The Role of Electron Transfer and Dithiol-Disulfide Interchange in Solute Transport in Bacteria

The Role of Electron Transfer and Dithiol-Disulfide Interchange in Solute

Transport in Bacteria

M. G. L. ELFERINK: K. J. HELLINGWERF:

B. POOLMAN: AND W. N. KONINGSa aDepartment of Microbiology

University of Groningen Kerklaan 30

9751 NN Haren, the Netherlands bDepartment of Physical Chemistry

University of Groningen Nijenborgh 16

9747 AG Groningen, the Netherlands

J. M. VAN DIJL: G. T. ROBILLARD~

Bacteria require for growth and survival several organic and inorganic solutes that have to be transported across their cytoplasmic membrane. The mechanisms and the energy requirement of the translocation processes have been investigated extensively since the pioneering studies of Cohen and Monod.' With respect to the energy requirement of solute transport, several schools of thought have dominated during the years. Since the postulation of Lipman in 19412 that ATP functioned as the molecular energy currency in living cells, it was generally assumed that transport of solutes across the cytoplasmic membrane against their concentration gradients required the hydroly- sis of ATP.3 In the course of these studies, it was observed that one and the same mechanism was not involved in solute translocation but that bacterial cells have several distinct mechanisms at their disposal. In 1964 Kundig, Ghosh, and Roseman4 found that a number of sugars can be transported by phosphoenolpyruvate-dependent group translocation systems. In gram-negative bacteria, transport of several solutes can be mediated by systems in which periplasmic binding proteins play an essential role.5 The energy for these translocation processes is supplied directly by ATP or a phosphorylated intermediate. ATP also appears to be the direct energy source for the translocation of ions like K+ or N a + via ion ATPases and in some anaerobes for the translocation of other solutes like glutamate.6

The majority of the solutes, however, appears to be translocated by diffusion processes. For membrane-permeable or lipophilic solutes, no specific membrane proteins are required for the translocation processes and transport can occur by passive diffusion. However, The translocation across the cytoplasmic membrane of membrane- impermeable solutes and the rapid translocation of membrane-permeant solutes requires the involvement of such specific transport proteins (carriers).

These diffusion processes have been studied most extensively. The pioneering studies of Kaback in isolated cytoplasmic membrane vesicles unequivocally demonstrated that ATP nor any other phosphorylated intermediate functioned as the direct energy source for facilitated solute transport.' These observations were initially explained by Kaback and coworkers with a model in which the carrier protein was depicted as an electron transfer chain intermediate.'

36 I

362 ANNALS NEW YORK ACADEMY OF SCIENCES

According to this model, electron transfer led to changes in the oxidation state of a dithiol/disulfide group in the carrier that led to conformational and affinity changes of the solute binding sites. In the oxidized form, the carrier had a high affinity, in the reduced form, a low affinity for its solute.

This idea was abandoned mainly for reason that it was shown that the carriers were not obligate electron transfer intermediates and evidence in favor of a chemiosmotic energy coupling of solute transport emerged. The first evidence for a chemiosmotic coupling of solute transport was presented by West and Mitchell,' Hirata et al.," and Hamilton." In the last fifteen years the relation between the proton motive force and solute transport has been studied extensively in a number of bacteria." The role of the electrochemical proton gradient between the bulk phases a t both sides of the cytoplasmic membrane as a driving force for solute transport in bacteria is now well established.

In the last five years results have been presented that could not readily be placed in the chemiosmotic context and that indicated that control mechanisms and other mechanisms of energy coupling could be involved in solute t r a n ~ p o r t . ' ~ This led to a revival of the original localized chemiosmotic concept of Wil l iarn~. '~

In this paper we will describe some properties of solute transport in the phototrophic organism Rhodopseudomonas sphaeroides and the facultative aerobic organism Escherichia coli. The results indicate that a direct interaction can exist between the carriers and the electron transfer systems and that redox transitions in the carrier protein take place during the solute translocation step. Possible interpretations of these results will be discussed.

