3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

Voltage changes involving photosystem II quinone–iron complex turnover

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q_A⁻ and the oxidized non-heme iron Fe³⁺ on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Δψ (negative charging of the proteoliposome interior) indicates that the iron–quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (τ ∼ 0.1 μs) of Δψ generation related to electron transfer between the redox-active tyrosine Y_Z and Q_A, an additional phase in the submillisecond time domain (τ ∼ 0.1 ms at 23°C, pH 7.0) and relative amplitude ∼ 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q_A and the secondary quinone Q_B. The rate of the additional electrogenic phase is decreased by about one-half in the presence of D₂O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.     

The enthalpy changes upon hydrolysis of guanosine triphosphate anhydride and ester bonds

The effects of N,N′-dicyclohexylcarbodiimide (DCCD), triphenyltin chloride (TPT), and 3,5-di-tert-butyl-4-hydroxybenzylidenemalonomtrile (SP6847) were tested on the light-dependent activities of Halobacterium halobium R₁mR which contains a new retinal protein pigment designated as halorhodopsin but no bacteriorhodospin. DCCD inhibited ATP synthesis either in the light- or in the dark-aerobic conditions without affecting the light-induced proton uptake (ΔH⁺). Although DCCD lowered the membrane potential under dark-anaerobic conditions, the potential increased in the light as high as the control (the light-dependent membrane potential increment Δψ became apparently larger in the presence of DCCD). TPT had negligible effect on ATP synthesis both in the dark or in the light but inhibited markedly ΔH⁺ and partly Δψ. After R₁mR was treated with DCCD, TPT abolished ΔH⁺ almost completely but Δψ only partly. The remaining Δψ was collapsed by SF6847 with a concomitant proton incorporation (pH increase). These results led to the following postulations: (i) In R₁mR, ATP is synthesized by a H⁺-ATPase coupled either to respiration and/or light energization by halorhodopsin; (ii) the majority of protons are incorporated in the light by a mechanism which differs from H⁺-ATPase but is driven by the Δψ generated by halorhodopsin; (iii) TPT acts in this system as a chloride/hydroxide exchanger; (iv) the uncoupler SF6847 carries protons into cells in response to Δψ.     

Model for the production of L-tryptophan from L-serine and indole by immobilized cells in a three-phase liquid-impelled loop reactor

