temperature. While a significant activity increase was detected at 42 °C, the presence of urea and thiocyanate does not affect the protein activity in an irreversible way. In-solution SAXS measurements performed in the same experimental conditions showed that αB-crystallin forms near-spherical, hollowed, polydisperse oligomers, whose dimensions change above 42 °C. Moreover, in the presence of urea and thiocyanate, a global fit analysis confirms the high stability of αB-crystallin assemblies in relationship with their variable quaternary structure. In particular, the changes in the inner radius as well as the thickness and dispersion of the protein shell, account for the preservation of the chaperone-like activity.

Redox‐Regulation of Glutathione Synthesis in Plants

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamate-cysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes; however, kinetic analysis shows that AtGCL is functionally similar to the enzyme from other organisms. Buthionine sulfoximine and cystamine inactivate AtGCL, but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. Global fitting analysis of initial velocity data indicates that a random ter-reactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consist of a catalytic subunit and a regulatory subunit, AtGCL functions as a monomeric protein. In response to changes in redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. Because reducing agents mediate this change, an intramolecular disulfide bond is likely involved. Thus, regulation of AtGCL does not require association with a regulatory subunit, as in the mammalian system, for activation. This model for controlling AtGCL activity explains observations that oxidative stimulus induces glutathione synthesis without increased gene expression in plants. Similar redox signal-induced conformational switching by disulfide formation occurs in other proteins, including Yap1, thioredoxins, and OxyR.

Formation and Dissociation of Rhodamine 800 Dimers in Water: Steady-State and Ultrafast Spectroscopic Study

We investigated the fundamental photophysics and photochemistry of a cationic dye rhodamine 800 (R800) in water using steady-state and ultrafast time-resolved spectroscopies. In the ground state, the monomer and dimer coexist in equilibrium, which causes significant concentration dependence of UV-visible (vis) absorption spectra. We determined the equilibrium constant as well as the molar absorption spectra of the monomer and dimer from a global fitting analysis of the UV-vis spectra. The obtained pure dimer spectrum indicates that it is a nonparallel H-dimer. In contrast to the absorption spectra, the steady-state fluorescence spectra do not show any noticeable concentration dependence. The fluorescence lifetime was determined as 0.73 ns regardless of the concentration, and the fluorescence of R800 in water was solely attributed to the monomer. In femtosecond time-resolved absorption measurements, we observed the S(n) <-- S1 absorption bands of the monomer and the dimer, as well as the ground-state bleaching signals. It was found that the S1 dimer dissociates to produce the S1 monomer (and the S0 monomer) or relaxes to the S0 dimer with a time constant of as short as 3.0 ps, which brings about the absence of dimer fluorescence.

Structural Changes during the Photocycle of Photoactive Yellow Protein Monitored by Ultraviolet Resonance Raman Spectra of Tyrosine and Tryptophan

Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50-->Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.

Sub-μ-second Time-Resolved Absorption Spectroscopy of a Polar Carotenoid Analogue, 2-(All-trans-retinylidene)indan-1,3-dione; Formation of the Dication by Direct Triplet-Excited Sensitization

Sub-micro-second time-resolved difference absorption spectra of a polar carotenoid analogue, 2-(all-trans-retinylidene)indan-1,3-dione (hereafter, we will call RetInd), were recorded in tetrahydrofuran at room temperature upon anthracene-sensitized triplet excitation. In addition to the typical Tn <-- T1 absorption spectrum of anthracene followed by that of RetInd, a novel transient species, which peaked at 670 nm, was detected. The lifetime and the population of the 670 nm species was not affected by the presence of oxygen but was quenched by the cation scavenger, triethylamine. Therefore, we have identified this species as a "cation". The transient 670 nm species was not generated by direct photoexcitation of RetInd in the absence of a triplet sensitizer. Therefore, this species was not generated via the T1 species of RetInd but rather via an "invisible state" of RetInd, which is generated by direct energy or electron transfer from T1 anthracene. This proposed pathway was confirmed by a singular-value decomposition followed by a global fitting analysis. The "cation" of RetInd shows vibrational structure in its absorption spectrum, and its lifetime was determined to be 15 micros. Chemical oxidation of RetInd in 2,2,2-trifluoroethanol (dichloromethane) produced a broad absorption band around 880 (1013) nm, which could be transformed into a shoulder around 640 (675) nm upon addition of increasing amounts of the oxidant, FeCl3. The former absorption band can be assigned to a radical cation, while the latter to a dication. Because of the spectral similarity, the 670 nm species can be assigned to the dication, and the "invisible state" is ascribed to the radical cation of RetInd. This is the first direct evidence for the production of a dication of a biological polyene moiety generated in non-halogenated solution following anthracene-sensitized excitation.

