Stimulation of Menaquinone-Dependent Electron Transfer in the Respiratory Chain of Bacillus subtilis by Membrane Energization

At a pH of $<=$ 7, respiration of Bacillus subtilis cells on endogenoussubstrates shut down almost completely upon addition of an uncoupler(carbonyl cyanide m-chlorophenylhydrazone [CCCP]) and a K⁺-ionophore(valinomycin). The same effect was observed with cell spheroplastslacking the cell wall. The concentration of CCCP required for50% inhibition of the endogenous respiration in the presenceof K⁺-valinomycin was below 100 nM. Either CCCP or valinomycinalone was much less efficient than the combination of the two.The inhibitory effect was easily reversible and depended specificallyon the H⁺ and K⁺ concentrations in the medium. Similar inhibitionwas observed with respect to the reduction of the artificialelectron acceptors 2,6-dichlorophenolindophenol (DCPIP) andN,N,N',N'-tetramethyl-p-phenylenediamine cation (TMPD⁺), whichintercept reducing equivalents at the level of menaquinol. Oxidationof the reduced DCPIP or TMPD in the bacterial cells was notsensitive to uncoupling. The same loss of the electron transferactivities as induced by the uncoupling was observed upon disruptionof the cells during isolation of the membranes; the residualactivities were not further inhibited by the uncoupler and ionophores.We conclude that the menaquinone-dependent electron transferin the B. subtilis respiratory chain is facilitated, thermodynamicallyor kinetically, by membrane energization. A requirement foran energized state of the membrane is not a specific featureof succinate oxidation, as proposed in the literature, sinceit was also observed in a mutant of B. subtilis lacking succinate:quinonereductase as well as for substrates other than succinate. Possiblemechanisms of the energy-dependent regulation of menaquinone-dependentrespiration in B. subtilis are discussed.

INTRODUCTION

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Introduction

There is a classical phenomenon of a so-called respiratory controlin mitochondrial oxidative phosphorylation (35; reviewed inreference 18). Electron flow in the mitochondrial respiratorychain becomes slow as the membrane is energized (the state ofrespiratory control) and can be stimulated under these conditionsby addition of ADP, which turns on ATP synthesis, or by theuncouplers of oxidative phosporylation, which dissipate thetransmembrane electrochemical proton potential difference ( ${Delta}$ µH⁺).Although described initially for mitochondria, the effect ofrespiratory control is often assumed to apply to bacterial coupledrespiration as well (see, e.g., reference 43).

It is therefore interesting that respiration of the aerobicbacterium Bacillus subtilis is stimulated rather than suppressedby proton motive force. The fact that the respiration of B.subtilis cells can be inhibited by K⁺/H⁺ and Na⁺/H⁺ antiportersand protonophores was noticed about 10 years ago (3, 37, 46)and confirmed more recently by Shirawski and Unden (47). A potentiallyrelevant observation is that the succinate oxidase activityof B. subtilis cells decreases drastically during isolationof the membranes (36, 47); this effect could also be a consequenceof membrane deenergization.

The mechanism underlying the energy requirement for B. subtilisrespiration remains to be established. Acidification of cytoplasm(3) and loss of cytoplasmic K⁺ (46) were proposed to be involvedin the uncoupler-induced inhibition of respiration of bacterialcells. In contrast, Unden and collaborators (37, 47) assignedthe energy dependence of respiration specifically to protonmotive force-driven transmembrane electron transfer from succinateto menaquinone (MQ) through succinate:MQ reductase as describedbelow.

Succinate:quinone oxidoreductases (SQR) of bacteria and mitochondria(called also complex II of the respiratory chain) catalyze two-electronreduction of membrane-bound quinone (usually ubiquinone [UQ]or MQ) to quinol by succinate. Besides this, many bacteria containenzymes that are homologous to SQR but are more efficient catalyticallyin performing the reverse quinol:fumarate reductase (QFR) reaction(see reference 33 for classification). The midpoint redox potential(E_m) of the MQ-menaquinol couple (MQ-MQH₂) is ca. -80 mV atpH 7, which is some 110 mV more negative than the E_m of thesuccinate-fumarate couple (ca. +30 mV at pH 7 [9, 14, 37]).Therefore, the succinate:quinone reductase activity in MQ-containingbacteria such as B. subtilis (10) poses an obvious thermodynamicproblem (36).