Production of L-tryptophan from L-serine and indole catalyzed by Escherichia coli, immobilized in k-carrageenan gel beads, is technically feasible in the liquidimpelled loop reactor (LLR), using an organic solvent, e.g. n-dodecane.With L-serine in large excess intrinsic reaction kinetics is approximately first order with respect to indole, with a reaction constant of 8.5×10^–5 m³ kg _dw ^–1 s^–1.The overall process kinetics is jointly controlled by intrinsic kinetics and by intraparticle mass transfer resistance, which can be quantified using an effectiveness factor.Mass transfer of indole from the organic to the aqueous phase and from the aqueous to the gel phase are relatively fast and thus have negligible influence in the overall process kinetics, under the operational conditions tested. However, they may become important if the process is intensified by increasing the cell concentration in the gel and/or the gel hold-up in the reactor.A simple model which includes indole mass balances over the aqueous and organic phases, mass transfer and reaction kinetics, with parameters experimentally determined in independent experiments, was successful in simulating L-tryptophan production in the LLR.List of Symbols a, b, c coefficients of the equilibrium curve for indole between organic and aqueous phases - A, B, C, D, E, F auxiliary variables used in liquid-liquid mass transfer studies - a _x specific interfacial area referred to the volume of the aqueous phase (m^–1) - A _x interfacial area (m²) - a _Y specific interfacial area referred to the volume of the organic phase (m^–1) - A _Y interfacial area (m²) - C _b substrate concentration in the bulk of the aqueous phase (kg m^–3) - C _e substrate concentration in exit stream (kg m^–3) - C _E biocatalyst concentration referred to the aqueous phase (kg m^–3) - C _{E s} biocatalyst concentration referred to the volume of gel (kg m^–3) - C _s substrate concentration at the gel surface (kgm^–3) - d, e, f coefficients of the equilibrium curve for indole between aqueous and organic phases - dp particle diameter (m) - K ₂ kinetic constant (s^–1) - K ₁ kinetic constant K₂/K_M (kg^–1 m³ s^–1) - K _M Michaälis-Menten constant (kgm^–3) - K _X mass transfer coefficient referred to the aqueous phase (ms^–1) - K _Xa_X volumetric mass transfer coefficient based on the volume of the aqueous phase (s^–1) - k _Y mass transfer coefficient referred to the organic phase (ms^–1) - K _Ya_Y volumetric mass transfer coefficient based on the volume of the organic phase (s^–1) - N _X mass flux of indole from organic to aqueous Phase (kg m^–2s^–1) - N _Y mass flux of indole from aqueous to organic phase (kg m^–2s^–1) - Q _e volumetric flow rate in exit stream (m³s^–1) - Q _f volumetric flow rate in feed stream (m³s^–1) - _obs observed reaction rate (kg s^–1 m^–3) - intrinsic reaction rate (kg s^–1 m^–3) - Re Reynolds number - Sc Schmidt number - Sh Sherwood number - t time (s) - u superficial velocity (m s^–1) - V _max maximum reaction rate (kg s^–1m^–3) - V _S volume of the support (m³) - V _X volume of aqueous phase (m³) - V _Y volume of the organic phase (m³) - X indole concentration in the aqueous phase (kgm^–3) - Y indole concentration in the organic phase (kg m^–3 Greek Letters overall effectiveness factor - _e external effectiveness factor - _i internal effectiveness factor - Thiele module A fellowship awarded to one of us (D.M.R.)by INICT is gratefuly acknowledged.     

Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude–Nernst equation

Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1–10 μs time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (–26 A³) and reaction enthalpy (–0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, –1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of ∼4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe₄S₄)(Cys₄) with charge change from –1 to –2. The small volume change in PS II is explained by sub-μs electron transfer from Y_Z anion to P₆₈₀ cation, in which charge is only moved from the Y_Z anion to the Q_A with no charge separation or with rapid proton transfer from oxidized Y_Z to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P₆₈₀ cation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Models for Proton-Coupled Electron Transfer in Photosystem II

The coupling of proton and electron transfers is a key part of the chemistry of photosynthesis. The oxidative side of photosystem II (PS II) in particular seems to involve a number of proton-coupled electron transfer (PCET) steps in the S-state transitions. This mini-review presents an overview of recent studies of PCET model systems in the authors’ laboratory. PCET is defined as a chemical reaction involving concerted transfer of one electron and one proton. These are thus distinguished from stepwise pathways involving initial electron transfer (ET) or initial proton transfer (PT). Hydrogen atom transfer (HAT) reactions are one class of PCET, in which H⁺ and e ⁻ are transferred from one reagent to another: AH+B→A+BH, roughly along the same path. Rate constants for many HAT reactions are found to be well predicted by the thermochemistry of hydrogen transfer and by Marcus Theory. This includes organic HAT reactions and reactions of iron-tris(α-diimine) and manganese-(μ-oxo) complexes. In PS II, HAT has been proposed as the mechanism by which the tyrosine Z radical (Y_Z) oxidizes the manganese cluster (the oxygen evolving complex, OEC). Another class of PCET reactions involves transfer of H⁺ and e ⁻ in different directions, for instance when the proton and electron acceptors are different reagents, as in AH–B+C⁺→A–HB⁺+C. The oxidation of Y_Z by the chlorophyll P680 + has been suggested to occur by this mechanism. Models for this process – the oxidation of phenols with a pendent base – are described. The oxidation of the OEC by Y_Z could also occur by this second class of PCET reactions, involving an Mn–O–H fragment of the OEC. Initial attempts to model such a process using ruthenium-aquo complexes are described. An erratum to this article can be found at     