Global fit analysis including the ν9 + ν4 − ν4 hot band of CD 3CD 3

A previous frequency analysis including data from the torsional ( ν 4) stack of the ground vibrational state as well as the lowest-frequency perpendicular fundamental ν 9 of CD 3CD 3 has been extended to include new data for the weaker but infrared-active ν 9 + ν 4 − ν 4 hot band. Analysis is complicated by the small torsional splitting due to barrier height in both the ν 9 and hot band data. The combined data set includes more than 2600 frequencies, and was fitted to within experimental accuracy using a 34-parameter model Hamiltonian. Remarkably, no resonances were observed between the upper state and the bath states, and no new intervibrational interactions were required in analyzing the hot band data. The barrier dependence of several purely vibrational terms, however, was found to be important. This represents the first reported global fit analysis including an associated hot band of a degenerate vibrational fundamental for any ethane-like molecule.

Controlling Association of Vesicle Embedded Peptides by Alteration of the Physical State of the Lipid Matrix

We report here the reversible association of a designed peptide embedded in a lipid membrane through a stimulus-sensitive trigger that changes the physical state of the bilayer matrix. A peptide designed with the classical 4-3 heptad repeat of coiled coils, equipped with leucine residues at all canonical interface positions, TH1, was rendered membrane soluble by replacement of all exterior residues with randomly selected hydrophobic amino acids. Insertion of TH1 into large unilamellar phosphatidylcholine vesicles was followed by monitoring tryptophan fluorescence. Peptide insertion was observed when the lipids were in the liquid-crystalline state [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] but not when they were in the crystalline phase [1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)]. Formation of a trimeric alpha-helical bundle in lipid bilayers was followed by fluorescence resonance energy transfer. Global fit analysis revealed a monomer--trimer equilibrium with a dissociation constant of around 10(-5) [corrected] MF(2). A lipid mixture composed of DPPC and POPC exhibiting a phase transition at 34 degrees C between a crystalline/liquid-crystalline coexistence region and a completely miscible liquid-crystalline phase was used to control the formation of the trimeric peptide bundle. TH1 is phase excluded in crystalline DPPC domains below 34 degrees C, leading to a larger number of trimers. However, when the DPPC domains are dispersed at temperatures above 34 degrees C, the number of trimers is reduced.

Cytochrome b6 Arginine 214 of Synechococcus sp. PCC 7002, a Key Residue for Quinone-reductase Site Function and Turnover of the Cytochrome bf Complex

Quinone-reductase (Q(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q(i) site mechanism remains largely enigmatic. To investigate the bf Q(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew approximately 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q(i) site, mimicking the effect of the Q(i) site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b(6) oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b(6) oxidation at the Q(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b(6) Arg(214) as a key residue for Q(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg(214) lies near the Q(i) pocket and the newly discovered c(i) or x heme. We propose a model for Q(i) site function and a role for Arg(214) in plastoquinone binding.

Spectroscopic Properties of the Carotenoid 3‘-Hydroxyechinenone in the Orange Carotenoid Protein from the CyanobacteriumArthrospira maxima