THE ROLE OF ELECTRON TRANSFER IN SOLUTE TRANSPORT IN BACTERIA

In the phototrophic bacterium Rhodopseudomonas sphaeroides, the existence of a proton motive force alone is not sufficient for solute uptake; turnover of the cyclic electron transfer chain is also necessary." The initial rate of uptake of the amino acid alanine has been measured at a constant light intensity (= constant rate of cyclic electron transfer), but with varying magnitudes of the proton motive force. Under these conditions the rate of transport increases exponentially with the proton motive force. However, when the proton motive force is kept constant and the rate of electron transfer is varied, the rate of transport increases linearly with the light intensity (FIG. 1). At low light intensities, there is no uptake of alanine, even when the proton motive force is high. These results demonstrate that the electron transfer chain functions not only as a generator of a proton motive force, but it also directly influences the activity of the carrier.

Such an interaction between the electron transfer system and solute transport carriers is not specific for Rpr. sphaeroides. In a strain of Rps. sphaeroides in which the E. coli transport protein for lactose (the M-protein) was incorporated via genetic transformation a similar relation between the rate of cyclic electron transfer and lactose transport was observed.16 Kinetic analysis of the changes in the initial rate of both alanine and lactose uptake indicated that the regulation is due to a light- dependent change in the number of active carrier molecules in the membrane.

The role of the electron transfer chain in solute transport was investigated in more detail. For that aim a thermostated vessel was constructed in which amino-acid or lactose uptake, the A$ (with an ion-selective TPP+ electrode), and the respiration rate (with an oxygen electrode) could be recorded simultaneously and continuously.

ELFERINK et al.: SOLUTE TRANSPORT IN BACTERIA 363

Furthermore, all experimental conditions were such that the proton motive force is composed of a A$ only since the ApH is abolished with nigericin.

These studies demonstrated that similar interactions between the transport carrier proteins and the linear electron transfer chain exist in Rps. sphaeroides and also in E. coli.” However, in the latter organism, differences between the various transport systems are observed. The initial uptake rate of glutamate and of lactose were measured in E. coli cells as a function of the respiration rate (FIG. 2). Respiration was progressively inhibited by the addition of increasing amounts of potassium cyanide. The proton motive force (composed solely out of a A$) under these conditions remained

c o n s t a n t light intensi ty

4 t 100% P l ight

/,d 66%

Y 2 I c o n s t a n t A ’ 6

I ight i n t e n s i t y (a rb i t rary .uni t $1

FIGURE 1. The relation between V,,, and A$ a t constant light intensity and between Val, and light intensity a t constant A$ in cells of Rps. sphaeroides.

Alanine uptake and A$ were measured simultaneously in cells treated with 2 mM K-EDTA and 2 r M nigericin at different light intensities. The incubation medium contained: 50 m M potassium phosphate pH 8, 5 m M MgS04, 50 p k f [‘‘Clalanine, 4 p M tetraphenylphosphonium, cells at 0.64 mg protein/ml and various concentrations of valinomycin. Upper panel: the alanine uptake rate (Vala) as a function of A$ a t constant light intensity. Lower panel: V,, as a function of light intensity at constant A$ values. Data from the upper panel were replotted.

approximately constant. The initial rate of uptake of glutamate decreased proportion- ally with the rate of respiration and at low respiration rates no uptake occurred. Thus, glutamate uptake requires, in addition to a proton motive force, obligatory electron flow, like solute transport in Rps. sphaeroides. Lactose transport, on the other hand, decreased in E . coli with the respiration rate, but was not completely abolished in the absence of electron transport. At very low respiration rates, the rate of lactose accumulation was still significant.


To investigate the differences between glutamate and lactose uptake in more detail, the uptake of both solutes in aerobically grown E. coli cells was measured under anaerobic and aerobic conditions in the presence or absence of a potassium- valinomycin-induced diffusion potential (FIG. 3) Under anaerobic conditions, glutamate was not transported, not even when the A$ was high. But a much more striking observation was that under aerobic conditions, both glutamate and lactose were transported even when the A+ was zero. It should be noted that in these E . coli cells, electrons can be transferred from endogenous substrates to oxygen. Lactose transport did occur with a potassium diffusion potential under anaerobic conditions, but the initial rate of uptake and the steady-state accumulation level were much Iower than in the presence of oxygen. Lactose uptake could only be abolished when both a A$ and electron transfer were absent. These results indicate that both a proton motive force and electron transfer can-independently-provide energy for lactose transport in E. coli.