N2O, CH4 and CO2 emissions from seasonal tropical rainforests and a rubber plantation in Southwest China

The main focus of this study was to evaluate the effects of soil moisture and temperature on temporal variation of N₂O, CO₂ and CH₄ soil-atmosphere exchange at a primary seasonal tropical rainforest (PF) site in Southwest China and to compare these fluxes with fluxes from a secondary forest (SF) and a rubber plantation (RP) site. Agroforestry systems, such as rubber plantations, are increasingly replacing primary and secondary forest systems in tropical Southwest China and thus effect the N₂O emission in these regions on a landscape level. The mean N₂O emission at site PF was 6.0 ± 0.1 SE μg N m⁻² h⁻¹. Fluxes of N₂O increased from <5 μg N m⁻² h⁻¹ during dry season conditions to up to 24.5 μg N m⁻² h⁻¹ with re-wetting of the soil by the onset of first rainfall events. Comparable fluxes of N₂O were measured in the SF and RP sites, where mean N₂O emissions were 7.3 ± 0.7 SE μg N m⁻² h⁻¹ and 4.1 ± 0.5 SE μg N m⁻² h⁻¹, respectively. The dependency of N₂O fluxes on soil moisture levels was demonstrated in a watering experiment, however, artificial rainfall only influenced the timing of N₂O emission peaks, not the total amount of N₂O emitted. For all sites, significant positive correlations existed between N₂O emissions and both soil moisture and soil temperature. Mean CH₄ uptake rates were highest at the PF site (−29.5 ± 0.3 SE μg C m⁻² h⁻¹), slightly lower at the SF site (−25.6 ± 1.3 SE μg C m⁻² h⁻¹) and lowest for the RP site (−5.7 ± 0.5 SE μg C m⁻² h⁻¹). At all sites, CH₄ uptake rates were negatively correlated with soil moisture, which was also reflected in the lower uptake rates measured in the watering experiment. In contrast to N₂O emissions, CH₄ uptake did not significantly correlate with soil temperature at the SF and RP sites, and only weakly correlated at the PF site. Over the 2 month measurement period, CO₂ emissions at the PF site increased significantly from 50 mg C m⁻² h⁻¹ up to 100 mg C m⁻² h⁻¹ (mean value 68.8 ± 0.8 SE mg C m⁻² h⁻¹), whereas CO₂ emissions at the SF and RP site where quite stable and varied only slightly around mean values of 38.0 ± 1.8 SE mg C m⁻² h⁻¹ (SF) and 34.9 ± 1.1 SE mg C m⁻² h⁻¹ (RP). A dependency of soil CO₂ emissions on changes in soil water content could be demonstrated for all sites, thus, the watering experiment revealed significantly higher CO₂ emissions as compared to control chambers. Correlation of CO₂ emissions with soil temperature was significant at the PF site, but weak at the SF and not evident at the RP site. Even though we demonstrated that N and C trace gas fluxes significantly varied on subdaily and daily scales, weekly measurements would be sufficient if only the sink/ source strength of non-managed tropical forest sites needs to be identified.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Fed-batch fermentation of a methanol utilization plus (Mut⁺) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut⁺ Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (ν _gly, ν _MeOH) were established. A mixed feeding strategy with desired μ _gly/μ _MeOH =1, 2, 3, 4 (desired μ _MeOH set at 0.015 h⁻¹) was employed to study growth interactions and their effect on production. The results show that the optimal desired μ _gly/μ _MeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells. Electronic Publication     

Prototropic tautomerism of imidazolone in aqueous solution: a density functional approach using the combined discrete/self-consistent reaction field (SCRF) models

