Membrane Depolarization by Hexacyanoferrate (III), Hexabromoiridate (IV) and Hexachloroiridate (IV)

Three artificial electron acceptors of different E_o and charge,hexacyanoferrate (III) (K₃Fe(CN)₆), hexachloroiridate (IV) (K₂IrCl₆),and hexabromoiridate (IV) (K₂IrBr₆), were compared with respectto their rate of reduction by roots of Zea mays L., the concomitantproton secretion, and to the effect on plasmalemma depolarization. It has been shown that these plasma membrane impermeable electronacceptors were reduced by a plasmalemma reductase activity.At low concentrations proton secretion was slightly inhibited,at higher concentrations, however, the rate of proton secretionwas stimulated. The root cell plasmalemma showed a transientdepolarization after addition of all three electron acceptors.The depolarization was concentration-dependent for the iridatecomplexes but not for hexacyanoferrate (III). For both iridatecomplexes maximum depolarization was reached at 50 µmoldm^–3. A hypothetical model as an explanation of the redox dependentproton secretion will be given. Key words: Hexachloroiridate (IV), hexabromoiridate (IV), hexacyanoferrate (III), plasmalemma redox, membrane potential, Zea mays     

Electron donation to the plasma membrane redox system of cultured carrot cells stimulates proton release.

Membrane-permeable electron donors, duroquinol, diphenylcarbazide, pyrocatechol and tert-octylcatechol, promoted both reduction of an impermeant electron acceptor and proton transport with cultured carrot cells. These cells were preloaded with electron donors for 15, 30, 45 and 60 min. Aliquots of cells were removed at various times, washed free of excess electron donors and assayed for their effect on transplasma membrane redox with impermeable hexacyanoferrate (HCF III) as the electron acceptor and for simultaneous H+ excretion in the presence of hexacyanoferrate. All four electron donors stimulated HCF III reduction and associated H+ excretion. Below a rate of hexacyanoferrate reduction of 6 mumol/g dry wt. per min, the ratios of H+/e- were between 0.3 and 1 with low concentrations (0.1 mM) of the added electron donors. When hexacyanoferrate reduction exceeded 6 mumol/g dry wt. per min, proton release began to cascade to give ratios of 1 to 3, suggesting activation of an H(+)-ATPase or a proton transporter. This behavior by cultured carrot cells indicates that a certain threshold of proton concentration in a limited membrane domain must be reached in order for the proton channel to be opened.     

Modification of the Activity of the Plasma Membrane Redox System of Zea mays L. Roots by Vitamin K3 and Dicumarol

The correlation of the effects of vitamin K₃ and dicumarol (ananti-vitamin K in pharmaceutical applications) on the transplasmamembrane electrical potential difference of maize roots withthe reduction of the artificial electron acceptors hexacyanoferrate(III) or hexabromoiridate (IV) and the concomitant enhancementof acidification of the incubation medium was investigated. Vitamin K₃ depolarized the plasma membrane of Zea mays L. roots,while dicumarol had no significant effect on the membrane potential.Plants treated with vitamin K₃ for 30 min followed by intenserinsing showed higher reduction of hexabromoiridate (IV) thanhexacyanoferrate (III), as well as a stimulated acidificationof the incubation medium. Depolarization of the plasma membraneby hexacyanoferrate (III) or hexabromoiridate (IV) decreasedafter an incubation with vitamin K₃. Pretreatment with dicumarolcaused an inhibition of hexacyanoferrate (III) reduction andmedium acidification as well as depolarization by K₃. The reductionof hexabromoiridate (IV) was not affected by dicumarol pretreatment.The proton secretion associated with the reduction was slightlylowered. According to our results, it seems possible that vitaminK₃ acts as an electron acceptor for the plasmalemma electrontransport system of maize roots whereas dicumarol appears toinhibit electron and proton transport. Key words: Vitamin K3, dicumarol, plasmalemma redox system, Zea mays L., membrane potential     

The Effects of Vitamin K3 and Dicumarol on the Plasma Membrane Redox System and H+ Pumping Activity of Zea mays L Roots Measured over a Long Time Scale

