Redox Titration Curves Research Articles

Inquiry-based Investigation of the Influencing Factors on the Redox Titration Curves by Simulation

Inquiry-based Investigation of the Influencing Factors on the Redox Titration Curves by Simulation

A Refined Redox Titration Simulation Program for the Simple System

Commonly used methods to simulate the oxidation–reduction (redox) titration curves include the three-step method and the rigorous method. The simple three-step method simulates the redox titration curve with the assumption that the reaction is complete, which is widely used in undergraduate quantitative analysis courses. For the rigorous simulation method, the concentrations of the components are obtained throughout the titration by solving the equilibrium equation, and then the potential is obtained based on the Nernst equation. However, both methods have certain defects. In this work, a user-friendly program that uses an optimized rigorous algorithm to simulate the redox titration curve of a simple system with n1 = n2 = 1 at different potentials was built with LabVIEW. The program was then applied to the teaching of redox titration, and questionnaires from 15 students were used for evaluation. The results showed that the program improved students’ understanding of the principles of redox titration and deepened their graphical impression of Nernst equations.

Effect of Ca2+ on the redox potential of heme a in cytochrome c oxidase

Subunit I of cytochrome c oxidase (CcO) from mitochondria and many bacteria contains a cation binding site (CBS) located at the outer positively charged aqueous phase not far from heme a. Binding of Ca2+ with the CBS in bovine CcO inhibits activity of the enzyme 2–3 -fold [Vygodina, T., Kirichenko, A. & Konstantinov A.A. (2013) Direct Regulation of Cytochrome c Oxidase by Calcium Ions, PLoS One.8 e74436]. Here we show that binding of Ca2+ at CBS of bovine CcO shifts Em of heme a to the positive by 15–20 mV. Na+ ions that bind to the same site and compete with Ca2+ do not affect Em of heme a and also prevent and reverse the effect of Ca2+. No effect of Ca2+ or EGTA is observed on Em of heme a with the wild type bacterial oxidases from R.sphaeroides or P.denitrificans that contain tightly-bound calcium at the site. In the D477A mutant CcO from P. denitrificans that binds Ca2+ reversibly like the mitochondrial CcO, calcium shifts redox titration curve of heme a to the positive by ∼35–50 mV that is in good agreement with the results of electrostatic calculations; however, as shown earlier, it does not inhibit CcO activity of the mutant enzyme. Therefore the data do not support the proposal that the inhibitory effect of Ca2+ on CcO activity may be explained by the Ca2+-induced shift of Em of heme a. Rather, Ca2+ retards electron transfer by inhibition of charge dislocation in the exit part of the proton channel H in mammalian CcO, that is absent in the bacterial oxidases.

Electrostatics of the FeS clusters in respiratory complex I

Respiratory complex I couples the transfer of electrons from NADH to ubiquinone and the translocation of protons across the mitochondrial membrane. A detailed understanding of the midpoint reduction potentials ( E m ) of each redox center and the factors which influence those potentials are critical in the elucidation of the mechanism of electron transfer in this enzyme. We present accurate electrostatic interaction energies for the iron–sulfur (FeS) clusters of complex I to facilitate the development of models and the interpretation of experiments in connection to electron transfer (ET) in this enzyme. To calculate redox titration curves for the FeS clusters it is necessary to include interactions between clusters, which in turn can be used to refine E m values and validate spectroscopic assignments of each cluster. Calculated titration curves for clusters N4, N5, and N6a are discussed. Furthermore, we present some initial findings on the electrostatics of the redox centers of complex I under the influence of externally applied membrane potentials. A means of determining the location of the FeS cofactors within the holo-complex based on electrostatic arguments is proposed. A simple electrostatic model of the protein/membrane system is examined to illustrate the viability of our hypothesis.