The dehydrogenase partial activity of SQR, i.e., electron transferfrom succinate to artificial electron acceptors such as N-methylphenaziniumsulfate (PMS) or ferricyanide, is associated in both mitochondriaand bacteria with the water-soluble two-subunit peripheral partof the enzyme, which contains the dicarboxylate binding center,covalently bound FAD, and three different iron-sulfur centers(reviewed in references 14, 20, and 38). The ability to reducehydrophobic quinones is conferred to the enzyme by a so-calledmembrane anchor subunit, whose structure and composition canvary significantly in different organisms (14, 38). In B. subtilis,this subunit is a diheme cytochrome b (15), with the two porphyringroups arranged across the membrane on top of each other, aspredicted by mutagenesis studies (14, 16) and confirmed recentlyby the three-dimensional crystal structure of complex II fromWolinella succinogenes (32, 34). The low-potential heme b (approximatelyisopotential with MQ-MQH₂ [15, 50]) is located close to theouter periplasmic side of the membrane and is believed to bethe MQ reducing site (20, 39, 42, 47, 50). The high-potentialheme b located near the cytoplasmic side appears to be withinfast electron transfer distance from the terminal iron-sulfurcenter S-3 of the dehydrogenase part of the enzyme.

Such a "vectorial" arrangement of the two b hemes across themembrane implies that the membrane potential and proton motiveforce can promote reduction of the low-potential heme b by succinatevia the high-potential heme b and thus stimulate the thermodynamicallyunfavorable MQ reductase activity of SQR. According to thismodel, the reverse QFR reaction catalyzed by SQR or QFR shouldgenerate ${Delta}$ µH⁺ (31). Although they are consistent with thecrystal structure of complex II, these predictions remain tobe proved. Recent experiments with whole B. subtilis cells providesome evidence for electrogenicity of fumarate reduction withglucose as an electron donor (48). On the other hand, detailedstudies with a structurally similar diheme QFR from W. succinogenes,carried out both with the whole cells and with the purifiedliposome-reconstituted enzyme, did not reveal either membranepotential or transmembrane pH difference ( ${Delta}$ pH) generation coupledto the QFR reaction (6, 13, 27, 28, 41). No proton pumping couldalso be demonstrated for homologous but heme-deficient QFR fromEscherichia coli (see references 8, 22, and 42 for a discussionof a possibility of vectorial charge transfer in different typesof complex II).

In several recent works, the putative energy-linked reductionof MQ by succinate via the low-potential heme b has been invokedto explain the energy-dependent stimulation of electron transferin the respiratory chain of B. subtilis (39, 42, 47). On theother hand, the energy-dependent activation of SQR may wellbe a separate phenomenon, not related to the specific mechanismof the endergonic MQ reduction by B. subtilis SQR. For instance,proton motive force activates SQR in plant mitochondria (2),although the enzyme catalyzes exergonic reduction of UQ andits membrane anchor contains only one heme b.

In this work we have explored the effect of the uncoupling onelectron transfer in the respiratory chain of B. subtilis inmore detail. Special attention was paid to the proposal thatthe phenomenon is specifically associated with succinate oxidation(47). Both intact cells and isolated membranes were studiedwith different respiratory substrates as electron donors andeither oxygen or artificial redox dyes as electron acceptors.We find that deenergization of the B. subtilis coupling membraneeither by uncoupling agents or as a consequence of breakingthe cells during preparation of the membranes severely inhibitsrespiration with oxygen as well as reduction of 2,6-dichlorophenolindophenol(DCPIP) and oxidized N,N,N',N'-tetramethyl-p-phenylenediamine(TMPD⁺) at the level of MQH₂. However, contrary to earlier conclusions(37, 47), these effects are observed not only with succinatebut also with other physiological electron donors such as NADHor glycerol-3-phosphate (GP) as well as with endogenous substratesin cells grown on succinate, glycerol, or malate. Moreover,results very similar to those observed with wild-type bacteriawere obtained with a mutant strain deficient in SQR.

We conclude that the stimulating effect of energization on electrontransfer in the respiratory chain of B. subtilis is not associateduniquely with the succinate:quinone reductase complex of therespiratory chain. Accordingly, the widely discussed specificmolecular mechanism of the effect based on a unique structureof SQR (47) cannot explain the phenomenon. We propose that electrontransfer in the respiratory chain of B. subtilis, and perhapsin other MQ-dependent bacteria as well, may be kinetically controlled(stimulated) by membrane energization at the level of MQ reductionby the dehydrogenases. Possible mechanisms of energy-dependentstimulation of electron flow in the respiratory chain of B.subtilis that may be common for different MQ-dependent pathwaysare discussed.

