Separation Of Pathways Research Articles

High Yield of Long-Lived B-Side Charge Separation at Room Temperature in Mutant Bacterial Reaction Centers

The photosynthetic reaction center of Rhodobacter (Rb.) sphaeroides is a quasi-symmetric pigment protein complex. This near symmetry results in two potential pathways for light-induced electron transfer, labeled A and B, yet under most conditions charge separation occurs almost exclusively along the A-side cofactors in wild-type reaction centers. One exception is that upon excitation with blue light (390 nm) a transient B-side charge-separated state, BB+HB- (the cation of the B-side monomer bacteriochlorophyll and the anion of the B-side bacteriopheophytin), is formed that decays in picoseconds at room temperature but is stable at cryogenic temperatures. To characterize the nature of this reaction further, a series of mutations involving the introduction of potentially negative amino acids in the vicinity of P and the monomer bacteriochlorophylls, BA and BB, were used to alter the local electrostatic environment. The most dramatic effects were observed for reaction centers with the mutations L168 His to Glu and L170 Asn to Asp. These mutant reaction centers display a stable (hundreds of picoseconds) formation of BB+HB- not only using 390-nm excitation but also upon direct excitation of the lowest excited singlet states of the reaction center bacteriopheophytins and bacteriochlorophylls at 740 and 800 nm, respectively. Reaction centers mutated at three sitesL168 His to Glu, L170 Asn to Asp and M199 Asn to Aspwere also found to form a stable BB+HB- state following excitation at 390 nm but with an apparently lower yield. In the other mutant reaction centers studied, long-lived BB+HB- formation was not observed using any excitation wavelength, though 390-nm excitation resulted in BB+HB- formation followed by the decay of this state on the picosecond time scale, as is observed in wild type. Combining these results with past work demonstrates that there is a rich variety of photochemistry possible in the reaction center. Both the pathway and products of light-induced charge separation depend on the interplay between local electrostatic interactions and the nature of the excited states available at early times.

Equilibrium gradient methods with nonlinear field intensity gradient: a theoretical approach.

Equilibrium gradient methods belong to a family of separation techniques in which analytes are forced to unique equilibrium points by a force gradient and a counter force along the separation pathway. The basic theory for equilibrium gradient methods where the force gradient is induced by a field gradient is developed in this paper. The results indicate that peak capacity can be dynamically improved by using a nonlinear field-intensity gradient in which the first section is steep, and the following section is shallow. Using electromobility focusing (EMF) as an example, a separation model is established. EMF is an equilibrium gradient method that uses an electric field intensity gradient to induce a force gradient on charged analytes, such as proteins, and a constant hydrodynamic flow as an opposing force. Equations relating operating parameters with separation performance are given. Although simulation results show that a peak capacity of over 10,000 is theoretically possible using a single channel in a separation time just under 2 months, if 100 parallel separation units are utilized in an array format under the same operating conditions, the same peak capacity can be obtained in just over 12 h.

A two-dimensional study of the rupture of funicular liquid bridges

An analysis of the rupture behaviour of liquid bridges in simple granular systems is reported. Wet granules present a complex structure in which primary particles are held together at a microscopic scale by means of capillary forces mediated by a liquid binder. These capillary interactions control the mechanical properties of the particle assemblies as well as the kinetics and pathways of granule growth. The evaluation of the interaction between contacting particles by means of the interstitial liquid binder in its various possible configurations has recently been reported for two-dimensional systems of contacting particles (J. Colloid Interface Sci. 220 (1999) 42). In the current work particle separation forces and bridge rupture have been examined for the symmetric separation pathways in all possible wetted states of triplets of particles. The separation force and the energy of the system due to the liquid capillary action have been calculated during the process of separation, which is assumed to occur slowly through a sequence of equilibrium configurations. A wide range of behaviour is found for the rupture processes of the different liquid configurations.

Pathways of energy transformation in antenna reaction center complexes of Heliobacillus mobilis.