A systematic investigation of the proton transfer in the keto-amino/enol tautomerization of imidazolone was undertaken. Calculations in aqueous solution were performed using both combined discrete/self-consistent reaction field (SCRF) and SCRF methods. Complexes containing one to three water molecules around the hydrophilic site of imidazolone were used for the combined discrete/SCRF calculations. The DFT results predict that the barrier height for non-water-assisted intramolecular proton transfer is very high (214.8 kJmol^–1). Hydrogen bonding between imidazolone and the water molecule(s) will dramatically lower the barrier by a concerted multiple proton transfer mechanism. The proton transfer process through a eight-member ring formed by imidazolone and two water molecules is found to be more efficient and the calculated barrier height is ca. 61 kJmol^–1.Figure DFT calculations in aqueous solution predict the H-bonding between imidazolone(IZ) and the water molecule(s) will dramatically lower the tautomeric barrier by a concerted multiple proton transfer mechanism, in which an eight-member ring structure formed by IZ and 2H₂O is found to be more efficient and the barrier is 60.8 kJ mol^–1, much less than 214.8 kJ mol^–1 in the non-water-assisted mechanism.     

Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m⁻²) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA₃₂₅(t), as an indicator of redox changes in Q_A, and of the relative fluorescence quantum yield, F(t)/F_o, in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA₃₂₅, remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA₃₂₅ dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl₂, c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680^+·Q_A ^−● is almost invariant to this UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modulation of I A Potassium Current in Adrenal Cortical Cells by a Series of Ten Lanthanide Elements

The modulation of I _A K⁺ current by ten trivalent lanthanide (Ln³⁺) cations spanning the series with ionic radii ranging from 0.99 ? to 1.14 ? was characterized by the whole-cell patch clamp technique in bovine adrenal zona fasciculata (AZF) cells. Each of the ten Ln³⁺s reduced I _A amplitude measured at +20 mV in a concentration-dependent manner. Smaller Ln³⁺s were the most potent and half-maximally effective concentrations (EC₅₀s) varied inversely with ionic radius for the larger elements. Estimation of EC₅₀s yielded the following potency sequence: Lu³⁺ (EC₅₀= 3.0 μm) ≈ Yb³⁺ (EC₅₀= 2.7 μm) > Er³⁺ (EC₅₀= 3.7 μm) ≥ Dy³⁺ (EC₅₀= 4.7 μm) > Gd³⁺ (EC₅₀= 6.7 μm) ≈ Sm³⁺ (EC₅₀= 6.9 μm) > Nd³⁺ (EC₅₀= 11.2 μm) > Pr³⁺ (EC₅₀= 22.3 μm) > Ce³⁺ (EC₅₀= 28.0 μm) > La³⁺ (EC₅₀= 33.7 μm). Ln³⁺s altered selected voltage-dependent gating and kinetic parameters of I _A with a potency and order of effectiveness that paralleled the reduction of I _A amplitude. Ln³⁺s markedly slowed activation kinetics and shifted the voltage-dependence of I _A gating such that activation and steady-state inactivation occurred at more depolarized potentials. In contrast, Ln³⁺s did not measurably alter inactivation or deactivation kinetics and only slightly slowed kinetics of inactivated channels returning to the closed state. Replacement of external Ca²⁺ with Mg²⁺ had no effect on the concentration-dependent inhibition of I _A by Ln³⁺s. In contrast to their action on I _A K⁺ current, Ln³⁺s inhibited T-type Ca²⁺ currents in AZF cells without slowing activation kinetics. These results indicate that Ln³⁺ modulate I _A K⁺ channels through binding to a site on I _A channels located within the electric field but which is not specific for Ca²⁺. They are consistent with a model where Ln³⁺ binding to negative charges on the gating apparatus alters the voltage-dependence and kinetics of channel opening. Ln³⁺s modulate transient K⁺ and Ca²⁺ currents by two fundamentally different mechanisms. Received: 21 January 1997/Revised: 3 April 1998     

Mechanism of dithionite reduction of the R2 protein of E. coli and mouse ribonucleotide reductase: evidence for reduction of FeIII 2 prior to the tyrosyl radical