MATERIALS AND METHODS

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Materials and Methods

Strains and preparations. The following B. subtilis strains, kindly provided by Lars Hederstedtand Claes von Wachenfeldt (Lund University), were used: (i)the "wild-type" strain 168 A (trpC2), (ii) the SQR-lacking strainLUH-16 (trpC2 ${Delta}$ sdhCAB::ble), and (iii) a strain with two- to threefold-enhancedexpression of SQR and lacking the bd-type terminal oxidase,LUW-20/pBSD1400 (trpC2 ${Delta}$ cydABCD::tet, transformed by the low-copy-numberpHP13 plasmid carrying the sdhCAB DNA region). The cells weregrown in flasks at 37°C and pH 7.0, with high aeration,in medium containing 0.5% yeast extract (Difco), 50 mM potassiumphosphate, and 1 mM MgSO₄ and supplemented with 50 mM sodiummalate or 50 mM sodium succinate with or without 0.5% glycerol.The culture was harvested near the end of the exponential phaseof growth, when the optical density of the cell suspension at600 nm reached $~$ 1.5. Membranes were isolated essentially as describedpreviously (19). All of the chemicals used were commercial productsfrom conventional sources (Sigma, Serva, Fluka, or Merck).

Measurements. Oxygen consumption was measured with a Clark-type oxygen electrodein a 1.1-ml cell at 25°C. Reduction of DCPIP (50 µM,with or without 0.4 mM PMS) and TMPD⁺ (100 µM) by thecells and membranes was monitored in an SLM-Aminco DW-2000 UV-visiblespectrophotometer with a 3-nm slit width in 1-cm-optical-pathwayspectrophotometric cells thermostatted at 25°C with permanentstirring. Extinction of the oxidized DCPIP was monitored at609 nm in a split-beam mode ( ${varepsilon}$ =16 to 17 mM^-1 cm^-1 under ourconditions). A stock solution of the oxidized form of TMPD (TMPD⁺;Wurster's Blue) was prepared by mixing TMPD with a mixture ofpotassium ferrocyanide-ferricyanide at a ratio of 1:0.5:1.75.Extinction of the oxidized TMPD at 612 nm versus the 700-nmreference point was monitored in a dual-wavelength mode usingan approximate extinction coefficient of 9 mM^-1 cm^-1 as determinedfor these conditions. The cytochrome aa₃ concentration in themembranes was determined from dithionite (reduced) minus ferricyanide(oxidized) difference spectra of the samples, using ${Delta}$ ${varepsilon}$ _{600 nm -575 nm} = 20 mM^-1 cm^-1. When the aa₃ content of the cells wasto be determined spectrophotometrically, the cells were suspendedin 50 mM MOPS [3-(N-morpholino)propanesulfonic acid]-NaOH (pH7.0) and treated with lysozyme (1 mg/ml, 45 min, 37°C) and15 mM sodium EDTA (20 min). In order to obtain spheroplasts,the cells were subjected to a similar lysozyme treatment ina hypertonic medium with 0.8 M sucrose and 20 mM MgSO₄. To induceosmotic lysis, spheroplasts were placed in the same medium butwithout sucrose. Formation and lysis of spheroplasts were controlledvisually with a phase-contrast microscope.

RESULTS

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Results

Energy-dependent respiration of the cells. Figure 1 illustrates the effect of a protonophorous uncoupler,carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the K⁺-ionophorevalinomycin on the respiration of B. subtilis cells with endogenoussubstrates as electron donors. The combination of 10 µMCCCP and 5 µM valinomycin brought about an almost fullinhibition of oxygen consumption, while when added alone theseagents were much less efficient (Fig. 1, traces a and b). Wecould not reproduce the >90% inhibition of respiration byvalinomycin alone reported previously (47), provided that carewas taken to wash the reaction vessel thoroughly from the uncouplerused in the previous probes. The inhibitory effect of CCCP couldbe fully reversed by bovine serum albumin (Fig. 1, trace c)or charcoal (not shown), which adsorb the compound. Additionof the reduced TMPD (trace d) or 50 µM DCPIP (not shown)after the ionophores restored a high respiration rate, indicatingthat these mediators donate electrons to the respiratory chainbelow the inhibition site.

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FIG. 1. Inhibition of respiration of B. subtilis cells by CCCP and valinomycin. Oxygen consumption was monitored in 50 mM potassium phosphate-1 mM MgSO₄, pH 7.0. The initial downward deflections of the traces (dotted lines) result from addition of oxygen-depleted cell suspension. The results are shown for cells grown on malate. Traces a to d, wild-type (strain 168 A); trace e, SQR-deficient mutant (strain LUH-16). Additions: bacterial cells, up to an optical density at 600 nm of 0.1; CCCP, 10 µM; valinomycin (Valino), 5 µM; bovine serum albumin (BSA), 1 mg/ml; reduced TMPD, 0.5 mM.

In the presence of 5 µM valinomycin, 50% inhibition ofrespiration with endogenous substrates was attained at a CCCPconcentration of as low as 0.1 µM (Fig. 2) (actually,even less; see the legend to Fig. 2). A similar inhibition patternwas observed with pentachlorophenol, a less potent protonophoreuncoupler, taken at about a 10-fold-higher concentration. SCN^-(a membrane-penetrating anion) at 50 mM also could substitutefor K⁺-valinomycin. Among other ionophores tested, gramicidinD (5 µM), alamethicin (15 µM), and monensin (10µM) were found to inhibit respiration significantly (3-to 10-fold).