The effects of vitamin K₃ or dicumarol on plasma membrane boundhexacyanoferrate (III) and hexabromoiridate (IV) reductase activityand on the H⁺ pumping rate were investigated. Incubation withvitamin K₃ followed by intense rinsing stimulated the subsequentreduction of hexabromoiridate (IV) and hexacyanoferrate (III)as well as proton secretion induced by external electron-acceptors,while pretreatment with dicumarol inhibited proton secretioninduced by redox activity and hexacyanoferrate (III) reductionrate, but not the effects of hexabromoiridate (IV). A 30 minincubation in 0·2 mM K₃ or dicumarol, followed by rinsing,inhibited H⁺ secretion for about 2 d. Incubation for more than12 h in 0·1 mM dicumarol or 0·2 mM K₃ caused lethalinjury to the root cells. Key words: Vitamin K.₃, dicumarol, plasmalemma redox system, Zea mays L., proton pump     

Hormone action on transmembrane electron and h transport

A possible involvement of two different systems in proton translocation was investigated by simultaneous measurement of transmembrane electron flow and proton secretion in a pH-stat combined with a redoxstat. The pH gradient between cytoplasm and apoplast is probably maintained by an H⁺ -pumping ATPase and by a second proton extrusion system, which seems to be linked to a redox chain with NAD(P)H as electron donor. Indole acetic acid inhibits both e⁻ and H⁺ efflux, but only if the `electron draw' from the outside is not too high. The electron draw depends on the hexacyanoferrate level at the plasmalemma surface and on the Ca²⁺ concentration. The inhibiting effect of auxin on e⁻ and H⁺ efflux in the presence of hexacyanoferrate can be only detected at low levels of bivalent cations and of the artificial electron acceptor. The inhibition of e⁻ and H⁺ efflux by auxin requires high oxygen levels. The influence of auxin on both e⁻ and H⁺ transfer disappears below 2 kilopascals O₂, a level which does not influence respiration. Ethanol and fusicoccin do not increase the e⁻ flux, probably because the electron transfer from the plasma membrane to HCF III is the limiting step. If electron transfer is reduced by IAA pretreatment, ethanol increases e⁻ flux. Fusicoccin decreases e⁻ and increases H⁺ efflux if the rates have been lowered previously by indole acetic acid pretreatment. This effect depends on high oxygen levels and is reversible by lowering oxygen pressure. Auxin and Ca²⁺ change e⁻ flow and H⁺ ejection in a 1:1 ratio.     

A glucose-activated electron transfer system in the plasma membrane stimulates the H(+)-ATPase in Penicillium cyclopium.

Hyphal cells of three fungal species of the genus Penicillium reduced the nonpermeable, external electron acceptor hexabromoiridate IV (HBI IV). In Penicillium cyclopium, the rate of HBI IV reduction by hyphal cells was drastically increased by the addition of beta-glucose. The stimulation showed high specificity for this sugar and did not require its uptake and cellular metabolism. Cell wall oxidases (e.g., glucose oxidase) did not seem to be involved in the reduction of HBI IV, as no measurable H2O2 was formed from added glucose and removal of oxygen had no effect. We propose that there is a glucose-binding component outside the plasma membrane which controls transmembrane electron fluxes in response to external glucose. Reduction of HBI IV was accompanied by rapid acidification of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified with an e-/H+ stoichiometry of > 1. In plasma membrane vesicles containing endogenous electron donors, the membrane-residing fluoroprobe Di-8-ANEPPS reported a transient depolarization of the membrane potential triggered by the external electron acceptor. Inhibitors of ATP-dependent proton pumping enhanced the extent of this depolarization, inhibited the subsequent normalization of membrane potential, and, in whole cells, reduced the amount of redox-triggered proton extrusion. From these and other findings, it is concluded that the observed trans-plasma membrane redox process activates the H(+)-ATPase via membrane depolarization and cytosolic acidification.     