The cyanobacterial water-soluble orange carotenoid binding protein (OCP) is an ideal system for study of the effects of protein environment on photophysical properties of carotenoids. It contains a single pigment, the carotenoid 3'-hydoxyechinenone (hECN). In this study, we focus on spectroscopic properties of hECN in solution and in the OCP, aiming to elucidate the spectroscopic effects of the carotenoid-protein interaction in the context of the function(s) of the OCP. The noncovalent binding of hECN to the OCP causes a conformational change in the hECN, leading to a prolongation of the effective conjugation length. This change is responsible for shortening of the S(1) lifetime from 6.5 ps in solution to 3.3 ps in the OCP. The conformational change and the hydrogen bonding via the carbonyl group of hECN result in stabilization of an intramolecular charge-transfer (ICT) state. No signs of the ICT state were found in hECN in solution, regardless of the solvent polarity; spectral bands in transient absorption spectra of OCP-bound hECN exhibit features typical for the ICT state. Application of global fitting analysis revealed further effects of binding hECN in the OCP. The S(1) state of hECN in the OCP decays with two time constants of 0.9 and 3.3 ps. Modeling of the excited-state processes suggests that these two components are due to two populations of hECN in the OCP that differ in the hydrogen bonding via the carbonyl group. These results support the hypothesis that the OCP functions as a photoprotective shield under excess light. Mechanistically, the broadening of the hECN absorption spectrum upon binding to OCP enhances filtering effect of hECN. Furthermore, the binding-induced conformational change and activation of the ICT state that leads to a shortening of hECN lifetime effectively makes the protein-bound hECN a more effective energy dissipator.

Arabidopsis thaliana Glutamate-Cysteine Ligase: FUNCTIONAL PROPERTIES, KINETIC MECHANISM, AND REGULATION OF ACTIVITY

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of gamma-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki approximately 1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress.

The effect of calcination on photocatalytic activity of TiO 2 particles: femtosecond study

The transient absorption properties of TiO 2 nanocrystalline anatase powder samples calcinated at different temperatures were studied by femtosecond diffuse reflectance spectroscopy. The measurements were performed in TiO 2 aqueous suspensions in the presence and absence of thiocyanate ions, SCN −. Transient absorption decay behaviour of the TiO 2 particles was independent of calcination temperature. Both the shapes of the transient absorption spectra and the decay kinetics were practically the same for all the samples. The multiexponential global fitting analysis applied to the transient absorption data of the TiO 2/SCN − system revealed the spectra and the lifetimes of the transient species. After the hole transfer from TiO 2 to SCN − the consecutive formation of a weekly coupled dimeric thiocyanate radical anion, (SCN⋯SCN) −, and its structural stabilization to (SCN) 2 − were observed with time constants of a few picoseconds and few tens to hundreds of picoseconds, respectively. The time constants were dependent on the calcination temperature of the TiO 2 particles being the faster the lower was the calcination temperature. Also the relative amounts of the formed (SCN⋯SCN) − and (SCN) 2 − were dependent on the calcination temperature of the TiO 2 samples. The extent of the initial fast hole transfer reaction correlated with the calcination temperature being the more intensive the lower was the calcination temperature. On the other hand, relatively more (SCN) 2 − were formed at longer timescales the higher was the calcination temperature.

High‐ResolutionChandraSpectroscopy of γ Cassiopeiae (B0.5e)

gamma Cas has long been famous for its unique hard X-ray characteristics. We report herein on a 53 ks Chandra HETGS observation of this target. An inspection of our spectrum shows that it is quite atypical for a massive star, with abnormally weak Fe XXV, XXVI lines, Ly-alpha lines of H-like species from Fe XVII, XXIII, XXIV, S XVI, Si XIV, Mg XII, Ne X, O VII, VIII, and N VII. Also, line ratios of the rif-triplet of for a few He-like ions XVII are consistent with the dominance of collisional atomic processes. Yet, the presence of Fe and Si fluorescence K features indicates that photoionization also occurs in nearby cold gas. The line profiles indicate a mean velocity at rest and a broadening of 500 km/s. A global fitting analysis of the line and continuum spectrum finds that there are 3-4 plasma emission components. The dominant hot (12 keV) component and has a Fe abundance of 0.22 solar. Some fraction of this component (10-30%) is heavily absorbed. The other 2-3 components, with temperatures 0.1, 0.4, 3 keV, are warm, have a nearly solar composition, a lower column absorption, and are responsible for most other emission lines. The strength of the fluorescence features and the dual-column absorption model for the hot plasma component suggest the presence near the hot sites of a cold gas structure with a column density of 10^23 cm^-2. Since this value is consistent with theoretical estimates of the vertical disk column of this star, these attributes suggest that the X-rays originate near the star or disk. It is possible that the Fe anomaly in the hot component is related to the First Ionization Potential effect found in coronal structures around active cool stars. This would be yet another indication that the X-rays -rays are produced in the immediate vicinity of the Be star.