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FIG. 2. Concentration dependence of the CCCP inhibitory effect on respiration of B. subtilis cells. Endogenous respiration of malate-grown 168A cells was measured in 50 mM Tris-HCl-1 mM MgSO₄-5 µM valinomycin, pH 7.0. Other conditions were as described for Fig. 1. Note that at below 0.5 µM, the actual CCCP concentration in the probe is decreased significantly by adsorption of the compound on the surface of the vessel (N. Azarkina, unpublished); therefore, the true concentration at which 50% inhibition is attained is probably even lower than the uncorrected observed value of 0.1 µM quoted in the text.

All of the experiments carried out with the wild-type B. subtiliscells were duplicated with an SQR-deficient strain, LUH-16,without any noticeable difference in the results (e.g., cf.traces a and e in Fig. 1). Therefore, the inhibition of respirationinduced by the uncoupling is not associated specifically withthe succinate-oxidizing pathway of the respiratory chain asproposed previously (47). Accordingly, respiration of wild-typecells grown on malate was found to be only slightly less sensitiveto CCCP and valinomycin than respiration of cells grown on 50mM succinate. In contrast, when the cells were grown on glucose,about half of the oxygen consumption was not sensitive to CCCPplus valinomycin. However, in this case the uncoupler-resistantpart also is not inhibited by cyanide, and its mechanism isnot fully clear. It is possible that the uncoupler-insensitiverespiration on glucose, as opposed to the uncoupler-sensitivesuccinate oxidation described previously (47), could originatesimilarly in the cyanide-resistant electron flow. The resultsshown below were obtained mainly with malate-grown cells, ifnot stated otherwise.

The inhibitory effect of uncoupling agents on respiration maydepend on the exact experimental conditions. The nature of thepH buffer used in the experiments (e.g., Tris or phosphate)did not show any noticeable effect on the results. However,the data were quite sensitive to the concentrations of K⁺ andH⁺ in the medium (Fig. 3a and b, respectively). At pH 7, KCl(but not NaCl) potentiated the inhibition imposed by valinomycinbut markedly counteracted the inhibition induced by valinomycinplus CCCP. The effects of K⁺ saturated at about 200 mM KCl,which is close to concentration of K⁺ in the cytoplasm of B.subtilis (40). The inhibition by valinomycin plus CCCP changedsteeply from a virtually complete block at a pH of $<=$ 7 to a moderateeffect similar to that induced by valinomycin alone at a pHof above 7.5, which is the normal cytoplasmic pH value (30).A very similar pH dependence has been shown for the inhibitionimposed by monensin. These observations are in agreement withthe data in references 3 and 46. We give these results mainlyto characterize more fully the phenomenology of the effect;however, the data are consistent with the role of both the transmembraneelectrical potential difference ( ${Delta}$ ${psi}$ ) and ${Delta}$ pH components of theproton motive force in the energy dependence of respiration.

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FIG. 3. K⁺ and H⁺ dependence of B. subtilis cell respiration in the presence of uncouplers. (a) Dependence on the concentration of KCl (circles) and NaCl (triangles). Oxygen consumption by the wild-type cells was measured as described for Fig. 1 but in 50 mM Tris-HCl-1 mM MgSO₄, pH 7.0. The data obtained in the presence of valinomycin (Valino) and valinomycin-CCCP are indicated by open and filled symbols, respectively. (b) pH dependence measured in 50 mM potassium phosphate-1 mM MgSO₄. Data were obtained with no additions (open circles) or in the presence of valinomycin (triangles), CCCP (squares), or valinomycin plus CCCP (filled circles).

Energy-dependent reduction of DCPIP and TMPD⁺ by the cells. In a second set of experiments, the effect of the uncouplingon the reduction of DCPIP and TMPD⁺ by endogenous donors inB. subtilis cells was investigated. Studies on both mitochondrialand B. subtilis SQR (15, 51, 52) as well as on bacterial type-IINADH:quinone oxidoreductases (see, e.g., reference 7) indicatethat in membrane preparations these artificial electron acceptorsreceive electrons mostly from the membrane-bound quinols ratherthan directly from the dehydrogenase metal or flavin redox centers.It can be seen that as in the case of respiration, consecutiveaddition of CCCP and valinomycin inhibits the reduction of theartificial acceptor (Fig. 4; cf. Fig. 1, traces a and e). Thesame final level of inhibition was achieved with the inverseorder of the additions. Once again, no significant differencesbetween the wild-type and SQR-deficient cells were found. Oxidationof the reduced DCPIP and TMPD by the cells, monitored spectrophotometrically,was resistant to CCCP plus valinomycin (but was fully sensitiveto cyanide), which corroborates the data obtained by measurementof oxygen consumption as described in the previous section.

