Determination of the redox potential and diffusion coefficient of the protein plastocyanin using optically transparent filar electrodes

Direct and mediated electrolysis of the protein plastocyanin at a gold filar electrode is described. The filar electrode used is of a unique design that allows potentiometric measurements, steady-state voltammetry and absorption spectrophotometry to be performed on a few microliters of solution containing 0.1-1.0 mM protein. As a result, we have determined the formal potential and diffusion coefficient of the blue copper protein, plastocyanin, to be 372 +/- 5 mV vs. normal hydrogen electrode and 8.9 X 10(-7) cm2 X s-1, respectively. The same value of the formal potential is obtained from a steady-state current experiment, an equilibrium spectrophotometric experiment, and a twin-electrode steady-state spectrophotometric experiment. The fact that the diffusion coefficient is measured under conditions of steady-state current, results in significant improvement in signal to background over techniques that monitor a transient current, while the potential is changing.     

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Interaction of beta-cyclodextrin with cadmium(II) ions

Reduction of Cd(II) on a dropping mercury electrode was used to study interaction of β-cyclodextrin with Cd(II) ions. It was found that Cd(II) forms Cdβ-CD(OH)₂²⁻ hydroxy-complex with the anion of β-cyclodextrin in alkaline solutions (pH>11), the logarithm of stability constant being 10.4±0.1 (20 °C; I=1.0). The calculated value of the diffusion coefficient equal to 1.0×10⁻⁶ cm²/s shows a large size Cd(II) complex species formation in alkaline solutions containing β-CD.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

Glucose-sensing carbon paste electrode containing polyethylene glycol-modified glucose oxidase

Polyethylene glycol-modified glucose oxidase (PEG-GOD) was prepared. Carbon paste (CP) containing PEG-GOD retained enzyme activity of 0·02 U cm⁻². Anodic and cathodic peak currents of modified GOD in CP matrix were observed on the differential pulse voltammograms at the potential of −0·36 and −0·36 V vs. Ag/AgCl, respectively. The addition of glucose to a test solution brought about an increase in the anodic current on the PEG-GOD-based electrode at the potential as low as 0·0 V vs. Ag/AgCl. The current increase was proportional to the concentration of glucose up to 50 mM.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700 reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6–8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42–45 and Nos. 59–61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42–45 and Nos. 59–61. Modification of plastocyanin at residues Nos. 42–45 showed no effect on the rate of P-700⁺ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700⁺ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59–61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 ± 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42–45 or Nos. 59–61.     

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e⁻_aq) and carboxyl anion radical (CO₂) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e⁻_aq with the negatively charged ferricytochrome c-551 (17 nM⁻¹ · s⁻¹) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM⁻ · s⁻). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e⁻_aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10⁴ s⁻¹) observed after fast e⁻_aq reduction is attributed to a small conformational change. The rate of reaction of CO₂ with ferricytochrome c-551 (0.7 nM⁻¹ · s⁻) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis

A new mediated amperometric biosensor for fructose is described. The sensor is based on a commercially available D-fructose dehydrogenase. The enzyme is incorporated in a carbon paste matrix containing Os(bpy)₂Cl₂ as redox mediator that achieves electron transfer at 0·1 V (versus Ag/AgCl) with maximum apparent current densities of 1·2 mA/cm². The dependence of the steady-state current on the loading of the mediator and the enzyme, other electrode construction parameters, the operating potential, the pH and the temperature was studied. In the steady-state mode the response current was directly proportional to D-fructose concentration from 0·2 to 20mM with a detection limit of 35 μM (signal-to-noise ratio, S/N, 3). In the flow injection analysis mode the response current was directly proportional to D-fructose concentration from 0·5 to 15 M with a detection limit of 115 μM (S/N 3). The sensor was used for the determination of fructose in food samples in a flow injection system and validated with a commercial enzyme kit.     

Hydrodynamic properties of the fractions of mannan formed by Rhodotorula rubra yeast

Aqueous solutions of fractions of an extracellular linear mannan formed by Rhodotorula rubra yeast have been investigated by hydrodynamic methods (high-speed sedimentation, translation isothermic diffusion and viscometry). The molecular weight was determined according to Svedberg ( ) and the polydispersity parameters of the initial sample were also determined (M_w/M_n = 1·20 and M_z/M_w = 1·21). Relationships between the molecular weight (M) and s_o, D_o and [η] in the range were: [η] = 2·33 × 10⁻² M^0.75, D_o = 1·65 × 10⁻⁴ M^0·58, s_o = 2·24 × 10⁻¹⁵ M^0·43. The equilibrium rigidity and hydrodynamic diameter of chains representing mannan molecules were evaluated.     

Biogas-plant effluent as an organic fertiliser in fish polyculture

Biogas-plant effluent collected from a KVIC model biogas-plant fed on cattle waste was utilised in fish polyculture. Biogas-plant effluent was applied at 0·15% concentration at 3-day intervals. The growth rate of Labeo rohita was 4·52 ±0 ·75 g fish⁻¹ day⁻¹, of Cirrhina mrigala 3·36 ± 0·48 g fish⁻ day⁻¹ and of Cyprinus carpio was 1·82 ± 0·41 g fish⁻¹ day⁻¹. Total fish production was 13·44 ± 0·77 kg 0·002 ha⁻¹ year⁻¹ (6653 kg ha⁻¹ year⁻¹) without any supplementary fish-feed.     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.     