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

The mechanism of carrier-mediated sucrose uptake by the dermal transfer cells of developing Vicia faba L. cotyledons was studied using excised cotyledons and isolated transfer cell protoplasts. Addition of sucrose resulted in a transitory alkalinization of the bathing solution whereas additions of glucose, fructose or raffinose had no effect. Dissipating the proton motive force by exposing cotyledons and isolated transfer cell protoplasts to an alkaline pH, carbonylcyanide m-chlorophenylhydrazone, weak acids (propionic acid and 5,5-dimethyl-oxazolidine-2,4-dione) or tetraphenylphos-phonium ion resulted in a significant reduction of sucrose uptake. The ATPase inhibitors, erythrosin B (EB), diethylstilbestrol (DES) and N,N-dicyclohexylcarbodiimide (DCCD) were found to abolish the sucrose-induced medium alkanization as well as reduce sucrose uptake. Cytochemical localization of the ATPase, based on lead precipitation, demonstrated that the highest activity was present in the plasma membranes located in wall ingrowth regions of the dermal transfer cells. The presence of a transplasma-membrane redox system was detected by the extracellular reduction of the electron acceptor, hexacyanoferrate III. The reduction of the ferric ion was coupled to a release of protons. The redox-induced proton extrusion was abolished by the ATPase inhibitors EB, DES and DCCD suggesting that proton extrusion was solely through the H⁺-ATPase. Based on these findings, it is postulated that cotyledonary dermal transfer cells take up sucrose by a proton symport mechanism with the proton motive force being generated by a H ⁺ -ATPase. Sucrose uptake by the storage parenchyma and inner epidermal cells of the cotyledons did not exhibit characteristics consistent with sucrose-proton symport.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - EB erythrosin B - Em membrane potential - FC fusicoccin - HCF II hexacyanoferrate II - HCF III hexacyanoferrate III - Mes 2-(N-morpholino)ethanesulfonic acid - pmf proton motive force - TPP⁺ tetraphenylphosphonium ion The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

Plasmalemma redox activity in the diatom thalassiosira: a possible role for nitrate reductase

Plasmalemma redox activity in the diatom Thalassiosira is competitively inhibited by antiserum prepared against algal nitrate reductase (NR), and fluorescent labeling experiments reveal the binding of NR antiserum to the cell surface. Furthermore, the external electron acceptor Cu bathophenanthroline disulfonate causes immediate inhibition of intracellular primary amine production. A model is proposed in which plasmalemma-bound nitrate reductase reduces extracellular electron acceptors and intracellular nitrate and also acts as a trans-plasmalemma proton pump.     

Inhibition of trans-membrane hexacyanoferrate III reductase activity and proton secretion of maize (Zea mays L.) roots by thenoyltrifluoroacetone

Summary Intact plants can reduce external oxidants by an appearingly trans-membrane electron transport. In vivo an increase in net medium acidification accompanies the reduction of the apoplastic substrate. Up to now, several NAD(P)H dehydrogenases,b-type cytochromes, and a phylloquinone have been identified and partially purified from plant plasma membranes. The occurrence of a quinone in the plasma membrane of maize roots supports the hypothetical model of a proton-transferring redox system, i.e., an electron transport chain with a quinone as mobile electron and proton carrier. In the present study the trans-membrane electron transport system of intact maize (Zea mays L.) roots was investigated. Flow-through and ionostat systems have been used to estimate the electron and proton transport activity of this material. Application of 4,4,4-trifluoro-1-(2-thienyl)-butane-1,3-dione (thenoyltrifluoroacetone) inhibited the reduction of ferricyanide in the incubation solution of intact maize roots up to 70%. This inhibition could not be washed off by rinsing the roots with fresh incubation medium. The acidification of the medium induced after ferricyanide application was inhibited to about 62%. The effects of thenoyltrifluoroacetone on proton fluxes in the absence of ferricyanide have been characterized in a pH-stat system. The net medium acidification by maize roots was inhibited up to 75% by thenoyltrifluoroacetone in the absence of ferricyanide, while dicumarol inhibited net acidification completely. The inhibition of H⁺-ATPase activity was estimated with plasma membrane vesicles isolated by phase partitioning and treated with 0.05% (w/v) Brij 58. ATP-dependent proton gradients and P_i release were measured after preincubation with the effectors. The proton pumping activity by those plasma membrane vesicles was inhibited by dicumarol (53.6%) and thenoyltrifluoroacetone (77.8%), while the release of P_i was unaffected by both inhibitors.Abbreviations Brij 58 polyoxyethylene 20-cetyl ether - duroquinone tetramethyl-p-benzoquinone - HCF III hexacyanoferrate III - TTFA thenoyltrifluoroacetone - vitamin K₁ 2-methyl-3-phytyl-1,4-naphthoquinone - vitamin K₃ 2-methyl-1,4-naphthoquinone     

Induction of elongation in maize coleoptiles by hexachloroiridate and its interrelation with auxin and fusicoccin action