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FIG. 4. Uncoupling-induced inhibition of DCPIP reduction in wild-type and SQR-deficient B. subtilis cells. The reduction of DCPIP by endogenous substrates in malate-grown cells was monitored in the same buffer as used for Fig. 1. (a) Wild-type strain 168 A; (b) SQR-deficient strain LUH-16. The reaction was started by addition of 50 µM DCPIP, which gives the initial increment of absorption. Dotted lines, reaction in the absence of further additions. Valino, valinomycin.

Interestingly, the DCPIP reduction appears to be inhibited byvalinomycin alone much more strongly than is oxygen consumption.This can be explained by some protonophoric activity of DCPIP,which has ionizable groups with pK_a values of around 6 and 7in the oxidized and reduced forms, respectively (9). Indeed,we have observed that, similarly to the uncoupler CCCP, DCPIPinhibits endogenous respiration drastically when added in combinationwith valinomycin.

Reduction of electron transfer rates upon isolation of membranes. Table 1 compares the specific rates of respiration as well asof DCPIP and TMPD⁺ reduction observed in B. subtilis membraneswith those in the intact cells. In the membranes, all of theactivities are strikingly low (10- to 70-fold lower than inthe intact cells) and, notably, fully resistant to further inhibitionby the uncouplers (10 µM CCCP plus 5 µM valinomycin).Similar low values are manifested by the uncoupled cells. Theinhibition of succinate oxidation agrees with the data in references36 and 47. However, the same inhibition is observed also withNADH and glycerophosphate as substrates, in contrast to theprevious results (37, 47).

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TABLE 1. Electron transfer activities in B. subtilis cells and membranes

The rates of PMS reduction by different substrates (Fig. 5;Table 1) measured with DCPIP as the final electron acceptor(1) were found to be similar in the cells and membranes, inagreement with previous results (47). Therefore, the dehydrogenaseactivities per se are not likely to be impaired during isolationof membranes. Once again, very similar results were obtainedwith the wild-type and SQR-deficient strains (Table 1; see thelegend to Fig. 5), showing that the effect is not specificallyassociated with SQR.

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FIG. 5. Comparison of DCPIP reduction by intact cells and membranes from B. subtilis. DCPIP reduction was measured as described for Fig. 4 with malate-grown 168 A cells (curves 1 and 2) and membranes prepared from these cells (curves 3 and 4). The reaction mixture contained 100 mM MOPS-NaOH buffer (pH 7.0) with 0.2 mM EDTA and, in experiments with the cells (curves 1 and 2), 10 mM malate. PMS at 0.4 mM was added in the case of curves 2 and 4. The reaction was initiated by addition of either cells (curves 1 and 2) or, in the case of membranes, NADH (1 mM in curve 3 and 50 µM in curve 4). Curves 3 and 4 have been corrected for nonenzymatic DCPIP reduction by NADH. The curves obtained with cells and membranes are normalized to the same concentration of cytochrome aa₃ (0.022 µM).

To determine at which stage(s) of the membrane isolation therespiratory chain becomes inhibited, spheroplasts were firstprepared from the cells in the presence of 0.8 M sucrose toprevent osmotic shock (see Materials and Methods). In the courseof this procedure, $~$ 90% of the cells lose their cell wall. Spheroplastshave been found to respire on the endogenous substrates as activelyas the untreated cells, and their respiration revealed the samehigh sensitivity to CCCP plus valinomycin. Notably, added NADH(1 mM) had no effect on the oxygen consumption by spheroplasts,indicating that their membrane is intact. When the medium wasreplaced with a hypotonic one (without sucrose), the spheroplastsunderwent visually detectable lysis, and, concomitantly, a drasticdrop in respiration rate was observed (a similar observationfor B. subtilis cell homogenates was reported previously [47]).Addition of NADH to osmotically shocked spheroplasts stimulatedoxygen consumption but to no more than 15% of the initial rateobserved in the cells. Moreover, oxidation of the added NADHwas cyanide sensitive but was not inhibited by CCCP plus valinomycin.

DISCUSSION

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Discussion

Electron transport in the respiratory chain of B. subtilis requires ${Delta}$ µH⁺. Our data corroborate earlier observations (3, 37, 46, 47) indicatingthat electron transfer in the respiratory chain of B. subtilisrequires an energized state of the membrane. In this work weaimed to design experiments so as to rule out a number of possiblealternative explanations for the inhibitory effect of uncouplersthat were not excluded by the conditions of the previous studies.

