Membrane potential of Plasmodium-infected erythrocytes

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.     

Synaptosomes from Rat Brain: Morphology, Compartmentation, and Transmembrane pH and Electrical Gradients

Abstract: Morphological studies of synaptosomes isolated from rat brains show that approximately 68% of the synaptosomes in these preparations contain synaptic vesicles (range, 62–72.5%). Approximately 30% of the synaptosomes contain mitochondria, and only less than 20% of the total mitochondria in good preparations are free and not enclosed in synaptic structures. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5% for synaptosomes isolated from anesthetized animals and 11% for synaptosomes isolated from unanesthetized animals. These numbers bracket the value of 8.7% obtained from electron micrographs. The volume percent of intrasynaptic vesicles is 1.5% as calculated from electron micrographs. The pH gradient between the extracellular pH and the mean intracellular pH is -0.45, as measured by equilibrium distributions of methylamine and dimethylamine, and -0.05, as determined by equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione and trimethylacetic acid. Analysis of these data shows that there cannot be a large pH gradient (alkaline inside) across the mitochondria, nor can the synaptic vesicle compartment be very large (<1.85%). Equilibrium distribution of [³H]triphenylmethyl-phosphonium ion in synaptosomal preparations gives a calculated apparent potential of -85 mV, in agreement with our previous value. Analysis of these data using the measured volumes of mitochondrial and intrasynaptic vesicular compartments (8.7 and 1.5%, respectively) gives a maximum possible trans mitochondrial membrane potential of -59 mV.     

Nernst Potential Measurements on the Leaf Cells of the Moss, Hookeria lucens

A study has been made of the ion distributions in the leaf cellsof the moss, Hookeria lucens. The cells were subjected to chemicalanalysis and electrical potential measurements. The observedinternal concentrations were 210 mM/1 for potassium, 3.9 mM/1for sodium, 22 mM/1 for calcium, and 27 mM/1 for chloride. Awide range of electrical potential values were obtained. Whilea majority of these values were grouped around —145 mV,a significant number were found at —200 mV. Nernst potentialsfor the various ions were calculated and compared with the observedelectrical potentials.     

Factors determining the plasma-membrane potential of lymphocytes.

1. Lymphocytes from pig mesenteric lymph node have low permeability to K+ (Rb+), Na+ and Cl-. None of these ions is in Nernst equilibrium with the plasma-membrane potential (delta psi p). 2. delta psi p can be calculated from the transmembrane distribution of the permeant cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to abolish uptake into intracellular mitochondria. In normal culture medium delta psi p is 56 mV. 3. A similar potential is found in T-enriched pig cells and in mouse thymocytes. 4. The contribution of electrogenic (Na+ + K+)ATPase to delta psi p is about 7 mV. 5. The remainder of the lymphocyte delta psi p is a polyionic potential set up by K+ and Cl- with a permeability coefficient for Cl- of similar magnitude to that for K+.     

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: role in the initiation of sperm motility

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H-tetraphenylphosphonium (TPP) and 14C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.     

Proton electrochemical potential of the inner mitochondrial membrane in isolated perfused rat hearts, as measured by exogenous probes

The membrane potential (delta psi) and delta pH of the inner mitochondrial membrane were studied in isolated perfused rat hearts using exogenous labelled probes and tissue fractionation in non-aqueous media. The mitochondrial delta psi, measured by means of the subcellular distribution of [3H]triphenylmethylphosphonium (TPMP+), was 125 +/- 7 mV (negative inside) in hearts beating at 5 Hz and 150 +/- 3 mV (negative inside) in hearts beating at 1.5 Hz. The mitochondrial membrane delta pH, measured by means of the subcellular distribution of low concentrations of [1-14C]propionate, was 0.63 +/- 0.06 pH units (alkaline inside) in hearts beating at 5 Hz and 0.53 +/- 0.12 pH units (alkaline inside) in hearts beating at 1.5 Hz. The implication of proton and electron gradients in the regulation of cellular respiration is discussed. In combination with previous evidence on adenylate distribution in the isolated perfused rat heart, the results indicate that the mitochondrial electrogenic adenylate translocator is in near equilibrium with delta psi.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

Influence of extracellular pH on the modulatory effect of zinc ions on Kv1.3 potassium channels.