The conversion of excitation energy in the antenna reaction center complex of Heliobacillus mobilis was investigated at 10 K as well as at 275 K by means of time-resolved absorbance difference spectroscopy of isolated membranes in the (sub)picosecond time range. Selective excitation of the primary electron acceptor, chlorophyll (Chl) a 670, and of the different spectral pools of bacteriochlorophyll (BChl) g (BChl g 778, BChl g 793, and BChl g 808) was applied. At 10 K, excitation at 770 or 793 nm resulted on the one hand in rapid energy transfer to BChl g 808 and on the other hand in fast charge separation from excited BChl g 793 ( approximately 1 ps). Once the excitations were on BChl g 808, the bleaching band shifted gradually to the red, from 806 to 813 nm, and charge separation from excited BChl g 808 occurred by a very slow process ( approximately 500 ps). The main purpose of our experiments was to answer the question whether an "alternative" pathway for charge separation exists upon excitation of Chl a 670. Our measurements showed that the amount of oxidized primary donor (P798(+)) relative to that of excited BChl g produced by excitation of Chl a 670 was considerably larger than upon direct excitation of BChl g. This indicates the existence of an alternative pathway for charge separation that does not involve excited antenna BChl g. This effect occurred at 10 K as well as at 275 K. The mechanism for this process is discussed in relation to different trapping models; it is concluded that charge separation occurs directly from excited Chl a 670.

Dynamics of energy conversion in reaction center core complexes of the green sulfur bacterium Prosthecochloris aestuarii at low temperature.

Excited-state and electron-transfer dynamics at cryogenic temperature in reaction center core (RCC) complexes of the photosynthetic green sulfur bacterium Prosthecochloris aestuarii were studied by means of time-resolved absorption spectroscopy, using selective excitaton of bacteriochlorophyll (BChl) a and of chlorophyll (Chl) a 670. The results indicate that the BChls a of the RCC complex form an excitonically coupled system. Relaxation of the excitation energy within the ensemble of BChl a molecules occurred within 2 ps. A time constant of about 25 ps was ascribed to charge separation. Absorption changes in the 670 nm region, where Chl a 670 absorbs, were fairly complicated. They showed various time constants and were dependent on the wavelength of excitation and they did not lead to a simple picture of the electron acceptor reaction. Energy transfer from Chl a 670 to BChl a occurred with a time constant of 1.5 ps. However, upon excitation of Chl a 670 the amount of oxidized primary electron donor, P840(+), formed relative to that of excited BChl a was considerably larger than upon direct excitation of BChl a. This indicates the existence of an alternative pathway for charge separation which does not involve excited BChl a.

Microstructure, deformation and cracking characteristics of thermal spray ferrous coatings

Abstract The microstructure and local mechanical characteristics of thermal spray ferrous coatings have been determined. The emphasis has been on coatings made by the high velocity oxyfuel (HVOF) process, especially the role of Al alloy additives. The oxide phase present in the material and preferred pathways for local cracking and separation have been determined. Thin intersplat oxide layers emerge as preferential sites. These oxides are amorphous and the cracks extend along the oxide/α–Fe interfaces with low local fracture toughness, in the range 0.2–1 MPa√m. These low toughness pathways govern coating deterioration.

Investigating a back door mechanism of actin phosphate release by steered molecular dynamics

In actin-based cell motility, phosphate (Pi) release after ATP hydrolysis is an essential biochemical process, but the actual pathway of Pi separation from actin is not well understood. We report a series of molecular dynamics simulations that induce the dissociation of Pi from actin. After cleavage from ATP, the singly protonated phosphate (HPO4(2-)) rotates about the ADP-associated Ca2+ ion, turning away from the negatively charged ADP towards the putative exit near His73. To reveal the microscopic processes underlying the release of Pi, adhesion forces were measured when pulling the substrate out of its binding pocket. The results suggest that the separation from the divalent cation is the rate-limiting step in Pi release. Protonation of HPO4(2-) to H2PO4- lowers the electrostatic barrier during Pi liberation from the ion. The simulations revealed a propensity of charged His73+ to form a salt bridge with HPO4(2-), but not with H2PO4-. His73 stabilizes HPO4(2-) and, thereby, inhibits rapid Pi release from actin. Arg177 remains attached to Pi along the putative back door pathway, suggesting a shuttle function that facilitates the transport of Pi to a binding site on the protein surface.