(i) The effect is not due to direct inhibition of the electron transfer chain by the ionophores. Uncouplers, such as CCCP, are known to inhibit directly themitochondrial respiratory chain at relatively high concentration(see, e.g., Fig. 68 of reference 49), presumably acting as UQantagonists. This is not likely to be the case in our experiments,as evidenced in particular by the very low (submicromolar) concentrationsof CCCP required to inhibit respiration in the presence of valinomycin.The widely diverse structures of the ionophores used in thiswork that exert the inhibitory effect also argue against thispossibility. Moreover, an inhibition similar to that inducedby the uncoupling agents takes place upon breaking the cellcoupling membrane, and the residual electron transfer activitiesin the membranes are no longer uncoupler sensitive (37).

(ii) The effect does not involve transport of substrates. In the earlier studies, the effect of uncoupling on the respirationof B. subtilis cells was investigated with added substrates,such as glucose, lactate, citrate, or succinate (37, 46, 47).Therefore, the inhibitory effect of the uncouplers could bedue to inhibition of the energy-dependent uptake of these compounds(23, 47, 57). This explanation is ruled out by the experimentsin this work that were performed with freshly harvested cellsoxidizing endogenous substrates.

(iii) The effect is reversible. The effect of energization could be indirect. One could visualizethat a drop in ${Delta}$ µH⁺ triggers a cascade of regulatory reactionswhich eventually shuts down the respiratory chain by some kindof long-term regulatory effect, involving, for instance, proteolysisor phosphorylation. The ease with which the inhibitory effectis reversed by albumin or charcoal, which resorb the uncoupler,makes such a possibility unlikely.

(iv) Local acidification of the periplasmic space is not involved in the effect. The cytoplasmic coupling membrane of B. subtilis cells doesnot directly face the ambient water phase but is surroundedby a cell wall. It was reported that in B. subtilis cells, protonpumping across the cytoplasmic membrane results in positivecharging of the cell wall, acidification of the periplasmicspace, and, accordingly, modification of electrostatic interactionsof periplasmic components, including several enzymes, with themembrane (24, 25, 53). All of these effects were abolished byprotonophorous uncouplers, and a similar mechanism could beinvolved in the energy-dependent modulation of respiration.As we have shown, removal of the cell wall has no effect onthe respiration and its sensitivity to the uncouplers. Hence,this possibility can also be excluded.

Those control experiments allow us to assume provisionally thatit is indeed proton motive force across the coupling membranethat stimulates electron transfer in the respiratory chain ofB. subtilis cells. The effects of CCCP, pentachlorophenol, valinomycin,KSCN, alameticin, gramicidin, and monensin, either alone orin combination, as well as the K⁺ and pH dependencies of thephenomenon, are consistent with contributions of both the ${Delta}$ ${psi}$ and ${Delta}$ pH constituents of ${Delta}$ µH⁺. It is, however, difficult to discriminateultimately whether it is an acidic shift on the cytoplasmicside of the membrane (3) or dissipation of ${Delta}$ pH across the membranethat accounts for the H⁺-dependent component.

Which step in the respiratory chain is blocked by deenergization? The inhibition of respiration induced by the uncouplers or bydisruption of the cell membrane appears to be associated withthe dehydrogenase-MQ part of the respiratory chain. This isevidenced by inhibition of reduction of DCPIP and TMPD⁺, bothof which are believed to accept electrons in the membrane-boundrespiratory chain at the level of UQ (MQ-7 in case of B. subtilis)(51, 52, 54), and it agrees with the inhibition of the succinate:MQreductase activity observed previously (36, 47).

It is noteworthy that the succinate:DCPIP reductase activitywas emphasized to be insensitive to the uncoupling in reference47. This apparent discrepancy with our data is due to confusionin terminology. The "succinate-DCPIP-reductase activity" asmeasured in (47) is actually a classical succinate dehydrogenaseassay (see, e.g., references 15 and 37), with 0.4 mM PMS asthe immediate electron acceptor for the dehydrogenase and DCPIPserving as the final colored electron acceptor for PMS. In agreementwith the previous study (47), both the membranes and the cellsreveal quite a high rate of PMS-mediated reduction of DCPIP(Table 1; Fig. 5). However, reduction of DCPIP or TMPD⁺ by endogenoussubstrates in the absence of PMS (which was not assayed in theprevious study [47]) is strongly inhibited by uncoupling.

At the same time, oxidation of the reduced TMPD and DCPIP throughthe terminal oxidases is not inhibited by deenergization. Apparently,the inhibition site is located above the point(s) at which thosedyes feed electrons in the respiratory chain and, so terminaloxidases are not the targets of the inhibitory effect. In addition,we have screened B. subtilis strains deficient in differentterminal oxidases and the bc₁ complex and have found all ofthem to show a similar inhibitory effect of the uncoupling onrespiration (unpublished work in collaboration with L. Hederstedt).On the basis of these data, specific association of the inhibitionwith any particular oxidase does not seem to be likely.