The interaction between the electron transfer chains and transport carriers is not restricted to H+/symport systems but also occurs in Na'/methyl @-D-thiogalactopyra- noside (TMG) symport via the melibiose transport carrier of E. coli. This is a strong argument against the involvement of local proton pools or local proton pathways in the direct interaction.

In order to obtain more information about the mechanism that is the basis for the direct interaction, the lactose transport system has been studied in more detail in cytoplasmic membrane vesicles of E. coli. In these vesicles !inear electron transfer was

c C .- E

Respiration rate lnmol 0 mg-lmirrl)

FIGURE 2. The initial rate of lactose and glutamate uptake and the magnitude of A$ as a function of the respiration rate in cells of E. coli.

Cells of E. coli were pretreated with 2 m M K-EDTA and 2 pLM nigericin. Lactose uptake, glutamate uptake, A+ and respiration were measured simultaneously a t increasing KCN concentrations. The incubation medium contained: 50 m M potassium phosphate pH 8, 5 m M MgSO,, 200 p M [14C]lactose or 50 p M [14C]Na-glutamate, 4 pM TPP', cells a t 1.1 mg protein/ml. The KCN concentration was varied between 0 and 5 mM. 0 initial rate of lactose uptake; 0 initial rate of glutamate uptake; A membrane potential measured simultaneously with lactose uptake; A membrane potential measured simultaneously with glutamate uptake. (From M. G. L. Elferink and K. J. Hellingwerf, unpublished results.)

2

ELFERINK et al.: SOLUTE TRANSPORT IN

I I

BACTERIA 365

t

FIGURE 3. The effects of a potassium diffusion potential on glutamate and lactose transport in E. coli cells under aerobic and anaerobic conditions.

E . coli cells were pretreated with EDTA and suspended in 100 mM potassiumphosphate pH 8, 5 mM MgSO,, 40 p M valinomycin at a protein concentration of 75 mg/ml. The uptake experiment was started by a dilution of the cell suspension 1: 100 in 100 mMsodium phosphate pH 8, 5 mM MgSO,, 4 p M TPP+, 200 p M ['4C]lactose or 50 p M [14C]Na-glutamate, respectively. For the control experiments, cells were diluted in potassium phosphate buffer with the same additions.

0, uptake in the presence of a potassium diffusion potential; 0, uptake in the absence of a potassium diffusion potential. The values of the recorded A# are given in mV. (From M. G. L. Elferink and K. J. Hellingwerf, unpublished results.)

initiated with D-lactate as electron donor. The initial rate of lactose accumulation decreased with decreasing respiration rate when electron transfer was inhibited with increasing amounts of the lactate dehydrogenase (LDH) inhibitor oxamate. The proton motive force (A$) under these conditions decreased only slightly (FIG. 4).

In parallel experiments the A$ was titrated with valinomycin and the rate of lactose uptake was measured as a function of the proton motive force (FIG. 5). Under these conditions, the respiration rate remained approximately constant. The rate of lactose uptake decreased with the proton motive force, but in the absence of a proton motive force a significant rate of lactose uptake was still observed. This rest activity could only be abolished with electron transfer inhibitors or anaerobiosis. From these results it is also evident that the observed decrease of the rate of lactose uptake upon inhibition of electron flow (FIG. 4) cannot be explained by the slight decrease of the proton motive force. When electron flow in the respiratory chain is inhibited with oxamate a t the level of lactate dehydrogenase, the components of the electron transfer chain will become oxidized. Similar experiments to those presented in FIGURE 4 were performed with the terminal oxidase inhibitor cyanide. Under these conditions, the components of the


TC .- -i' 20 0, E

0 200 Loo 600

Respration rate hm01 o2 q-trnir-4

FIGURE 4. The effect of the rate of lactate oxidation, varied with oxamate, on lactose uptake and the electrical potential in E . coli membrane vesicles.

Membrane vesicles of E. coli ML 308-225, preincubated with 1 pMnigericin were resuspended to 0.3 mg protein/ml in 50 mM K-phosphate buffer pH 7.0 with 5 mM MgSO, and 3 p M TPP+. Energization was started by adding D,L-laCtate to a final concentration of 20 mM. The respiration rate was titrated with varying concentrations (up to 370 p M ) of the lactate dehydrogenase inhibitor oxamate. The membrane vesicles were incubated with oxamate for four minutes before adding D,L-laCtate. Finally [14C]lactose (0.2 mM) was added to start the uptake experiment.