 Dithionite has been found to reduce directly (without mediators) the Escherichia coli R2 subunit of ribonucleotide reductase. With dithionite (∼10 mM) in large excess, the reaction at 25  °C is complete in ∼10 h. Preparations of E. coli R2 have an Fe^III ₂ (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme Fe^III ₂ . . . Tyr*, which has a tyrosyl radical at Tyr-122. In the pH range studied (7–8) the kinetics are biphasic. Rate laws for both phases give [S₂O₄ ^2–] and not [S₂O₄ ^2–]^1/2 dependencies, and saturation kinetics are observed for the first time in R2 studies. No dependence on pH was detected. The kinetics (25  °C) of the first phase are reproduced in separate experiments using only met-R2, with association of S₂O₄ ^2– to met-R2, K=330 M^–1, occurring prior to electron transfer, k _et=4.8×10^–4 s^–1, I=0.100 M (NaCl). The second phase assigned to the reaction of Fe^III ₂ . . . Tyr* with S₂O₄ ^2– gives K=800 M^–1 and k _et=5.6×10^–5 s^–1. Bearing in mind the substantially smaller reduction potential for Fe^III ₂ compared to Tyr*, this is a quite remarkable finding, with implications similar to those already reported for the reaction of R2 with hydrazine, but with additional information provided by the saturation kinetics. The similarity in rates for the two phases (∼fourfold difference) suggests that reduction of Fe^III ₂ is occurring in both cases, and since S₂O₄ ^2– is involved a two-equivalent change is proposed with the formation of Fe^II ₂ . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising Tyr* by the Fe^II ₂ is rapid, and further decay of Fe^IIFe^III is also fast. There is no stable mouse met-R2 form, and the single-phase reaction with dithionite gives saturation kinetics with K=208 M^–1 and k _et=1.7±10^–3 s^–1. Mechanistic implications, including the applicability of a pathway for electron transfer via Fe_A, are considered. Received: 25 February 1998 / Received: 20 August 1998     

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B_A⁻ and β⁻, where β is a bacteriochlorophyll substituting the native bacteriopheophytin H_A) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin H_B in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm⁻¹. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or B_A with accumulation of the P⁺β⁻ and/or P⁺B_A⁻ states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼150 cm⁻¹ and distinct low-frequency oscillations at 20–100 cm⁻¹; also, the amplitude of the oscillations at 150 cm⁻¹ is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm⁻¹ mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P⁺H_B⁻ state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.     

Monitoring of <Emphasis Type="Italic">E. coli</Emphasis> immobilization on modified gold electrode: A new bacteria-based glucose sensor

Electrochemical impedance spectroscopy (EIS) technique has proved to be an effective method for monitoring the immobilization of various bioactive species such as enzymes, DNA, whole cells, and so forth. In this work we describe the development of an electrochemical whole cell based biosensor. Biotinylated fluorescent E. coli are immobilized onto a cysteamine, Sulfo-NHS-LC-biotin, and avidin modified gold electrodes. Immobilized bacteria are clearly observed using confocal microscopy. Electrochemical measurements are based on the charge-transfer kinetics of [Fe (CN)₆]^3−/4− redox couple. The experimental impedance data were modelised with a computer. SAM assembly and the subsequent immobilization of bacteria on the gold bare electrodes greatly increased the electrontransfer resistance (R _et ) and reduced the constant phase element (CPE). It’s interesting to note, the hard immobilization of bacteria on the surface of electrode and do not remove during measurements. The effect of glucose addition was studied in the range of 10⁻⁷ μM to 10 μM. The relation between the evolution of R _et and D-glucose concentration was found to be linear for values ranging from 10⁻⁵ μM to 10⁻¹ μM and reached saturation for higher concentrations. Such biosensor could be applied to a more fundamental study of cell metabolism and drugs effect.     

