The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.     

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.     

Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem.

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.     

Protonmotive force and catecholamine transport in isolated chromaffin granules.

The effect of the transmembrane potential (delta psi) and the proton concentration gradient (delta pH) across the chromaffin granule membrane upon the rate and extent of catecholamine accumulation was studied in isolated bovine chromaffin granules. Freshly isolated chromaffin granules had an intragranular pH of 5.5 as measured by [14C]methylamine distribution. The addition of ATP to a suspension of granules resulted in the generation of a membrane potential, positive inside, as measured by [14C]thiocyanate (SCN-) distribution. The addition of carboxyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a proton translocator, resulted in a reversal of the potential to negative values (measured by [3H]tetramethylphenylphosphonium (TPMP+)) approaching -90 mV. Changing the external pH of a granular suspension incubated with FCCP produced a linear perturbation in the measured potential from positive to negative values, which can be explained by the distribution of protons according to their electrochemical gradient. When ammonia (1 to 50 mM) was added to highly buffered suspensions of chromaffin granules there was a dose-dependent decrease in the transmembrane proton gradient (delta pH) and an increase in the membrane potential (delta psi). On the other hand, thiocyanate or FCCP, at varying concentration, produced a dose-related collapse of the membrane potential and had no effect upon the transmembrane proton gradient. The addition of larger concentrations of catecholamines caused a decrease in the transmembrane proton gradient and an increase in the membrane potential. Time-resolved influx of catecholamines into the granules was studied radiochemically using low external catecholamine concentrations. The accumulation of epinephrine or norepinephrine was over one order of magnitude greater in the presence of ATP than in its absence. The rate and extent of amine accumulation was found to be related to the magnitude of the membrane potential at fixed transmembrane proton concentration (delta pH) values. Likewise, the accumulation was related to the magnitude of the delta pH at fixed membrane potential values. These results suggest that the existence of both a transmembrane proton gradient and a membrane potential are required for optimal catecholamine accumulation to occur.     

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.     

Bioenergetic properties of alkalophilic Bacillus sp. strain C-59 on an alkaline medium containing K2CO3.

Alkalophilic Bacillus sp. strain C-59 could grow well on an alkaline medium containing K2CO3, as well as Na2CO3, but did not grow on K+-depleted medium. Right-side-out membrane vesicles, energized in the absence of Na+, however, could not take up [14C]methylamine actively, while vesicles equilibrated with 10 mM NaCl actively took up [14C]methylamine. The uptake of [14C]serine was also stimulated by the addition of Na+, and the imposition of a sodium gradient caused transient uptake. These results indicated that an Na+/H+ antiporter was involved in pH homeostasis and generation of an electrochemical sodium gradient in strain C-59 even though a growth requirement for Na+ was not evident. The efflux of 22Na+ from 22Na+-loaded vesicles was more rapid at pH 9.5 than at pH 7 in the presence of an electron donor. On the other hand, vesicles at pH 7 showed more rapid efflux than at pH 9.5 when the antiporter was energized by a valinomycin-mediated K+ diffusion potential (inside negative).     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

The effect of insulin on plasma-membrane and mitochondrial-membrane potentials in isolated fat-cells.

1. A recently developed technique for the measurement of plasma-membrane and mitochondrial-membrane potentials in intact cells by using the distribution of 86Rb+ and [3H]methyltriphenylphosphonium+ has enabled us to characterize a novel insulin effect on fat-cell mitochondria. For control cells the plasma-membrane and mitochondrial-membrane potentials were 75 mV and 152 mV respectively. Insulin (10 mu units/ml) caused a 9 mV hyperpolarization of the plasma membrane and a 19 mV depolarization of the mitochondrial membrane. 2. The insulin-dependent mitochondrial depolarization was observed at physiological insulin concentrations (10 mu units/ml) and was apparent when the cells metabolized a wide variety of substrates. 3. Evidence from the uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione by fat-cells was interpreted as indicating that the mitochondrial pH gradient was increased by insulin. 4. Insulin alters the balance between the electrical and pH-gradient components that form the mitochondrial protonmotive force. A model is proposed.     

Effects of K+ on the proton motive force of Bradyrhizobium sp. strain 32H1.

In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase.     

K+ and Cl- uptake by cultured oligodendrocytes

Cultured oligodendrocytes take up K+ triggered by an increase in [K+]o. Simultaneously [Cl-]i increases in the majority of the oligodendrocytes. This KCl uptake, which is not furosemide sensitive, can be explained by the following model. The first event is the entry of Cl- into the cell driven by the discrepancy between the membrane and Cl- equilibrium potential. As a consequence of the movement of negative charge across the membrane, K+ is driven into the cell. The prerequisites of this model, a passive Cl- distribution at resting membrane potential and a Cl- conductance of the membrane were found to exist in most cultured oligodendrocytes. The chloride equilibrium potential (-61 mV, SD +/- 10 mV) was slightly more positive than the membrane potential (-64 +/- 8 mV). Since cell input resistance determined with two independent electrodes increased by 11% (SD +/- 0.07) when [Cl-]o was reduced to 10 mM, part of the membrane conductance appears to be mediated by Cl-. Differences between membrane potential and Cl- equilibrium potential therefore will lead to Cl- fluxes across the membrane. In contrast with oligodendrocytes, [Cl-]i in astrocytes is significantly increased (from 20 to 40 mM) above the equilibrium distribution owing to the activity of an inward directed Cl- pump; this suggests a different mechanism of K+ uptake in these cells.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation.