0, initial rate of lactose uptake; 0 , steady-state level of lactose accumulation; A, electrical potential. (From J. M. van Dijl, M. G. L. Elferink, and K. J. Hellingwerf, unpublished results.)

respiratory chain will be more reduced. The results demonstrate that the rate of lactose uptake was similarly affected by the rate of electron flow indicating that the oxidation/reduction state of the electron transfer components does not determine the rate of lactose uptake. The turnover rate determines the rate of uptake, when the At) is the same. However, there is a difference in the steady-state level of lactose accumulation between a more oxidized and a more reduced electron transfer chain. This steady-state level of lactose accumulation is higher in the oxidized state indicating that efflux of lactose is inhibited when electron transfer chain components become more oxidized. An inhibition of lactose-efflux by oxamate, but not by cyanide, in membrane vesicles of E. coli was shown by Kaback and Barnes.'

A very attractive experimental possibility for investigating the role of electron transfer and the proton motive force in solute transport is offered by the presence of PQQ-dependent glucose dehydrogenase (GDH) in membrane vesicles of E. coli. In E. coli an upo-GDH is present, which is converted into the active holoenzyme upon addition of its prosthetic group pyrrolo-quinolinequinone (PQQ).'' This system is ideal for transport studies, because the enzyme is coupled to the respiratory chain and its activity can be increased by adding increasing amounts of PQQ to E. coli membrane vesicles. Therefore, the rate of electron flow and the redox state of the components of the electron transfer chain can be varied by adding varying amounts of PQQ.

ELFERLNK et al.: SOLUTE TRANSPORT IN BACTERIA 367

In FIGURE 6 the result of such an experiment is shown. At constant A+ value, the rate of lactose uptake decreased with the respiration rate, while Aj+Llactasc remained constant. At lower respiration rates (lower PQQ concentrations) both the A$ and Aplsctosc decreased. This decrease of the uptake rate is steeper since this is affected by both parameters involved in energization of transport.

In the experiments described above, D-lactate or glucose have been used as electron donors for energizing solute transport in E . coli membrane vesicles. Under both conditions a direct interaction between the solute carriers and the electron transfer chain occurs. Similar experiments were also performed with the electron donor succinate (FIG. 7). The rate of succinate-driven lactose accumulation was measured in the presence of different concentrations of the succinate dehydrogenase inhibitor malonate (to vary the rate of electron transfer) and in the presence of different concentrations of valinomycin (to vary the proton motive force). The results show that under these conditions the rate of lactose transport is independent of the rate of electron transfer and depends solely on the proton motive force.

These results suggest that the interaction between solute carriers and the electron transfer chain of E. coli occurs a t a level more electronegative than succinate dehydrogenase.

- -k 'E' 20 F 7

-8 E C -

J >

- > E -

Q

4 I r ' a

0 - 0 50 100

ay (mVI

FIGURE 5. The initial rate and steady-state accumulation level in E. coli membrane vesicles of lactose uptake as a function of the A+.

Lactose uptake, A+ and respiration rate were measured simultaneously at 3OoC and at increasing valinomycin concentrations with D,L-lactate as electron donor. The incubation medium contained: 50 mM potassium phosphate pH 7.0, 5 mM MgSO,, 3 p M TTP+, 20 mM D,L-lactate and the membrane vesicles suspension at a protein concentration of 0.3 mg/ml. The nigericin (1 pM) containing membrane vesicle suspension was pretreated with C-2 r M valinomycin. The uptake experiment was started by adding 200 pM ['4C]lactose to the vesicle suspension.

0, initial rate of lactose uptake; D, steady-state level of lactose accumulation.