The uncoupler-induced inhibition of electron transfer is thuslikely to occur at the level of MQ. Either reduction or oxidationof the quinone could be affected. Formally, one cannot fullyexclude the possibility that MQ is reduced by dehydrogenasesto MQH₂ in the deenergized membranes while oxidation of MQH₂by the cytochrome chain is impaired. Alternatively, it can bereduction of MQ by the dehydrogenases to MQH₂ or MQ^·^-that is energy dependent. Monitoring of the redox state of endogenousMQ in the energized and deenergized membranes is required todirectly resolve this alternative. However, it is not easy tovisualize how the reduced MQ can be protected from direct nonenzymaticoxidation by TMPD⁺ and DCPIP (this work) or by 2,3-dimethyl-1,4-naphthoquinone(47). Therefore, we assume provisionally that it is MQ reductionrather than oxidation that shuts down upon deenergization ofthe membrane.

The inhibition is not associated specifically with SQR. We confirm the finding of Schirawski and Unden that deenergizationimpairs succinate-dependent electron transfer to oxygen or artificialacceptors in the respiratory chain of B. subtilis (47). However,our results do not support their conclusion on the effect beingspecific for succinate oxidation (47). Contrast to the resultsin references 37 and 47 and in accordance with those in reference3, the inhibitory effect of the uncoupling was observed withthe NADH-dependent substrates as well. Moreover, the same inhibitionwas obtained in this work with the B. subtilis mutant lackingSQR. Hence, the data used previously to substantiate the conclusionthat "menaquinone-dependent succinate dehydrogenase of bacteriacatalyzes reversed electron transport driven by the proton potential"(47) may not be relevant to the hypothesis. It is noted thatthe hypothesis of vectorial electron transfer through SQR isnot necessarily compromised by our results. The membrane potential-drivenreduction of MQ in B. subtilis SQR is predicted explicitly bythe structure of the enzyme and therefore remains an attractivepossibility (14, 32, 42), which, however, should not be confusedor identified with the effect of energy-dependent stimulationof respiration.

Possible mechanisms of the energy-dependent stimulation of electron transfer in B. subtilis. According to our observations, membrane energization is requiredfor any of the MQ-dependent electron transfer pathways in therespiratory chain of B. subtilis. It cannot be excluded formallythat each of the dehydrogenases is endowed with its own specificmechanism of energy-dependent reduction of MQ. However, sucha possibility does not look appealing. Notably, many of thedehydrogenases, such as type II NADH dehydrogenase (4) or GPdehydrogenase (T. Hedin and L. Hederstedt, unpublished data),are not integral membrane proteins and lack anchor subunitsor membrane-spanning helices that could sense or mediate theeffect of ${Delta}$ µH⁺.

MQ is the only common link for the different electron transferroutes in B. subtilis, and it is tempting to propose that MQitself can be the component whose reduction-oxidation is controlledby energization. Such a control could be imposed via specialMQ-binding adapter proteins located in the membrane. Protein-imposedcontrol over redox reactions of MQ could also be meaningfulfor a more general reason. B. subtilis is a relatively rareexample of a strictly aerobic bacterium with MQ as the onlyquinone species. Due to its low E_m (ca. -80 mV, versus ca. +90mV in the case of, e.g., UQ [42]), MQH₂ is much more autoxidizablethan ubiquinol (UQH₂). In animal mitochondria, reactions ofUQ in the respiratory chain are believed to be described bya simple mass action law (the so-called Q-pool model [29]).This model assumes the existence of homogenous pools of mobilefree UQ and UQH₂ dissolved in the phospholipid milieu of themembrane that shuttle between the dehydrogenases and cytochromebc₁ complexes. The UQ pool concept was also used to describerespiration through the branched electron transfer chains ofParacoccus denitrificans, although more complex behavior ofthe system was noticed in this case (43) and in plant mitochondria(26). The pool behavior may be problematic in B. subtilis, however,since free reduced MQH₂ is prone to rapid autoxidation. Therefore,a more specific control over MQ reduction-oxidation would befunctionally significant.

First, the time of MQH₂ free diffusion in the membrane betweenthe dehydrogenases and quinol-oxidizing enzymes should be minimized(e.g., by virtue of formation of functionally active supercomplexesbetween different dehydrogenases and quinol oxidases [5, 21,45]). Second, involvement of the special MQ-binding proteinsthat stabilize the reduced form(s) of MQ may be envisaged. Accordingto this hypothesis, there may be protein-bound, rather thanfree, forms of MQ and MQH₂ that serve as substrates for thedehydrogenases and quinol oxidases in B. subtilis. The stabilizationcould be either kinetic, e.g., sequestration of the quinol ringof bound MQH₂ inside the protein protecting it from collisionswith oxygen dissolved in the membrane, or thermodynamic, raisingthe effective E_m of the bound MQ-MQH₂ (or MQ-MQ^·^-) couple.