In the present study we applied the whole-cell patch-clamp technique to study the influence of extracellular pH (pH(o)) on the modulatory effect exerted by zinc ions (Zn(2+)) on voltage-gated potassium channels Kv1.3 expressed in human lymphocytes. Obtained data provide evidence that lowering of pH(o) from the 7.35 to 6.4 slowed significantly the current activation rate, shifted the activation midpoint by about 16 mV towards positive membrane potentials and reduced the current amplitude to about 0.55 of the control value. In contrast, raising the pH(o) from 7.35 to 8.4 did not affect significantly the activation midpoint and current amplitude. Application of Zn(2+) in the concentration range from 100 microM to 1 mM at pH(o)=6.4 slowed additionally the activation rate, shifted the activation midpoint by about 20 mV towards positive membrane potentials and reduced the current amplitude in a concentration-dependent manner. The total effect exerted by Zn(2+) and protons at pH(o) = 6.4 was more significant than the effect exerted by Zn alone. Both the magnitude of the shift and the degree of current inhibition by Zn(2+) were independent on pH(o) in the range from 6.4 to 8.4. The data might suggest that the effects exerted by protons and zinc ions occur independently on each other and probably involve different mechanisms. Changing the holding potential from -90 mV to -60 mV at pH(o)=7.35 abolished the Zn(2+)-induced inhibition of the current amplitudes at concentrations below 300 microM. At pH(o)=6.4 the total inhibition caused by Zn(2+) and protons was also diminished, however, a significant reduction was observed at 100 microM concentration. In contrast, changing the holding potential did not change the Zn(2+)- and proton-induced shift of the activation midpoint. Altogether, obtained data suggest that extracellular protons exert the modulatory effects that are additive to the effects exerted by Zn(2+) on the channels. Possible physiological significance of these additive effects is discussed.     

Two types of single inward rectifying potassium channels in rat myocardial cells

The patch-clamp method was used to examine inward rectifying potassium channels in the membrane of rat ventricular myocytes. Two types of inward rectifying channels strongly selective for K+ ions and with different conductance and kinetics coexist in rat myocardial cells. When the concentration of K+ was 140 mmol/l on the extracellular side of the patch, the conductance was 38.9 pS for type I channels and 25.7 pS for the type II. The type II channels had a detectable conductance (4 pS) at potentials positive than the potassium equilibrium potential. The mean open time was 18 ms at -60 mV patch membrane potential and 12 ms at -100 mV for type I channels, and 1.3 s at -60 mV and 0.94 s at -105 mV for type II channels, respectively. The opening probability of type II channels decreased with hyperpolarization. The type II channels can adopt several (about 10 or more) conductance states, which can occur either within one opening or as individual events.     

Origin of the membrane potential in plasmodial droplets of Physarum polycephalum. Evidence for an electrogenic pump

Spherical droplets, derived from Physarum plasmodia by incubation in 10 mM caffeine, seemed to be an excellent system for electrophysiological studies because they were large (less than or equal to 300 micrometer in diameter) and because they tolerated intracellular electrodes filled with 3 M KCl and 10 mM EDTA for a few hours. Intact plasmodia, by contrast, gave valid records for only a few minutes. Under standard conditions ([K+]o = 1 mM, [Na+]o = 5 mM, [Ca++]0 = 0.5 mM, [Mg++]o = 2 mM, and [Cl-]o = 6 mM at pH 7.0), the potential difference across droplet membranes was -80 to -120mV, interior negative. The membrane potential was only slightly sensitive to concentration changes for the above-mentioned ions, and was far negative to the equilibrium diffusion potentials calculated from the known internal contents of K, Na, Ca, Mg, and CL (29.4, 1.6, 3.7, 6.5, and 27.8 mmol/kg, respectively). Variations of external pH did have a strong influence on the membrane potential, yielding a slope of 59 mV/pH between pH 6.5 and 5.5. In this pH range, however, the equilibrium potential for H+ (assuming 6.2 less than or equal to pHi less than or equal to 7.0) was greater than 75 mV positive to the observed membrane potential. Membrane potential was directly responsive to metabolic events, being lowered by potassium cyanide, and by cooling from 25 to 12 degrees C. This ensemble of results strongly indicates that the major component of membrane potential in plasmodial droplets of Physarum is generated by an electrogenic ion pump, probably one extruding H+ ions.     

Determination of the intravesicular pH of fragmented sarcoplasmic reticulum with 5,5-dimethyl-2,4-oxazolidinedione.

The intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56. It was also demonstrated by direct measurements of pHe with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions. From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively.     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.     

A network model for biofilm development in Escherichia coli K-12

Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl^--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts. In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl^-. Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl^- ions needs to be >20 times higher outside the cell. When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.     

Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes

A number of cellular functions have recently been associated with alterations of the membrane potential in non-excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage-sensitive oxonol dye di-BA-C4(3) was developed. The fluorescent signal of di-BA-C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH-sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the "putative" rat osteoblasts (UMR 106) as -28.3 +/- 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 +/- 0.7 mM (n = 3) and 24 +/- 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.     