Kinetics and equilibria of Ga(III)---thiocyanate complex formation. Mechanism of ligand substitution reactions of Ga(III) in aqueous solution

The kinetics and equilibria of complex formation by Ga(III) with NCS⁻ in aqueous solution have been measured over a range of acidities and temperatures, the contributing paths to the reaction resolved, and their rate constants and activation parameters determined. The hydrolysis equilibria required to carry out this resolution of kinetic behaviour have also been measured.

Unlike the other reported complexation reactions of Ga(III) in aqueous solution, the separate reaction pathways can be assigned with no ambiguity. At 25 °C and ionic strength 0.5 M, the observed forward rate constant for the complex formation is described by {k₁ + k₂K_1h/[H⁺] + k₃K_1hK_2h/[H⁺]²} M⁻¹ s⁻¹. For these conditions, the first and second successive hydrolysis constants of Ga(H₂O)₆³⁺ are given by pK_1h = 3.69 ± 0.01 and pK_2h = 3.74 ± 0.04. The rate constants corresponding to the reactions of the species Ga(H₂O)₆³⁺, Ga(H₂O)₅(OH)²⁺ and Ga(H₂O)₄(OH)₂⁺ with NCS⁻ are k₁ = 57 ± 4 M^{−1 −1}, k₂ = (1.08 ± 0.01) × 10⁵ M⁻¹ s⁻¹ and k₃ = 3 × 10⁶ M⁻¹ s⁻¹ respectively. The complexation equilibrium quotient [GaNCS²⁺]/([Ga³⁺][NCS⁻]) has been independently determined by spectrophotometric titration to be 20.8 ± 0.3 M⁻¹ at 25 °C and ionic strength 0.5 M.

These kinetic results lead to an interpretation of the data, and a reinterpretation of other data for aquo-Ga(III) complex formation kinetics from the literature which support the assignment of a dissociative interchange mechanism for these reactions rather than the associative activation mode sometimes proposed.     

Diffusion of KCl in an amylose film

A method has been developed measuring the diffusion coefficient of KCl in amylose films. The films were soaked in potassium chloride solutions, then immersed in pure water and conductivity measured as a function of time. Different concentrations of the soaking solution were used and the measurements were made at several temperatures. The diffusion coefficient of KCl was found to be independent of the soaking solution KCl concentration, but found to increase with increasing temperature. The diffusion coefficient values were about one quarter of those found in water and varied from 4.8×10⁻¹⁰ to 11×10⁻¹⁰ m² s⁻¹. The activation energy of diffusion was close to that found in water. Two values for the activation energy were obtained, 20.1 and 14 kJ mol⁻¹, indicating a change in the film structure at 45 °C. Amylose films swelled equally in KCl-solutions and water. The thickness of amylose films doubled and the increase in mass was 100–200% corresponding the decrease of amylose content from about 87 to 37%, when the conditions changed from normal humidity conditions to water.     

Influence of phosphorus concentration on the growth kinetics and stoichiometry of the microalga Scenedesmus obliquus

The growth of the freshwater microalga Scenedesmus obliquus was studied at 30°C in a mineral culture medium with phosphorus concentrations of between 0 and 372 μ . The values for the specific growth rates, between and , fitted a semistructured substrate-limitation model with μ_m1 = 0·0466 h⁻¹, μ_m2 = 0·0256 h⁻¹ and . The specific uptake rate of phosphorus reached a maximum value of q_Sm1 = 658·01 × 10⁻⁴ μmol P mg⁻¹ biomass h⁻¹.     

Kinetic studies on 1:1 electron transfer reactions involving blue copper proteins : 8. Reactions of plastocyanin and azurin with cytochrome c and high potential iron-sulfur protein

Rate constants determined by the stopped-flow method for four protein-protein reactions at 25°C, pH's in the range 5.8–7.5. I = 0.10 M (NaCI), are as follows: cytochrome c(II) with plastocyanin, PCu(II). 1.5 × 10⁶ M⁻¹ sec⁻¹, pH 7.6; high-potential iron-sulfur protein (Hipip) with PCu(II), 3.7 × 10⁵ M⁻¹sec⁻¹. pH 5.8; cytochrome c(II) with azurin, ACu(ll). 6.4 × 10³ M⁻¹sec⁻¹, pH 6.1; Hipip with ACu(II), 2.2 × 10⁵ M⁻¹sec⁻¹, pH 5.8. Activation parameters have been determined for all four reactions; they indicate higher enthalpy requirements and less negative entropy requirements for the PCu(II) as opposed to ACu(II) reactions. Equilibrium constants K for association prior to electron transfer are < 150 M⁻¹ for the cytochrome c(II) reduction of PCu(II) (estimated charges 8 + and 9-,respectively), and < 300 M⁻¹ for the other reactions, indicating no favorable interactions. Rate constants have been analyzed in terms of the simple Marcus theory, which has previously given an excellent fit to thirteen protein-protein reactions considered by Wherland and Pecht. No similar correlation exists in the present studies, and calculated rate constants differ by orders of magnitude from experimentally determined values.