ROLE OF DITHIOL-DISULFIDE INTERCHANGE IN SOLUTE TRANSPORT

A large number of reports has appeared on the involvement of sulfhydryl groups in the function of membrane proteins such as carriers involved in solute transport, energy-transducing enzymes and receptor proteins. Dithiol-disulfide interconversions have been reported to play an essential role in many solute transport and energy- transducing systems in bacteria, mitochondria, and chloroplast^.'^-^^ Moreover, many reports have demonstrated that the sensitivity of various transport systems to SH reagents is increased either by addition of substrates or by energization of the membrane or by both (for review see F ~ n y o ~ ~ ) . Such sulfhydryl reagent sensitivity has also been reported for the phosphoenolpyruvate-dependent glucose transport sys- tem25-27 and the lactose transport system of Escherichia ~oli?*,*~

It has been discussed in the introduction that these two transport systems operate by completely different mechanisms. The lactose transport system is a secondary transport system that translocates lactose in symport with protons. The PEP- dependent glucose transport system (glucose PTS) couples the transport of glucose to

50 150 Respiration rate (nmd O2 mq-' mi n-' )

FIGURE 6. The initial rate of lactose uptake, steady-state accumulation level, and A$ as a function of the respiration rate in E. coli membrane vesicles energized by PQQ-dependent glucose oxidation.

Lactose uptake, A$, and respiration rate were measured simultaneously a t 3OOC. The incubation medium contained: 50 m M potassium phosphate pH 7.5, 5 mM MgSO,, 3 g M TPP' and the membrane vesicles a t 0.7 mg protein/ml. The membrane vesicles were pretreated with 1 ~ L M nigericin. After five minutes of incubation with increasing amounts of PQQ (0-12.5 p M ) 20 m M glucose was added. After 2 min, when the A$ was maximal, the uptake experiment \*as started by adding 200 g M ['4C]lactose to the membrane vesicle suspension.

A, electrical potential; 0, initial rate of lactose uptake; 0, steady-state level of lactose accumulation. (From J. M. van Dijl, M. G. L. Elferink, and K. J. Hellingwerf, unpublished results.)

ELFERINK et al.: SOLUTE TRANSPORT IN BACTERIA

n

Kespiration raie 1 vaunomycin J UM J (nmol O2 rnsl rnin-1 )

FIGURE 7. The initial rate of lactose uptake and A+ as a function of the respiration rate and the valinomycin concentration in E. coli membrane vesicles energized with succinate.

Lactose uptake, A$ and respiration rate were measured simultaneously at 30OC. The incubation medium contained 50 mM potassium-phosphate pH 7.5, 5 mM MgSO,, 3 p M TPP+, 10 m M potassium succinate, and the membrane vesicle suspension at 0.6 mg protein/rnl. The respiration rate was titrated with 0-3.7 mM malonate (left panel). The membrane vesicles were preincubated with 1 p M nigericin and 0-20 ~Mvalinomycin (right panel).

A, A+; 0, initial rate of lactose uptake. (From J. M. van Dijl, M. G. L. Elferink, and K. J. Hellingwerf, unpublished results.)

. . . . . ,,--.. . . n . * . ,. . .I" - the hyarorysis or YEY via a series or phospnoryi group transfer reactions.- Kecently, we have presented evidence for the involvement of redox-sensitive dithiol groups in the membrane-bound enzyme I1 of the glucose PTS and a control of the activity of the enzyme by the redox state of these groups. The redox state of these dithiol-disulfide groups is determined by the proton motive force across the cytoplasmic membrane and by the redox potential of the en~ironment .~ '

Changes in the binding of lactose to the carrier have also been shown to be dependent on the proton motive force across the membrane. In the absence of a proton motive force, the binding affinity is low; in the presence of a proton motive force, the binding affinity is high.32 Recently we investigated the mechanism controlling the activities of these carriers.

The accumulation of L-proline and lactose in right-side-out membrane vesicles of E. coli can be energized by ascorbate-phenazine methosulfate, D-lactate or an artificially imposed chlorate or potassium diffusion potential. This uptake is inhibited to the uptake levels observed under nonenergized conditions by lipophilic oxidants like plumbagin (0.5 mM), phenazine methosulfate (PMS) (5 mM), menadione (0.6 mM), and 1,2-naphthoquinone (0.3 mM).33 Strong inhibitions of uptake were also observed with polar oxidants like ferricyanide (10 mM) provided that the membrane vesicles were preincubated for several minutes with these The inhibitions exerted by these oxidants could not be explained by a decreased flow of electrons to oxygen and/or a decreased proton motive force. For instance in the presence of 10 m M ferricyanide, the rate of D-lactate oxidation was decreased to only 15% and the proton motive force was decreased from - 124 mV to - 11 3 mV. In all cases the addition of dithiothreitol (DTT) to the membrane vesicle suspension fully restored the transport


activity (FIG. 8). These data demonstrate that the activity of the carriers for L-proline and lactose of E. cofi can be regulated by altering the redox potential in the membrane in a similar way as was demonstrated for the glucose-PTS. Kinetic studies of L-proline transport energized by a chlorate diffusion potential in the presence of different concentrations of plumbagin demonstrate that oxidation of the carriers leads to a decrease of the V,,, and thus to inactivation of the carriers (S. Wijma and W. N . Konings, unpublished results). These observations are in agreement with results obtained by Neuhaus and Wright.”