Electrostatic Interactions Between FeS Clusters in NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

The redox properties of the cofactors of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli were studied by following the changes in electron paramagnetic resonance (EPR) and optical spectra upon electrochemical redox titration of the purified protein. At neutral pH, the FMN cofactor had a midpoint redox potential ( E m) approximately -350 mV ( n = 2). Binuclear FeS clusters were well-characterized: N1a was titrated with a single ( n = 1) transition, and E m = -235 mV. In contrast, the titration of N1b can only be fitted with the sum of at least two one-electron Nernstian curves with E m values of -245 and -320 mV. The tetranuclear clusters can also be separated into two groups, either having a single, n = 1, or more complex redox titration curves. The titration curves of the EPR bands attributed to the tetranuclear clusters N2 ( g = 2.045 and g = 1.895) and N6b ( g = 2.089 and g = 1.877) can be presented by the sum of at least two components, each with E m (app) approximately -200/-300 mV and -235/-315 mV, respectively. The titration of the signals at g = 1.956-1.947 (N3 or N7, E m = -315 mV), g = 2.022, and g = 1.932 (Nx, -365 mV) and the low temperature signal at g = 1.929 (N4 or N5, -330 mV) followed Nernstian n = 1 curves. The observed redox titration curves are discussed in terms of intrinsic electrostatic interactions between FeS centers in complex I. A model showing shifts of E m due to the electrostatic interaction between the centers is presented.

Cooperative interaction of high-potential hemes in the cytochrome subunit of the photosynthetic reaction center of bacterium Ectothiorhodospira shaposhnikovii

Cooperative interaction of the high-potential hemes (C(h)) in the cytochrome subunit of the photosynthesizing bacterium Ectothiorhodospira shaposhnikovii was studied by comparing redox titration curves of the hemes under the conditions of pulse photoactivation inducing single turnover of electron-transport chain and steady-state photoactivation, as well as by analysis of the kinetics of laser-induced oxidation of cytochromes by reaction center (RC). A mathematical model of the processes of electron transfer in cytochrome-containing RC was considered. Theoretical analysis revealed that the reduction of one heme C(h) facilitated the reduction of the other heme, which was equivalent to a 60 mV positive shift of the midpoint potential. In addition, reduction of the second heme C(h) caused a three- to four-fold acceleration of the electron transfer from the cytochrome subunit to RC.

A General Approach for Calculating Speciation and Poising Capacity of Redox Systems with Multiple Oxidation States: Application to Redox Titrations and the Generation of pε-pH Diagrams

This work describes a general approach for calculating the redox speciation of systems with multiple oxidation states. The calculational approach provides solutions for the distribution functions (andI±I»I€I·I±;) and degree of oxidation (η) of redox systems as a function of pe or redox potential. From these parameters the poising capacity (ρ) or electron buffer capacity of multicomponent redox systems can be calculated. This allows direct calculation of redox titration curves without segmentation or approximations. The titration curves can be calculated for individual species or for multiple redox species typical of many environmental systems. A contour surface of poising capacity as a function of both pe and pH allows automated generation of pe–pH diagrams. All of these calculations are readily implemented using spreadsheet software making them accessible for classroom instruction.

Isolation and Analysis of Tetraheme-Bound-Cytochrome from Photosynthetic Reaction Centers of Rhodopseudomonas viridis

A tetraheme cytochrome (BCytc) was isolated from the photosynthetic reaction centers (RC) of Rhodopseudomonas viridis while maintaining the redox activity. BCytc was removed from the H-subunit-detached RC by polyacrylamide electrophoresis using an alkyl ether sulfate mixed with sodium dodecyl sulfate. Redox titration of BCytc showed a simple one-step redox titration curve and a lowered midpoint potential than that of one in RC. Direct electron transfer between BCytc and electrode surfaces, such as indium tin oxide, was successfully performed, indicating a potential for molecular electronic material.

Algorithm for plotting titration curves

de Moura's algorithm for plotting redox titration curves seems to be good for systems that are not dependent on pH or product concentration, but is not applicable to those situations that is student is more likely to encounter.