Ionic mechanism for the generation of horizontal cell potentials in isolated axolotl retina

The ionic mechanism of horizontal cell potentials was investigated in the isolated retina of the axolotl Ambystoma mexicanum. The membrane potentials of both receptors and horizontal cells were recorded intracellularly while the ionic composition of the medium flowing over the receptor side of the retina was changed. The membrane potential of the horizontal cell is highly depender side of the retina was changed. The membrane potential of the horizontal cell is highly dependent on the extracellular concentration of sodium. When the external ion concentration of either chloride or potassium was changed independently of the other, there were shifts in the membrane potential of the horizontal cell which could not be explained by changes in the equilibrium potential of these ions. If the external concentrations of both potassium and chloride ions were varied so that the product of their external concentrations did not change, the shift in the membrane potential of the horizontal cell was in the direction predicted by the Nernst equation. The results are consistent with the suggestion that in the dark the receptors release a synaptic transmitter which increases primarily the sodium conductance of the horizontal cell postsynaptic membrane.     

The energetics of ion distribution: the origin of the resting electric potential of cells

The relation between the energies of ion movement and ATP hydrolysis is unknown in tissues with widely varying electric potentials. Consequently, we measured the concentration of the nine major inorganic ions in the extra- and intracellular phases in heart, liver, and red cells with resting electrical potentials, E(N), of -86, -28, and -6 mV, respectively, under six different physiological conditions. We calculated the Nernst electric potential and the energy of ion movement between the phases. We found that the energy of ATP hydrolysis was essentially constant, between -54 and -58 kJ/mol, in all tissues and conditions. In contrast, as E(N) decreased, the energies of the Na+ and K+ gradients decreased, with slopes approximating their valence. The difference between the energies of Na+ and K+ gradients remained constant at 17 kJ/mol, which is approximately one third of the energy of ATP hydrolysis, demonstrating near-equilibrium of the Na+/K+ ATPase in all tissues under all conditions. All cations, except K+, were pumped out of cells and all anions, except Cl- in liver and red cell, were pumped into cells. We conclude that the energy of ATP was expressed in Na+/K+ ATPase and its linked inorganic ion transporters to create a Gibbs-Donnan near-equilibrium system, an inherent part of which was the electric potential.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium.

The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.     

Analysis of sodium and potassium redistribution during sustained permeability increases at the innervated face of Electrophorus electroplaques

Cholinergic agonists cause an increase in the membrane permeability of Na and K at the innervated face of Electrophorus electroplaques. Therefore, sustained exposure to agonist reduces Na and K concentration gradients. There gradients are monitored with voltage-clamp sequences and pharmacological treatments that selectively measure the Nernst potentials for individual ions. EK is normally near--90 mV but moves toward zero during bath application of agonist. Depolarizations by bath- applied agonist measure primarily this shift of EK, not short- circuiting of EK by the agonist-induced conductance. After a rapid jump of agonist concentration, there is a fast (millisecond) depolarization due to the conductance increase, followed by a much slower additional "creep" due to the shift in EK. Sodium replaces the lost intracellular potassium: ENa, normally very positive, also moves toward zero. The shifts in EK and ENa are normally reversible but become permanent after blockade of the Na-K pump. In the presence of agonist, the shifts can be driven further by passing current of the appropriate polarity. Similar ion redistribution occurs with other drugs, such as batrachotoxin and nystatin, which induce prolonged increases in Na permeability. The redistributions cause little net change in the reversal potential of the neurally evoked postsynaptic current.     

Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen.

Apparent oxidation-reduction potentials at pH 7.0 and 25 degrees C were determined using the H2-hydrogenase system with ferredoxins from the following sources: Clostridium pasteurianum, -403 mV; C tartarovorum, -424 mV; C. acidi-urici, -434 mV; Peptococcus aerogenes, -427 mV; Chromatium D, -482 mV (pH 8.0); B. polymyxa, Fd I, -377 mV, and Fd II, -422 mV; and spinach, -428 mV. The pH dependence of these values was variable, ranging from -2 to -24 mV/pH unit increase for different ferredoxins. Over the range of buffer concentrations between 0.05 and 0.2 M, the potentials did not vary significantly. The number of electrons transferred during reduction (as determined by integrations of EPR spectra and by dithionite titration) is 2 for the first five proteins, while potentiometric data for all the cases fit a Nernst equation for which n = 1. The E degrees' value for the redox indicator methylviologen at pH 7.4 was found to be -460 mV, according to both the H2-hydrogenase system and cyclic voltammetry, significantly different from the value previously reported at higher pH's. Additionally, the presence of C. pasteuranum ferredoxin appears to shift the E degrees value of methylviologen to even more negative values. An analysis of sources of error inherent with potential determinations with H2 and hydrogenase is presented. The electronic and EPR spectra of P. aerogenes ferredoxin, for which the x-ray structure has been published, are given here. It appears that the determination of potentials of ferredoxin and other low-potential porteins with the H2-hydrogenase system affords certain experimental advantages over alternative methods currently employed with these and similar substances.