Investigating a back door mechanism of actin phosphate release by steered molecular dynamics

In actin-based cell motility, phosphate (Pi) release after ATP hydrolysis is an essential biochemical process, but the actual pathway of Pi separation from actin is not well understood. We report a series of molecular dynamics simulations that induce the dissociation of Pi from actin. After cleavage from ATP, the singly protonated phosphate (HPO42−) rotates about the ADP-associated Ca2+ ion, turning away from the negatively charged ADP towards the putative exit near His73. To reveal the microscopic processes underlying the release of Pi, adhesion forces were measured when pulling the substrate out of its binding pocket. The results suggest that the separation from the divalent cation is the rate-limiting step in Pi release. Protonation of HPO42− to H2PO4− lowers the electrostatic barrier during Pi liberation from the ion. The simulations revealed a propensity of charged His73+ to form a salt bridge with HPO42−, but not with H2PO4−. His73 stabilizes HPO42− and, thereby, inhibits rapid Pi release from actin. Arg177 remains attached to Pi along the putative back door pathway, suggesting a shuttle function that facilitates the transport of Pi to a binding site on the protein surface. Proteins 1999;35:262–273. © 1999 Wiley-Liss, Inc.

Copper(II) complexes of tetraaza macrocycles bearing pendant arms: syntheses, structures and properties

A new difunctionalized tetraaza macrocycle, 1,8-bis(2-hydroxyethyl)-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane, H2L2 has been synthesized in one step by the reaction of ethylene oxide and C-rac-5,5,7,12,12, 14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (L1). The crystal structure of [Cu(H2L2)][ClO4]2 1 shows the metal ion in tetragonally elongated octahedral geometry, co-ordinated by the oxygen atoms of the pendant arms in the trans-axial position. Copper(II) complexes were also synthesized and characterized from other 1,8-disubstituted macrocyclic ligands L3, L4, HL5, H2L6, L8 and H2L9. The molecular structure of [CuL3][ClO4]2 2 (R1 = R2 = CH2CH2CN) and [CuL5]ClO4, 4 (R1 = H, R2 = CH2CO2–) shows the macrocycle in trans-I configuration with square planar and distorted square pyramidal geometries respectively at the metal centers. The structures of complexes [CuL8][ClO4]2 6 [L8 = 1,8-bis(2-cyanoethyl)-4,7,11,14-tetramethyl-1,4,8,11-tetraazacyclotetradecane] and [Cu(H2L9)][ClO4]2 7 (corresponding CH2CO2H derivative) show the macrocyclic ligand in the trans-III configuration. Interestingly the two pendant acetic acid groups of H2L9 in 7 remain protonated and co-ordinate through the carbonyl oxygen atoms in trans-octahedral geometry. The cyclic voltammetric studies of the complexes revealed that the copper(I) oxidation state is highly stabilized by the ligands L3 and L8. The pH dependent co-ordination geometry changes of complexes 4, [CuL6] 5 (R1 = R2 = CH2CO2–) and 7 based on the changes in their electronic spectra are discussed. The kinetics of acid promoted dissociation reactions of complexes 2 and 6 has been studied in HCl–NaCl solutions (I = 5.0 M). The possible mechanisms of the reactions, the factors influencing the rate and the relative importance of the solvent separation and protonation pathways are discussed.