A simple expression for the redox titration curve

Titration curves are often discussed in piecemeal fashion: one approximate description for the part before the equivalence point, a different approximation for the part beyond this point and, finally, yet another formula for the equivalence point itsell We use examples of some simple redox titrations to illustrate that it is not really necessary to make the customary approximations, because the entire redox titration curve can be described exactly as the simple ratio of a formation curve and a dissociation curve.

EPR determination of interaction redox potentials in a multiheme cytochrome: cytochrome c3 from Desulfovibrio desulfuricans Norway

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40–50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.

EPR determination of interaction redox potentials in a multiheme cytochrome: cytochrome c3 from Desulfovibrio desulfuricans Norway.

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.

Fluorescence decline as a function of redox potential and actinic light intensity in spinach thylakoids

Excitation of isolated thylakoids with sufficiently strong actinic light increases the fluorescence quantum yield up to a maximum level, F max, followed by a slower decline under certain experimental conditions. In this study the latter effect was analyzed as a function of the ambient redox potential and the actinic light intensity. Two different types of fluorescence decrease were found. (a) In the presence of specific quinones widely used as redox mediators a fast and comparatively small decrease (30% of F max), referred to as ΔF SQ, was observed at moderate redox potentials (−300 < E m < + 200 mV). ΔF SQ disappears at positive values with E m, 7.5 = + 110 mV, whereas the decrease at negative redox potential depends on the midpoint potential of the quinone. (b) A more pronounced fluorescence decline was observed at redox potentials below −300 mV, which comprises 65–70% of the maximum fluorescence. The full expression of this effect, referred to as ΔF max LP, requires markedly higher actinic light intensities than ΔF max SQ. The extent of ΔF max LP as a function of the redox potential is dependent on the presence of redox mediators. In their absence the full expression of ΔF max LP can be only observed below −400 mV. Based on the hypothesis of Pheo − photoaccumulation being responsible for the fluorescence decline at low redox potentials (Klimov, V.V., Klevanik, A.V. and Shuvalov, V.A. (1977) FEBS Lett. 82, 182–186), a reaction scheme is presented that qualitatively describes the time course of ΔF LP at different actinic light intensities and redox potentials. Based on this analysis, the rate of Pheo ⨪ reoxidation is inferred to be limited by the reaction center apoprotein acting as a barrier to redox equilibration. The implications for the interpretations of redox titration curves are briefly discussed.

Photoelectric effects in bacterial chromatophores. Comparison of spectral and direct electrometric methods

Generation of photoelectric potential in chromatophores of Rhodopseudomonas sphaeroides has been measured (i) spectrophotometrically, using electrochromic shift of carotenoid absorption band or (ii) electrometrically, by means of two electrodes separated by a collodion film covered on one side with chromatophores. A 15 ns laser flash was used to induce a single turnover of photosynthetic reaction centers. It was found that results obtained by both methods are similar in (i) direction of electric vector (the chromatophore interior positive) and (ii) redox titration curves ( E m = 10mV). The magnitudes of the photopotential were about 60 and 25 mV, when monitored with spectral and electrometric techniques, respectively. In both cases, the rise times of the photopotentials were faster than time resolution of the techniques used. Decay of the response of carotenoids was found to be slower than that in the collodion film system. The addition of ubiquinone Q 10 into the decane solution of asolectin used to impregnate the collodion film led to slowing down of the decay. The carotenoid response decay could be accelerated by FCCP or o-phenanthroline. In the latter case, the shape of the decay curve coincides with decay of the photopotential measured in the collodion film system. It is suggested that decane extracts secondary ubiquinone from chromatophores attached to the collodion film. Such an unfavorable effect can be strongly decreased by added ubiquinone

Studies on the mechanism of the fluorescence decline induced by strong actinic light in PS II particles under different redox conditions