The demonstration of an auxin-stimulated NADH-oxidase in the plasma membrane (Brightman et al. 1988. Plant Physiol. 86: 1264–1269) has led to the suggestion that the plasma membrane redox system is involved in the mechanism of auxin action. To evaluate the relevance of this concept in vivo, the influence of micromolar concentrations of hexachloroiridate (IV), an impermeable electron acceptor for the plant plasma membrane redox system, on elongation growth of excised, abraded maize coleoptile ( Zea mays L. cv. Golden Bantam) segments was studied. It was found that the substance induced a rapid growth response if the experiment was carried out in an unbuffered solution. This effect was entirely prevented by a 2 m M phosphate buffer. Nevertheless, the acid-growth-theory does not seem sufficient to explain this effect, since proton extrusion is induced without a lag, whereas increased growth rates commence after a lag phase of 40 min.
If growth is stimulated by a pretreatment with fusicoccin or auxin, hexachloroiridate IV transiently inhibits growth. The kinetics of the response are then determined by the concentrations of hexachloroiridate and auxin or fusicoccin. These results are compatible with the view that the plasma membrane redox system is somehow involved in the control of elongation growth.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Rates of vectorial proton transport supported by cyclic electron flow during oxygen reduction by illuminated intact chloroplasts

The light-dependent quenching of 9-aminoacridine fluorescence was used to monitor the state of the transthylakoid proton gradient in illuminated intact chloroplasts in the presence or absence of external electron acceptors. The absence of appreciable light-dependent fluorescence quenching under anaerobic conditions indicated inhibition of coupled electron transport in the absence of external electron acceptors. Oxygen relieved this inhibition. However, when DCMU inhibited excessive reduction of the plastoquinone pool in the absence of oxygen, coupled cyclic electron transport supported the formation of a transthylakoid proton gradient even under anaerobiosis. This proton gradient collapsed in the presence of oxygen. Under aerobic conditions, and when KCN inhibited ribulose bisphosphate carboxylase and ascorbate peroxidase, fluorescence quenching indicated the formation of a transthylakoid proton gradient which was larger with oxygen in the Mehler reaction as electron acceptor than with methylviologen at similar rates of linear electron transport. Apparently, cyclic electron transport occured simultaneously with linear electron transport, when oxygen was available as electron acceptor, but not when methylviologen accepted electrons from Photosystem I. The ratio of cyclic to linear electron transport could be increased by low concentrations of DCMU. This shows that even under aerobic conditions cyclic electron transport is limited in isolated intact chloroplasts by excessive reduction of electron carriers. In fact, P₇₀₀ in the reaction center of Photosystem I remained reduced in illuminated isolated chloroplasts under conditions which resulted in extensive oxidation of P₇₀₀ in leaves. This shows that regulation of Photosystem II activity is less effective in isolated chloroplasts than in leaves. Assuming that a Q-cycle supports a H⁺/e ratio of 3 during slow linear electron transport, vectorial proton transport coupled to Photosystem I-dependent cyclic electron flow could be calculated. The highest calculated rate of Photosystem I-dependent proton transport, which was not yet light-saturated, was 330 mol protons (mg chlorophyll h)^–1 in intact chloroplasts. If H⁺/e is not three but two proton transfer is not 330 but 220 mol (mg Chl H)^–1. Differences in the regulation of cyclic electron transport in isolated chloroplasts and in leaves are discussed.     

Studies on hydrogenase activity and chlorobenzene respiration in <Emphasis Type="Italic">Dehalococcoides</Emphasis> sp. strain CBDB1

Hydrogen oxidation and electron transport were studied in the chlorobenzene-utilizing anaerobe Dehalococcoides sp. strain CBDB1. While Cu²⁺ and Hg²⁺ ions irreversibly inhibited hydrogenase activity in intact cells, Ni²⁺ ions inhibited reversibly. About 80% of the initial hydrogenase activity was inactivated within 30 s when the cells were exposed to air. In contrast, hydrogenase was active at a redox potential of +10 mV when this redox potential was established anoxically with a redox indicator. Viologen dyes served both as electron acceptor for hydrogenase and electron donor for the dehalogenase. A menaquinone analogue, 2,3-dimethyl 1,4-naphthoquinone, served neither as electron acceptor for the hydrogenase nor as electron donor for the dehalogenase. In addition, the menaquinone antagonist 2-n-heptyl-4-hydroxyquinoline-N-oxide had no effect on dechlorination catalyzed by cell suspensions or isolated membranes with hydrogen as electron donor, lending further support to the notion that menaquinone is not involved in electron transport. The ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone did not inhibit dechlorination by cell suspensions, indicating that strain CBDB1 does not require reverse electron transport. The ATP-synthase inhibitor N,N-dicyclohexylcarbodiimide inhibited the dechlorination reaction with cell suspensions; however, the latter effect was partially relieved by the addition of tetrachlorosalicylanilide. 1,2,3,4-Tetrachlorobenzene strongly inhibited dechlorination of other chlorobenzenes by cell suspensions with hydrogen as electron donor, but it did not interfere with either hydrogenase or dehalogenase activity.     