New Evidence Supporting the Interfacial Model of Electrically Conductive Polymer Composites. 2. Indication of Phase Separation during Ageing of Polyaniline-Poly(ethylene-co-vinylacetate) Composites

Phase separation in conductive polyaniline-poly(ethylene-co-vinyl acetate) (PANI-EVA) composite films, resulting in formation of PANI enriched (lower side) and PANI deficient (upper side) layers was found during storage of these composites. A distinct difference in the conductivity of the two sides of the films reaching a half order of magnitude was detected. It was shown that phase separation and conductive pathways formation resulted from flocculation of PANI particles during ageing of the films. Subsequently, the total electrical conductivity of the composites of lower PANI content (up to 2.5 wt. %) was substantially enhanced (ca. five orders of magnitude). The observed conductivity jump and phase separation phenomena have been discussed using mathematical terms. Moreover, for the first time a mathematical interpretation of the evolution in percolation behaviour of PANI-EVA composites over time is presented. Coefficients of correlation (rxy) between average conductivity, σ, (y) and PANI content (x) in the composites were calculated to be ca. 0.85 - 0.99. Besides, the shape of percolation curves (σ vs. PANI content) was found to change during storage of the films. The unstable conducting properties (both conductivity jump and phase separation phenomena) of PANI-EVA composites over time revealed their dissipative nature, and have to be regarded as evidence supporting the dynamic interfacial model of conductive polymer composites.

New Evidence Supporting the Interfacial Model of Electrically Conductive Polymer Composites. 2. Indication of Phase Separation during Ageing of Polyaniline-Poly(ethylene-co-vinylacetate) Composites

Phase separation in conductive polyaniline-poly(ethylene-co-vinyl acetate) (PANI-EVA) composite films, resulting in formation of PANI enriched (lower side) and PANI deficient (upper side) layers was found during storage of these composites. A distinct difference in the conductivity of the two sides of the films reaching a half order of magnitude was detected. It was shown that phase separation and conductive pathways formation resulted from flocculation of PANI particles during ageing of the films. Subsequently, the total electrical conductivity of the composites of lower PANI content (up to 2.5 wt. %) was substantially enhanced (ca. five orders of magnitude). The observed conductivity jump and phase separation phenomena have been discussed using mathematical terms. Moreover, for the first time a mathematical interpretation of the evolution in percolation behaviour of PANI-EVA composites over time is presented. Coefficients of correlation (rxy) between average conductivity, σ, (y) and PANI content (x) in the composites were calculated to be ca. 0.85 - 0.99. Besides, the shape of percolation curves (σ vs. PANI content) was found to change during storage of the films. The unstable conducting properties (both conductivity jump and phase separation phenomena) of PANI-EVA composites over time revealed their dissipative nature, and have to be regarded as evidence supporting the dynamic interfacial model of conductive polymer composites.

Fluorescence excitation spectra of membrane-bound photosynthetic reaction centers of Rhodobacter sphaeroides in which the tyrosine M210 residue is replaced by tryptophan: evidence for a new pathway of charge separation

The B A and H A absorption bands of membrane bound reaction centers of Rhodobacter sphaeroides, in which the tyrosine M210 residue has been replaced by tryptophan appear to be only weakly present in the flourescence excitation spectrum of P ∗ emission. In contrast, these contributions are clearly observable in the P +Q A − excitation spectrum. Upon reduction of Q A, the B A and H A bands in the excitation spectrum are largely recovered. These findings are discussed in terms of a model in which, following excitation of the B A or H A pigments a direct path exists for charge separation that does not involve the excited low-exciton state of P. Similar observations were made in two other mutants in which tyrosine M210 was replaced by leucine or phenylalanine.