Conceivably, the MQ-binding adapter proteins could also mediatethe energy-driven stimulation of MQ-dependent electron transfer.First, the energy of ${Delta}$ µH⁺ can be utilized for thermodynamicpromotion of MQ reduction to MQH₂, as discussed, for instance,for SQR (42). In general, such an effect implies that upon reduction,MQ receives electrons from the dehydrogenases at the inner sideof the membrane while being protonated from the outer aqueousphase (e.g., via a proton channel in the adapter protein). Second,membrane energization could simply lower the kinetic barrierof the reaction and thus be part of a regulation mechanism.For instance, in plant mitochondria, SQR is activated by protonmotive force despite the absence of a thermodynamic barrierbetween the enzyme and UQ (2). It must be emphasized that inB. subtilis, there is no explicit thermodynamic problem in theoverall reduction of MQ to MQH₂ by dehydrogenases of substratesother than succinate, as their E_ms are typically below thatof the MQ-MQH₂ couple. Therefore, we would favor the more ergonomickinetic regulation mechanism over the ${Delta}$ µH⁺-consuming thermodynamicallydriven quinone reduction.

Reduction of MQ to the unstable semiquinone radical upon additionof the first electron is likely to be the limiting step of theoverall process due to the very low E_m of the MQ/MQ^·^-transition. The reaction can be facilitated by a specific tightbinding of the semiquinone intermediate, as is typical of the2e^- $->$ 1e^- junctions in the respiratory and photosynthetic electrontransfer chains (42). It can be speculated that in B. subtilis,the major peripheral dehydrogenases such as type II NADH dehydrogenaseor GP dehydrogenase cannot stabilize MQ^·^- themselvesand that this function requires MQ-binding adapter proteinswhich are in turn regulated by proton motive force.

First, energization could modulate conformation of the adapterprotein, opening the binding crevice for MQ. Second, energizationcould increase affinity of the adapter protein for MQ^·^-.The latter effect can be induced, in particular, by membranepotential-driven protonation of a residue in vicinity of theMQ^·^--binding site from periplasm via a proton channel.Energy-dependent proton delivery from the periplasmic side willchange electric charge near the binding site, e.g., by virtueof protonation of ionized carboxylate. For instance, stabilizationof ubisemiquinone anion in E. coli fumarate reductase has beenobserved upon mutation of a nearby anionic Glu-29 to neutralLeu (17). Accordingly, binding of the semiquinone anion willbe greatly enhanced and the E_m of the MQ-MQ^·^- transitionwill be raised relative to those for the deenergized membrane.

UQ has been shown recently to modulate the structure of theso-called uncoupling proteins (UCP 1, 2, and 3) in the mitochondrialmembrane (11, 12), with the effect being specific for the oxidized(UQ) form (12). These findings provide a precedent of a linkagebetween the redox state of bound quinone and the structure ofa nonredox proton-conducting integral membrane protein sensitiveto ${Delta}$ µH⁺ and thus may be relevant to the hypothesis of thequinone adapter proteins. Interestingly, a fraction of UQH₂that is resistant to aerobic oxidation through the respiratorychain due to interaction with a yet-unidentified protein (A.M. Wagner, unpublished data) has been found in plant mitochondria(44, 56).

Finally, the energy-dependent stimulation of respiration maynot be a unique feature of B. subtilis. Relevant effects weredescribed earlier for Micrococcus luteus (M. lysodeikticus)(55) and for two more bacteria (47). In recent work, we haveobserved the uncoupler-induced inhibition of respiration inmore than 20 other bacteria, most of which were gram positiveand contained MQ (N. Azarkina, E. Strom, and A. A. Konstantinov,unpublished data). Evidence for membrane potential-dependentmodulation of UQ pool behavior in P. denitrificans has beenobtained (43), and an energy requirement for active respirationhas been observed recently for some animal mitochondria (A.I. Shestopalov and B. S. Kristal, unpublished data). Hence,the ${Delta}$ µH⁺-dependent regulation of electron flow throughthe respiratory chain at the level of coenzyme Q may be a phenomenonof wide physiological significance.

ACKNOWLEDGMENTS

The work was supported by grants from the Russian Fund for BasicResearch (00-04-48251, 99-04-48095, 01-04-49330, and 01-04-06274),Howard Hughes Medical Institute International Research ScholarAward 55000320, and the INTAS Young Scientist fellowship program(YSF 00-140).

FOOTNOTES

* Corresponding author. Mailing address: A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobjovy Gory, 119992 Moscow, Russia. Phone: (7) (095) 939-55-49. Fax: (7) (095) 939-03-38. E-mail: konst{at}genebee.msu.su.

REFERENCES

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