The redox process involves the conversion of dithiols to disulfide or vice versa in the carrier proteins. In the proline carrier, two sets of dithiol/disulfides play a role; one set is located at the outer surface, the other a t the inner surface of the cytoplasmic membrane.34 This conclusion is based on the following observations. In right-side-out membrane vesicles, electron transfer in the respiratory chain leads to the generation of a proton motive force, inside negative and alkaline, and to the conversion at the outer surface of a disulfide to a dithiol as is shown by the increased inhibition of proline transport by the membrane-impermeable dithiol reagent thorin. The inhibition exerted by thorin was completely reversed by dithiol threitol. A similar but irreversible inhibition was observed with the membrane-impermeable SH reagent glutathione hexane maleimide. Pretreatment of the membrane vesicles with ferricyanide or with

5l No Additions

- / - A ~ Ap +Piurnbagin+ DTT

No Additipns K-K + urn aatn

Min FIGURE 8. Effects of 0.5 mM plumbagin and 10 mM dithiothreitol on uptake of L-proline by membrane vesicles of E. coli energized by a valinomycin-induced potassium diffusion potential. Membranevesicles (35 mg proteinlml) in 50 mMpotassium phosphate pH 8 were incubated with 10 pM valinomycin for 30 min and with the redox reagents for five minutes each before dilution 100-fold into 50 mMTris maleate pH 8 containing 10 mM MgSO, and 3.5 rM [~-‘~C]proline or 50 mM potassium phosphate pH 8 containing 10 mM MgSO, and 3.5 pLM [~-‘~C]proline (K - K). Experiments were performed at room temperature. No additions: with a potassium gradient; plumbagin + DTT: with a potassium gradient, incubated with 0.5 mM plumbagin and then with 10 mM dithiothreitol; DTT: with a potassium gradient, incubated with 10 mM dithiothreitol; No additions K -+ K: no potassium gradient; + plumbagin: with a potassium gradient, incubated with 0.5 mM plumbagin. (From G . T. Robillard and W. N. Koning~’~ with permission.)

ELFERINK et al.: SOLUTE TRANSPORT IN BACTERIA 371

20( [r 0 t- U

3

4 J

z E 15c

3

U U a

100

5(

~

THORIN + DTT NO ADD

TH ORIN + G SM +

DTT > THORIN

0

GSM

1 2 3 4 5 TIME ( M I N I

FIGURE 9. Protection of thorin against glutathione hexane maleimide inactivation of proline transport in membrane vesicles of E. coli. Right-side-out membrane vesicles (1.4 mg protein/ml in 50 mM potassium phosphate pH 7 and 10 mM MgSOI) were incubated under aerobic conditions in the presence of ascorbate (20 m M ) PMS (100 g M ) with 1 mM thorin for 5 min at 26OC and if indicated subsequently for another 5 min with 5 mM glutathione hexane maleimide (GSM). Where indicated, 10 mMDTT was added to stop the reaction of GSM. When sequential additions are involved, theorder of addition is indicated by the order listed in the figure. Uptake of [~-'~C]proline (3.5) pM) was started immediately after the end of the reactions. (From Poolman et nl." with permission.)

thorin protected against glutathione hexane maleimide inhibition since the transport activity of L-proline was fully restored after DTT treatment (FIG. 9). Both SH reagents therefore appear to react with the same SH groups of a dithiol located a t the outer surface.