The proton cycle of a deciduous forest ecosystem in the Netherlands and its implications for soil acidification

Investigations into the proton cycle of a forest ecosystem in the Netherlands revealed an intermediate rate of soil acidification: 4.5 × 10² keq km^-2 yr^-1 of which 2/3 is caused by external proton sources. The high retention of NH₄-N in the biomass is the dominant source of protons. This retention of accounts for 90% of the external and for 59% of the total proton source, while atmospheric input of free acidity only accounts for 4% of total proton production. Next to this, Ca release by weathering is the main proton sink, accounting for 72% to total proton consumption. The proton transfer processes have caused very acid conditions of the upper soil horizons (pH 2.9–3.5) which resulted in the mobilization of aluminium as inorganic monomeric (toxic) Al up to maximum concentrations of 1500 μmol L^-1 (40 mg Al³⁺ L^-1).     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Kinetic spectrophotometric method for the determination of ramipril in pharmaceutical formulations

The objective of this research was to develop a kinetic spectrophotometric method for determination of ramipril in pure form and pharmaceutical formulations. The method was based on the reaction of carboxylic acid group of the drug with a mixture of potassium iodate (KIO₃) and potassium iodide (KI) in aqueous medium at room temperature. The reaction is followed spectrophotometrically by measuring the increase in absorbance at 352 nm as a function of time. The initial-rate and fixed-time methods were adopted for constructing the calibration curves. Both the calibration curves were linear in the concentration range of 10.0–70.0 μg mL⁻¹. The detection limits were 0.02μg mL⁻¹ and 0.15-μg mL⁻¹ for initial rate and fixed time methods, respectively. The proposed methods are validated statistically and through recovery studies. The point and interval hypothesis tests have been performed confirming that there is no significant difference between the proposed methods and the reference method. The experimental true bias of all samples is less than ±2%. The methods have been successfully applied to the determination of ramipril in tablets and capsules. Published: October 27, 2005     

Kinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release

 The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn₇MT and Cd₇MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn₇MT and pyridylazoresorcinol for Cd₇MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for the two phases are independent of the ZI and Zn₇MT concentrations. The complex NEM concentration dependence of each phase, k _f, obs=k ^f ₁+k ^f ₂ [NEM] and k _s, obs=k ^s ₁+k ^s ₂ [NEM], demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylation can be reversed using 2-mercaptoethanol to compete for the protein-bound NEM and regenerate the Zn-binding capability of alkylated MT. An explanation of these observations, based on the reversibility of cysteine alkylation by NEM, was developed and tested. The reactions of Cd₇MT are less complete than those of Zn₇MT and occur more slowly. ¹¹¹Cd-NMR studies of the partially alkylated ¹¹¹Cd₇MT reveal that reaction with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral signatures of the α and β clusters, although very little cadmium has been removed from the protein. This finding substantiates the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of the metal ions, and rules out the possibility of cooperative reactions at either cluster. Received: 5 August 1996 / Accepted: 24 October 1996     

Transformation of indole and quinoline by Desulfobacterium indolicum (DSM 3383)

Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the 2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The kinetic parameters for indole were an apparent maximum specific transformation rate (V _Amax) of 263 μmol mg total protein⁻¹ day⁻¹ and an apparent half-saturation constant (K _Am) of 139 μM. The V _Amax for quinoline was 170 μmol mg total protein⁻¹ day⁻¹ and K _Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline. Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q_A⁻Q_B→Q_AQ_B⁻ (k_AB⁽¹⁾) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k_AB⁽¹⁾, attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q_B [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (−), 1601 (−) and 1467 (+) cm⁻¹, characteristic of Q_A reduction. The time evolution of the spectra shows reoxidation of Q_A⁻ and concomitant reduction of Q_B with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q_B⁻ occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm⁻¹ [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q_B in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q_A⁻ to Q_B and the identification of Glu-L212 as the main proton acceptor in the state Q_AQ_B⁻.