Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain.

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 ± 1) × 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study.

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

Copper-psychoactive drug complexes: a voltammetric approach to complexation by 1,4-benzodiazepines.

Copper complexation by the 1,4-benzodiazepines medazepam, diazepam, flurazepam, nitrazepam, and clonazepam was investigated using differential pulse polarography and cyclic voltammetry at a mercury electrode in 0.10 M KNO3 and pH 7.0 +/- 0.1. Because the 1,4-benzodiazepines are easily reduced at a mercury electrode through the two-electron reduction of the 4,5-azomethine functional group, copper reduction, as well as that of the ligands, was analyzed under varying experimental conditions. In most situations adsorption phenomena occurred and their influence on voltammetric signals had to be carefully analyzed. The voltammetric behavior was then interpreted in terms of complex formation. The results showed that all benzodiazepines can act as ligands toward copper(II) ions, forming 1:1 and 1:2 complexes with similar stabilities. The stoichiometric acidity constants of the benzodiazepines under study were also determined by potentiometric titration in water-ethanol medium and 0.10 M KNO3 and then extrapolated to 0% concentration of ethanol.

Gradient flow titration for the determination of fluoride ion in natural waters.

The determination of fluoride ions in water samples was accomplished by using a gradient flow titration. A standard commercial combined electrode is used in a cell configuration that combines the gradient chamber and the electrode in a single unit. The methodology developed gives results with a relative standard deviation of about 3%. The average recoveries after spiking natural samples with fluoride are in the range 100-102%. The method was used successful in determining the fluoride concentration in water samples.

Thermodynamics of uptake of cadmium by Chlorella marina.

From voltammetric titration curves of living Chlorella marina cells with cadmium ion, at constant pH, the surface binding capacity of the alga was determined and interpreted in terms of average conditional equilibrium constants and of differential equilibrium function. Since the alga can live in estuaries, the influence of ionic strength and competition of alkaline and alkaline earth cations has been considered. Cadmium complexation with dead algae killed by heat or using formaldehyde has also been studied for comparison.