Light-Induced Polar pH Changes in Leaves of Elodea canadensis: II. Effects of Ferricyanide: Evidence for Modulation by the Redox State of the Cytoplasm

The effect of an extracellular electron acceptor, ferricyanide, on the light-induced polar leaf pH changes of the submerged angiosperm Elodea canadensis in light and in darkness was determined. The rate of transmembrane ferricyanide reduction was stimulated by increased light intensity and was inhibited by inorganic carbon, indicating that changes in the redox state of the chloroplast were reflected at the plasma membrane. The addition of ferricyanide inhibited the light-induced polar leaf pH reaction. This effect could be balanced by increasing the light intensity. In the dark, the acidification induced by ferricyanide was not influenced by diethylstilbestrol at concentrations that completely inhibited the polar leaf pH changes. This indicates that the ferricyanide-induced H⁺ extrusion and the H⁺ transport during the polar reaction were mediated by different mechanisms.     

Computer simulation study of pH-dependent regulation of electron transport in chloroplasts

A mathematical model is presented that describes the key steps of photosynthetic electron transport and transmembrane proton transfer in chloroplasts. Numerical modeling has been performed with due regard for regulatory processes at the donor and acceptor parts of photosystem (PS) I. The influence of pH-dependent activation of the Calvin cycle enzymes and energy dissipation in PS II (nonphotochemical quenching of chlorophyll fluorescence) on the light-induced redox transients of P₇₀₀, plastoquinone, and NADP as well as on the changes in intrathylakoid pH and ATP level is examined. It is demonstrated that pH-dependent regulatory processes alter the distribution of electron fluxes on the acceptor side of PS I and the total rate of electron flow between PS II and PS I. The light-induced activation of the Calvin cycle leads to significant enhancement of the electron flow from PS I to NADP⁺ and attenuation of the electron flow to molecular oxygen.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Electron transport and triplet formation in membranes of the photosynthetic bacterium Heliobacterium chlorum

The spectroscopic and thermodynamic properties of the electron-transport components of the photosynthetic bacterium Heliobacterium chlorum were studied by means of absorbance-difference spectroscopy. Upon flash illumination of membranes of H. chlorum photooxidation of the primary electron donor, P-798, was observed. In about 15% of the reaction centers P-798⁺ was reduced by cytochrome c-553, while in the remaining reaction centers P-798⁺ reduction occurred via a back reaction with a reduced electron acceptor. Titration experiments indicated a midpoint potential of −440 mV for the electron acceptor. At low redox potentials the formation of the triplet of P-798 was observed after a flash. The triplet was formed in about 30 ns by a back reaction with a reduced electron acceptor and decayed with a time constant of 35 μs. The yield of triplet formed in a flash was 30%. Upon continuous illumination at low redox potentials the accumulation in the reduced state of an electron acceptor was observed. The difference spectrum of this acceptor indicates that it is an iron-sulfur center. The yield of triplet formation was independent of the redox state of the iron-sulfur center, which indicates that the center is not located in the main electron-transport chain. A scheme with three acceptors in the main electron-transport chain is presented to accomodate our results and those of others.     

Dehydrogenase and electrochemical activity of <Emphasis Type="Italic">Escherichia coli</Emphasis> extracts

The dehydrogenase activity of Escherichia coli BB cell extracts was studied at different growth stages in the presence of different substrates and triphenyl tetrazolium chloride as an electron acceptor. It was shown that the highest degree of reduction of triphenyl tetrazolium chloride was observed during exponential growth of the bacteria when potassium isocitrate was used as a substrate. It was found that extracts of the bacteria during the exponential phase of growth on an inert glassy carbon electrode in a three-electrode liquid electrochemical cell manifested electrochemical activity in the presence of potassium citrate and methylene blue or potassium hexacyanoferrate(III) as redox mediators.     

Generation of a proton gradient in Desulfovibrio vulgaris

Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H₂, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl₂ as an inhibitor of periplasmic hydrogenase inhibited H₂ and formate oxidation with sulfur compounds, and lactate oxidation in H₂-grown, but not in lactate-grown cells. H₂ oxidation was sensitive to O₂ concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H₂ pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP⁺) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl₂ or carbon monoxide. The extrapolated H⁺/H₂ ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O₂ to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H⁺/H₂ ratios of 3.9 with H₂, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H⁺/H₂, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.