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The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short-circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborought) and from Megasphaera elsdenii were applied. The observed pH values in reversed micelles were not dependent on the type of hydrogenase. Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sulfosuccinate types of reversed micelles were used in combination with viologens having distinguishable valencies. It was observed that, in the cationic-reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic-reversed micelles. Furthermore, the dissociation constant was independent of the concentration of viologen semiquinone in the reversed micelle, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a · pHrm + b, between the pH of the bulk water and the pH as measured in the revered micelle (pHrm). In all these cases the value of α is smaller than unity, the value of b ranges between 1.6–2.7. For DAP the pHrm is always around 7. In DAP-reversed micelles, the counter-ion propionate probably serves as an internal buffer. Using cytochrome c3 as pH indicator in combination with N,N′-di(3-aminopropyl)-4,4′-bipyridinium)4+ to take care of electron transfer, in cetyltrimethylammonium-bromide-reversed micelles the pHrm is about the same as indicated by the viologen; in SDS-reversed micelles the pHrm is always lower than that indicated by N,N′-di(3-aminopropyl)-4,4′-pyridinium4+. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. vulgaris hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly. [ABSTRACT FROM AUTHOR]

1. Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c. After the pulse. methyl viologen radicals are formed and the kinetics of these radicals with cytochrome C3 are studied. The reaction between cytochrome C3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome C3 molecule was Z = ± 4.7 ± 0.7. 2. After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome C3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, α With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0 - - 0.250-0.088α (in V). 3. An estimation is made of the energy of interaction between the haems due to electrostatic interactions (Δ∊ < 32 mV) and due to ionic strength effects ( - 12 mV < Δ∊ < 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome C3 molecule are dependent on the degree of reduction of the other haems in the molecule. 4. The reaction of cytochrome C3 with MV+ or with ethanol radicals (EtOH+) has been compared with the reactions of horse-heart cytochrome c and of metinyoglobin with the same radicals. The reaction ot MV or EtOH - with horse-heart cytochrome c is found to be diffusion controlled: the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy. [ABSTRACT FROM AUTHOR]

Studies the effects of pH and redox potential on the hydrogen production activity of the hydrogenase from Megasphaera elsdenii. Ionization constant; Apparent midpoint potential; Concentrations of methyl viologen semiquinone; Concentration of flovodoxin hydroquinone; Hydrogen oxidation activity.

Describes a spectrophotometric method to study the effects of pH and redox potential on the hydrogen production activity of hydrogenase. Changes in the ratio of reduced/oxidized redox mediator as a function of pH and redox potential; Conditions for determining the pH dependence of the hydrogen production activity.

The catalytic activities of Megasphaera elsdenii hydrogenase are stimulated by salts. The stimulation is due to the anion: the more chaotropic the anion, the greater the effect. Dithionite-reduced and dye-oxidised preparations of hydrogenase are inactivated by reaction with oxygen. The inactivation of the reduced enzyme by excess oxygen follows pseudo-first-order kinetics; the reaction order for the oxidised enzyme has not been established. The rate of oxygen-inactivation is decreased by bovine serum albumin. The hydrogen production activity decreases in the presence of dimethylsulphoxide and ethylene glycol. The hydrogen oxidation activity is stimulated by dimethylsulphoxide, and the activity remains linear with time at concentrations up to 50% (v/v). Above 70% dimethylsulphoxide the steady-state activity of hydrogenase is abolished for both types of activity. The enzyme is more stable in a hydrogen atmosphere than in an argon atmosphere, and the oxidized enzyme is more stable than the reduced enzyme. The enzyme is isolated in the presence of dithionite and it is therefore reduced. When the enzyme is oxidized by treatment with 2,6-dichloroindophenol or with (bi)sulphite, its activity increases by up to 65%; this activation is not reversed when the enzyme is re-reduced. The increase in activity is associated with a change of the redox potential of the incubation medium to a less negative value; half of the maximum activation occurs at — 0.41 V. The electron paramagnetic resonance spectrum of the dithionite-reduced hydrogenase resembles that of a reduced ferredoxin-type of spectrum with two 4Fe-4S clusters. The spectrum of the oxidized enzyme is similar to that of Chromatium high-potential iron-sulphur protein. No redox potentials can be ascribed to these spectra since the redox system changes upon freezing to liquid helium temperatures. [ABSTRACT FROM AUTHOR]

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R. S. & Shain, I. (1964) Anal. Chem. 36, 706–723], using the Gauss-Legendre method for calculation of the integral equations. The experimental data obtained for this multi-redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid-point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described. [ABSTRACT FROM AUTHOR]

The electron transfer kinetics between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di-(n-aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second-order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic-strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer-controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility). [ABSTRACT FROM AUTHOR]