A proton motive force (delta (-) microH+) of 70 to 130 mV was measured across the membrane of Mycoplasma gallisepticum cells. The membrane potential was measured utilizing the lipid-soluble cation tetraphenylphosphonium. The method was validated by showing that in the presence of valinomycin the ratio of the concentrations (in/out) of tetraphenylphosphonium agreed well with those for K+ and Rb+. The pH gradient was calculated from the measured distribution ratio of benzoic acid. The proton motive force was approximately the same in cells harvested at early exponential, midexponential, and stationary phases of growth. The proportion of pH gradient to membrane potential varied with external pH. In the absence of glucose, cells incubated in an isosmotic NaCl solution showed low adenosine triphosphate and delta (-) microH+ levels and a tendency to swell and lyse compared with cells incubated with added glucose. It is concluded that energy is required for normal cell volume regulation.     

The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.     

The Quinol:Fumarate Oxidoreductase from the Sulphate Reducing Bacterium Desulfovibrio gigas: Spectroscopic and Redox Studies

The membrane bound fumarate reductase (FRD) from the sulphate-reducer Desulfovibrio gigas was purified from cells grown on a fumarate/sulphate medium and extensively characterized. The FRD is isolated with three subunits of apparent molecular masses of 71, 31, and 22 kDa. The enzyme is capable of both fumarate reduction and succinate oxidation, exhibiting a higher specificity toward fumarate (K _m for fumarate is 0.02 and for succinate 2 mM) and a reduction rate 30 times faster than that for oxidation. Studies by Visible and EPR spectroscopies allowed the identification of two B-type haems and the three iron–sulphur clusters usually found in FRDs and succinate dehydrogenases: [2Fe-2S]^2+/1+ (S1), [4Fe-4S]^2+/1+ (S2), and [3Fe-4S]^1+/0 (S3). The apparent macroscopic reduction potentials for the metal centers, at pH 7.6, were determined by redox titrations: –45 and –175 mV for the two haems, and +20 and –140 mV for the S3 and S1 clusters, respectively. The reduction potentials of the haem groups are pH dependent, supporting the proposal that fumarate reduction is associated with formation of the membrane proton gradient. Furthermore, co-reconstitution in liposomes of D. gigas FRD, duroquinone, and D. gigas cytochrome bd shows that this system is capable of coupling succinate oxidation with oxygen reduction to water.     

The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature.

The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.     

The electrochemical proton gradient in Mycoplasma cells

The electrochemical proton gradient, delta mu H+ generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, delta pH, and the membrane potential, delta psi, were measured using several methods. The determination of the delta pH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extra-cellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of - 85 mV, interior negative. The delta mu H+ in M. mycoides var. Capri cells is thus about - 115 mV. When the external pH was altered from 7.7 to 5.7 delta psi decreased from - 90 mV to - 60 mV. On other hand although the internal pH decreased, delta pH was found to increase from 0.2 to 1.0 pH unit. Since the changes in delta psi were largely compensated by the changes in delta pH, delta mu H+ remained practically constant at about - 115 mV throughout the pH range tested. Finally, inhibition of delta pH by N,N'-dicyclohexylcarbodiimide, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.     

Direct electrochemical oxidation of Clostridium pasteurianum ferredoxin: Identification of facile electron-transfer processes relevant to cluster degradation

Rapid oxidation processes relevant to the degradation of [4Fe4S] clusters in Clostridium pasteurianum ferredoxin were studied via direct (unmediated) heterogeneous electron transfer at a pyrolytic graphite electrode. Differential-pulse voltammograms of native [4Fe4S] ferredoxin showed two well-defined oxidation peaks corresponding to apparent E-values of +793 and +1120 mV at 5°C. Direct involvement of the cluster was established through parallel experiments with the 2[4Fe4Se] derivative for which peak positions were shifted. Square-wave voltammetry showed that the product of the first electron transfer, which may correspond to the ‘super-oxidised’ [4Fe4S]³⁺ oxidation level, undergoes rapid degradation (t_{$\text{1}\text{2}$} < 1.6 ms at 5°C). The second oxidation process, as characterised by a significant (?100 mV) negative shift upon selenium substitution, very likely represents oxidation of S(Se) still associated with the protein and possibly contained within the remaining FES(Se) substructure.