Fluorescence changes induced by strong actinic light at room temperature were measured in isolated photosystem II (PS II) particles under different redox conditions. Comparative measurements of the absorption changes accompanying the light-induced fluorescence decay show that in the absence of Na 2S 2O 4 the formation of a chlorophyll cation radical (probably together with oxidized carotenoid) causes a quenching while in the presence of Na 2S 2O 4 photoaccumulation of the pheophytin anion radical (Pheo .−) takes place. The 695 nm band of the 77 K emission spectrum becomes specifically reduced if Pheo .− accumulates in the reaction center, whereas in the case of quenching by the cation radicals the ratio of the emission peaks at 685 nm and 695 nm remains constant. The present data favor the hypothesis that F-695 originates from the PS II reaction center [FEBS Lett. (1982) 147, 16–20]. If the primary plastoquinone acceptor (PQ A) stayed oxidized in the dark before the onset of the illumination. Pheo .− photoaccumulation was not observed. This effect is explained by the existence of at least one further redox component which is able to accept electrons efficiently from Pheo .−. The proposed model also explains the differences in the redox titration curves of the electrochromic bandshift and the inital fluorescence, respectively.

The effects of pH and redox potential on the hydrogen production activity of the hydrogenase from Megasphaera elsdenii.

The effects of temperature on the ionization constant (pK') and apparent midpoint potential (EB) of the unprotonated species of Megasphaera elsdenii flavodoxin hydroquinone shows that, above 15 degrees C, delta pK' . K-1 = -9.7 X 10(-3) and delta EB . K-1 = -0.6 mV. The effects of pH and redox potential on the hydrogen production activity with fixed concentrations of methyl viologen semiquinone (0.3 mM; artificial donor) and M. elsdenii flavodoxin hydroquinone (50 microM; natural donor) show that with decreasing pH the activity increases. Irrespective of the pH and electron donor, at increasing redox potential, a redox-potential-independent production activity is followed by a redox-potential-dependent production activity. This redox-potential-dependent behaviour of the hydrogen production activity represents an n = 2-type of redox titration curve with an 'apparent midpoint potential' which corresponds with the potential of the hydrogen electrode at that pH. The effect of pH on the manometrically determined hydrogen production activity (direct) is in good agreement with that determined spectrophotometrically (indirect; see preceding paper), with both electron donors tested. In contrast to predictions from the models for hydrogenase activity [van Dijk et al. (1980) Eur. J. Biochem.102, 317--330], a double-reciprocal plot of the kinetic data for M. elsdenii flavodoxin hydroquinone at pH 5.5 is non-linear. A slightly adapted kinetic model based on a similar mathematical formulation of its rate equation, to explain the effects of redox potential, proton and electron (donor) concentration on the hydrogenase activity is proposed. This model also explains, on a theoretical basis, the effects of pH and redox potential on the hydrogen production activity. The effect of pH on the hydrogen oxidation activity with methyl viologen and benzyl viologen as electron acceptors shows for both dyes an optimum at pH 9.7. The ratio of the activities with both viologens is constant over the ph range tested.

Potentiometric stripping analysis for mercury

In potentiometric stripping analysis for mercury, elemental mercury is deposited on a glassy carbon electrode surface by means of potentiostatic reduction. It is then oxidized by potassium permanganate added to the sample prior to analysis and the “redox titration curve” thus obtained is recorded on a high-input impedance recorder. Deaeration of the sample is unnecessary. The analytical range is 5 × 10 -9–10 -3 M mercury(II), the times needed for potentiostatic accumulation ranging from 64 min at 10 -8 M to 1 min at concentrations above 10 -6 M. The chemistry of the stripping process is discussed and an automatic instrument for potentiometric stripping analysis is described.