Excitation Wavelength Dependence of Energy Transfer and Charge Separation in Reaction Centers from Rhodobacter sphaeroides: Evidence for Adiabatic Electron Transfer

Femtosecond transient absorption spectroscopy has been used to investigate the excitation wavelength dependence of energy transfer and initial charge separation processes in reaction centers of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides (R-26) at room temperature. The Q{sub Y} transition bands of the bacteriopheophytins (H), bacteriochlorophyll monomers (B), and special pair (P) were selectively excited with pulses of 150 fs duration and 5 nm spectral bandwidth. Absorbance changes were analyzed over the entire wavelength region from 700 to 1000 nm. From this analysis we concluded the following: (1) As seen by others, energy transfer between H, B, and P is extremely fast, occurring on the 100-300 fs time scale. (2) The spectral evolution of the system is excitation wavelength dependent for picoseconds after excitation, implying that vibrational relaxation is not complete on the time scale of either energy transfer or charge separation and suggesting that the pathway of charge separation may be excitation wavelength dependent. (3) The absorbance change spectra of the initial excited states of B and H are not consistent with intensity borrowing between these bands, reopening the question of what gives rise to the complex absorbance changes normally associated with the H{sub A}{sup -} state. (4) Themore » 10-20 ps component of the stimulated emission decay is excitation wavelength dependent and spectrally different from the dominant 2-3 ps decay of the stimulated emission. 67 refs., 8 figs.« less

The relationship of HsEg5 and the actin cytoskeleton to centrosome separation.

Although centrosome separation is essential to the formation of a bipolar spindle, it can proceed along several different pathways. This raises questions as to the similarity between the mechanism(s) underlying these various forms of separation. To address this question we reinvestigated centrosome separation in HeLa cells using a variety of techniques. We present a refined description of the two major pathways of centrosome separation found in HeLa cells and demonstrate that each of these pathways has its own timing, protein requirements, morphological characteristics, and relationship to spindle assembly. The first pathway, which occurs in prophase cells, is dependent on an intact actin cytoskeleton, and when this pathway is completed prior to nuclear envelope breakdown, the microtubules associated with this process do not become part of the spindle. Thus, centrosome separation and spindle pole organization can occur as two separate events. The second centrosome separation pathway is found in cells in which separation occurs concurrent with prometaphase. In this case, centrosome separation and the formation of the mitotic spindle are integrated together and an intact actin cytoskeleton is not required. The relationship between these multiple pathways of centrosome separation and the distribution of the human kinesin-like protein HsEg5 was also investigated. This protein was found associated with all centrosomal microtubules present during both prophase and prometaphase centrosome separation, as well as with prophase centrosomes displaying independent movement in Cytochalasin-D treated cells. In addition, we demonstrate that this protein is associated with post-mitotic centrosome movement which involves a single centrosome. Thus, HsEg5 is a feature of individual centrosome function and does not require anti-parallel microtubule arrays.

Codependency: a feminist perspective.

1. Our understanding of psychological life has been underdeveloped and distorted because explanations have been created by only one half of the human species. The current broad and encompassing disease definition of codependency may devalue some of women's greatest strengths. 2. The disease model of codependency, it may be argued, is rooted in extant, reductionist models that suggest a developmental pathway of separation and individuation leading to an autonomous and independent maturity. 3. The feminist model of Growth in Connection proposes that the flowering of the self occurs within the development and maintenance of relationships in which accurate and mutual empathy is both the goal and the motivation toward growth.

Charge separation (coulomb explosion) processes of doubly charged cations of sandwich compounds in the gas phase: evidence for the junction of two C 5H 5 units

The charge separation reactions (coulomb explosion) of the ferrocene, cobaltocene, and nickelocene dications, generated by electron impact ionization in the gas phase, are reported. These sandwich dications display a common behaviour in their unimolecular decomposition reactions, which are characterized by two distinct charge separation pathways. One corresponds to the reaction [MC 10H 10] 2+ → [MC 5H 5] + + C 5H 5 +, and the other to the generation of C 10H 10 + and M + (M  Fe, Co, Ni). From the translational energy releases, associated with the charge separation processes, the intercharge distances of the exploding dications in their transition structures have been calculated. The results demonstrate that, irrespective of the nature of the transition metal ion M, the interchange distance for the formation of [MC 5H 5] +/C 5H + 5 is 5.0 Å and 6.2 Å for the generation of C 10H + 10 and M +.

Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation by static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane.

The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.