The electron transfer kinetics between the hydrogenase from Desulvovibrio vulgaris (strain Hildenborough) and three different viologen mediators has been investigated by cyclic voltammetry. The mediators methyl viologen, di(n-aminopropyl) viologen and propyl viologen sulfonate differ in redox potential and in net charge. Dependent on the pH both the one- and two-electron-reduced forms or only the two-electron-reduced form of the viologens are effective in electron exchange with hydrogenase. Calculations of the second-order rate constant k for the reaction between reduced viologen and hydrogenase are based on the theory of the simplest electrocatalytic mechanism. Values for k are in the range of 106-107 M-1 s-1 and increase in the direction propyl viologen sulfonate → methyl viotogen → di(n-ammopropyl) viologen. An explanation is based on electrostatic interactions. It is proposed that the electron transfer reaction is the rate-determining step in the catalytic mechanism. [ABSTRACT FROM AUTHOR]

The hydrogenases of Desulfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe-4 clusters, while M. elsdenii hydrogenase only release part of its Fe-S clusters. The resting D. vulgaris enzyme shows only a small 3 Fe-xS type of EPR signal (maximum 5% electron equivalent). This amount can be increased to approximately 25% by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high-potential iron-sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin-like spectra. [ABSTRACT FROM AUTHOR]

1. Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c. After the pulse. methyl viologen radicals are formed and the kinetics of these radicals with cytochrome C3 are studied. The reaction between cytochrome C3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome C3 molecule was Z = ± 4.7 ± 0.7. 2. After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome C3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, α With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0 - - 0.250-0.088α (in V). 3. An estimation is made of the energy of interaction between the haems due to electrostatic interactions (Δ∊ < 32 mV) and due to ionic strength effects ( - 12 mV < Δ∊ < 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome C3 molecule are dependent on the degree of reduction of the other haems in the molecule. 4. The reaction of cytochrome C3 with MV+ or with ethanol radicals (EtOH+) has been compared with the reactions of horse-heart cytochrome c and of metinyoglobin with the same radicals. The reaction ot MV or EtOH - with horse-heart cytochrome c is found to be diffusion controlled: the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy. [ABSTRACT FROM AUTHOR]

The aim of this study was to gain insight into the factors that affect the permeation of volatiles through starch films. These films were obtained by casting gelatinized starch/water/glycerol mixtures. The films were dried and conditioned under different conditions (temperature and relative humidity) resulting in films that vary in the degree of starch crystallinity and glycerol and water content. The permeation of two model volatiles (carvone and diacetyl) at 20 degrees C and at 30, 60, or 90% relative humidity (RH) was analyzed gravimetrically. Further, the solubility of the two model compounds (under conditions where the permeation experiments were carried out) was determined. From the obtained permeation and solubility data, the diffusion coefficients of these compounds in the different starch films were calculated. The crystallinity in the starch films increased with increasing water content of the films during preparation. The water content of the resulting films in turn increased with increasing glycerol and when the films were exposed to a higher RH during drying or conditioning. For films with the same composition, the flux for diacetyl was greater than for carvone. The solubilities of diacetyl and carvone were slightly dependent on the properties of the films. It was found that with increasing starch crystallinity the diffusion coefficient for both compounds decreases, which is probably due to the impermeability of starch crystallites. Interestingly, in films with about the same extent of crystallinity, the diffusion can be described with the free volume model, with water and glycerol determining the amount of free volume.

The release of a model volatile (diacetyl) from a system based on a starch matrix, in which the volatile is dispersed, was studied. Kneading was used to obtain a homogeneous mixture (melt) composed of starch, glycerol alpha-amylase, and diacetyl. Samples were then ground to powders. When the starch powders were exposed to 30% relative humidity (RH) at 20 degreesC, no degradation of the starch matrix occurred. The samples only showed an initial burst release of diacetyl (around 10% of the loaded dose), whereas the remaining amount of diacetyl was not released, most likely due to the glassy character of the matrix and the low solubility of diacetyl in the matrix. However, when the samples were incubated at 90% RH, due to the uptake of moisture by the particles full release of the entrapped volatile occurred. The release of diacetyl from the matrix without enzyme followed first-order kinetics and, as expected, the release rate increased with decreasing particle size. Due to absorption of water, the enzyme became active and starch degradation occurred. The initial release of diacetyl from amylase-containing matrixes followed first-order kinetics as well. However, once the matrix was degraded to a certain extent, the particles collapsed, which was associated with concomitant rapid increase in release. The time at which the particle collapse occurred decreased with increasing enzyme concentration in the matrix. In conclusion, it is demonstrated that the release of a volatile from starch matrixes can be modulated both by the amount of coencapsulated matrix-degrading enzyme and by the humidity of the environment.