The steady state activity of succinate dehydrogenase in the presence of opposing effectors

The extent of the deactivation of the mitochondrial succinate dehydrogenase by oxaloacetate is a function of the redox state of the enzyme. Oxidized enzyme is deactivated by much lower concentrations of oxaloacetate than those needed to deactivate reduced enzyme. An accurate method for measuring this relationship is the redox titration of the enzymic activity of succinate dehydrogenase, carried out in the presence of oxaloacetate. For each concentration of oxaloacetate a different redox titration curve was reported with the apparent mid-potential decreasing with increasing oxaloacetate. These results are compatible with a model which proposes that both oxidized and reduced enzymes can form the catalytically non-active complex with oxaloacetate, but that the complex formed the the oxidized enzyme is more stable than that formed by the reduced enzyme. When the oxaloacetate concentration is low, reduction of the enzyme will lower the fraction of the succinate dehydrogenase-oxaloacetate complex, a reaction which we observe as reductive activation of the enzyme. If this experiment is repeated in the presence of high concentration of oxaloacetate, no activation of the enzyme takes place, but the low stability of the reduced enzyme oxaloacetate complex is revealed by the rapid exchange of the enzyme-bound oxaloacetate with the free ligand. The rate of this exchange is extremely slow at high positive potential and becomes faster upon lowering of the poise potential. The reductive activation of the succinate dehydrogenase is regarded as a two step reaction. In the first step the reduced non-active complex releases the oxaloacetate and in the second step the active form of the enzyme is evolved. These two steps can be observed experimentally; Reductive activation at a redox potential higher than the mid-potential of the oxaloacetate-malate couple (minus 166 mV) is characterized by Ea = 18 Kca/mole, the final equilibrium level of activation decreases upon lowering of the temperature. Reduction activation of the enzyme at minus 240 mV is a very rapid reaction which goes to completion at all temperatures tested and has an activation energy of 12.5 Kcal/mole. The mechanism of the reductive activation and its possible role in the regulation of succinate dehydrogenase in the mitochondria is discussed.

Effect of membrane potential on equilibrium poise between cytochrome a and cytochrome c in rat liver mitochondria.

The object of this work was to test the suggestion that the equilibrium poise between cytochromea and cytochromec in mitochondria might be influenced by the membrane potential. 1. The midpoint potentials of cytochromes (c+c1) and cytochromea (CO present) were found to be 250 mV and 245 mV, respectively, by equilibrating rat liver mitochondria with mixtures of ferrocyanide and ferricyanide anaerobically in presence of antimycin A and measuring the redox state of the cytochromes spectrophotometrically. In absence of CO, cytochrome oxidase gave an anomalous redox titration curve with a “midpoint” at about 275 mV. 2. When the mitochondria were equilibrated with ferricyanide/ferrocyanide, the redox poise of cytochromea (CO present) and of cytochromes (a+a3) but not of cytochromes (c+c1) was dependent on the sign and magnitude of the membrane potential developed by treating the mitochondria as follows: by adding ATP, by chaging the composition of the suspension medium so as to vary the Donnan or Nernst potential, by adding valinomycin in a medium of low K+ ion content, or by adding a pulse of acid or alkali when the membrane was made permeable to protons with FCCP. 3. The findings agree with the suggestion that the respiratory chain is arranged across the cristae membrane with cytochromesc1 andc in contact with the outer phase and cytochromesa anda3 plugged through, so that the equilibrium distribution of electrons between thec anda cytochromes is influenced by the electric field across the membrane.

THE EFFECT OF REDOX POTENTIAL ON P870 FLUORESCENCE IN REACTION CENTERS FROM Rhodopseudomonas spheroides

We have prepared photosynthetic reaction centers from Rhodopseudomonas spheroides and have studied the fluorescence of the photochemical electron donor, P870. The yield of this fluorescence rises at low redox potential, presumably because the photochemical electron acceptor becomes reduced and the photochemical utilization of excitation energy is then prevented. The redox titration curve for this increase in the fluorescence has a shape corresponding to the transfer of one electron. The midpoint potential is -0.05 volt, independent of the pH from 6.5 to 8.8. The amplitude of a light-induced absorbance change at 280 nanometers varies with redox potential and shows, at pH 7.5, a midpoint potential of 0.00 volt. These studies indicate that the primary photochemical electron acceptor is not ubiquinone and is not the substance responsible for the absorbance change at 280 nanometers.