Zur Fernerkundung der Erdatmosphäre mittels Infrarotspektrometrie: Rekonstruktionstheorie und Anwendung

Remote sensing retrieval theory covers the methodology used to infer information on unknown parameters of the object under investigation from indirect remote measurements. Application of infrared spectrometry of the atmosphere leads to the inverse solution of the atmospheric radiative transfer equation. Its successful inversion depends on (1) a suitable forward model for simulation of atmospheric radiative transfer (2) the appropiate definition of the retrieval parameter vector, (3) an optimized vector containing the measurements, (4) the appropriate formulation of a constraint, and (5) a tool for stable numeric inversion. Experiments with limb emission spectrometers as well as uplooking emission and absorption spectrometers serve as examples for discussion of application of relevant retrieval schemes. With respect to retrieval theory, the following progress is reported and the following findings were gained: An optimized radiative transfer model for application within a retrieval program was developed. This model is both accurate and efficient. It includes all radiative processes relevant to application to atmospheric infrared spectrometry, including refraction, absorption, emission, scattering, line-coupling and non-local thermodynamic equilibrium. Objective selection of spectral gridpoints used for data analysis reduces the retrieval error and reduces computer resources needed. The more spectral data are considered, the more information is used for the retrieval of the target parameter. This is counterbalanced by the fact that radiance at the considered gridpoints depend on further parameters which may be uncertain and thus contribute to the error budget. Tentative retrieval of all these parameters from the measurement leads to an impracticable large number of unknowns. Therefore, an objective method for an optimized selection of spectral data was developed. It was shown that for limb measurements of medium spectral resolution the unconstrained solution of the inverse problem is instable if the vertical profile of the target parameter is represented on a discrete level model instead of a discrete layer model. Regularization is needed for level models, while retrieval of parameters represented by layers typically are stable. Solvability of ill-posed inversion problems in the context of remote sensing retrieval problems by implicit or explicit regularization was demonstrated. For explicit regularization in particular a smoothing constraint was investigated and considered useful. Implicit regularization we understand is the reduction of the size of the parameter vector by appropriate discrete representation of vertical profiles of atmospheric state parameters. It was shown that the global fit analysis of a limb sequence of spectra is not at all always superior over sequential onion peeling analysis, as commonly believed. In particular in the case of medium spectral resolution measurements the advantage of gobal fit with respect to accuracy disappears and the efficiency of onion peeling becomes significant. Methods developed were applied to the following experiments: The MIPAS (Michelson Interferometer for Passive Atmospheric Sounding) balloon-borne instrument; these were the first high spectral resolution limb emission measurements in a winter polar vortex. Vertical profiles of some trace gases relevant to ozone research were retrieved under polar night conditions for the first time. The MIPAS aircraft instrument; the MIPAS-FT (Aircraft Transall) instrument recorded the first scientifically useful high resolution spectroscopic measurements under positive elevation angles. These allowed retrieval of zenith column amounts of atmospheric constituents sampled along the flight trajectory independent of solar illumination.

Axial ligand binding and release processes in nickel(II)-tetraphenylporphyrin revisited: a resonance Raman study

Processes of axial ligand(s) binding and release for nickel meso-tetraphenylporphyrin (NiTPP) dissolved in mixed benzene/piperidine solvents have been quantitatively studied by resonance Raman spectroscopy in using both continuous-wave and pulsed low-power nanosecond excitations at 441.6 nm. Global-fit analysis of intensity changes in the Raman spectra has been performed, along with a mathematical modeling, by a rate equations approach, of the photoinduced ligand binding/release processes at the central Ni atom. The existence of a five-coordinate high-spin groundstate NiTPP(Pip) species has been confirmed, whose concentration amounts to not more than a few percent of the total porphyrin concentration at any benzene/piperidine ratio. We also found that this five-coordinate species does not substantially contribute to the photoinduced processes of axial ligand(s) binding and release. Four-coordinate NiTPP and six-coordinate NiTPPðPipÞ2 are the major species which can directly convert into each other. On photoexcitation of the NiTPPðPipÞ2 species, from one quarter to one half of bis-ligated molecules reversibly loose their axial ligands, while on NiTPP photoexcitation, less than 10% of non-ligated molecules reversibly attach axial ligands. � 2002 Published by Elsevier Science B.V.