Evidence for the involvement of dithiols located at the inner surface of the cytoplasmic membrane was obtained from studies with inside-out membrane vesicles. These inside-out membrane vesicles accumulate L-proline if a chlorate diffusion potential (inside negative) is imposed. Ferricyanide reversibly inhibits this proline uptake, which appears to be the result of oxidation of SH groups since ferricyanide also protects against the irreversible inhibition by glutathione hexane maleimide. A similar protection against glutathione hexane maleimide inactivation in these inside-out membrane vesicles could be achieved by imposition of a proton motive force by electron flow, inside positive and acid, indicating that a dithiol is converted to a disulfide upon energization. These results strongly indicate that two redox-sensitive dithiol groups play a role in the carrier of proline (and lactose). One center is located at the inner surface of the membrane and one at the outer surface. Establishing a proton motive force, interior negative and alkaline, converts the dithiol to disulfide on the inner


surface of the cytoplasmic membrane and disulfide to dithiol on the outer surface (FIG. 10). This is precisely the distribution of redox states expected on theoretical grounds for redox centers located in different sectors of the membrane in the presence of a proton motive force, inside negative and alkaline.36

A MODEL FOR PROTON-SOLUTE SYMPORT

The observations described above can be summarized as follows. In Rps. sphaeroides and E. coli, a direct interaction exists between the transport proteins and the electron transfer systems. This direct interaction can, in addition to the proton motive force, supply energy for solute transport. The tightness of the interaction varies for the different solutes. The site of interaction appears to be located at the electronegative site of the electron transfer system. The transport proteins contain redox-sensitive dithiol/disulfide groups at the inner surface and a t the outer surface of the membrane. The redox state of these groups is determined by the redox potential of the environment and by the proton motive force. The redox state of the dithiol/ disulfide group determines whether the high-affinity binding site in its vicinity is accessible or not. On the basis of these observations, the following scheme for solute transport is postulated (FIG. 11). The carrier is depicted as a nonobligatory intermediate a t an electronegative level of the electron transfer system. In the absence of a specific interaction with the electron transfer chain, the carrier can undergo the following cycle. A proton motive force, inside negative and alkaline, will bring the redox group at the outer surface in the reduced, dithiol form and solute can bind a t the high-affinity site. Under these conditions, the redox group at the inner surface will be oxidized and the binding site a t the inner surface will not be accessible. Solute binds at the binding site a t the outer surface. Binding of protons on base B- in the vicinity of the dithiol will cause a decrease both in the apparent pK of the dithiol and the midpoint potential of the redox transition resulting in the oxidation of this center. Hydrogens move across the membrane reducing the inner redox center and raising the pK of the inner base, BH. Consequently the base deprotonates, releasing protons to the inner solution. The change in redox states of the inner and outer sites causes a loss of the solute binding site a t the outer surface and exposure of a high-affinity site a t the inner surface. As a result solute is drawn through the channel and bound to the inner site. At this point protons and solute have been translocated, but the carrier is out of redox equilibrium. This can be reestablished by movement of electrons back to the outer

FIGURE 10. Relationship between A$ and the redox potential in the membrane. $ is the electrical potential and E is the redox potential. (Modified from Robillard and Koning~.’~)

ELFERINK ef al.: SOLUTE TRANSPORT IN BACTERIA 313

FIGURE 11. A redox mechanism for a proton solute symport system parallel coupled to the electron transfer system. (Modified from Robillard and Kon- ing~. '~)

Q = quinone, DH = dehydrogenase. A T (

I

redox center. The inner site becomes oxidized and releases the ligand, and the outer site becomes reduced to complete the cycle.

When the carrier interacts directly with the electron transfer system, electrons can be donated to the outer redox center bringing this center to the reduced form. The same sequence of events can occur as described above except for the final step in which the inner redox center becomes oxidized by transferring electrons to an intermediate of the electron transfer system. According to this scheme, electrons can be transferred within the carrier from one redox center to the other or via electron transfer chain intermediates. When the internal transfer is not possible, the carrier depends completely on the electron transfer system for activity. This could be the situation in glutamate transport in E. coli. Other carriers such as the lactose carrier of E. coli might operate in both modes.

The scheme presented in FIGURE 1 1 has been drawn for a situation in which one H' is translocated per solute. A translocation of two H+ per solute would require the transfer of two electrons within the carrier or via electron transfer intermediates.

Finally, it should be noticed that the redox model presented above has certain aspects in common but differs in other aspects with a redox model postulated in 1971 by Kaback and Barnes.'

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The Role of Electron Transfer and Dithiol-Disulfide Interchange in Solute Transport in Bacteria

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