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Coupling between a redox reaction, an interfacial proton flow and a countercurrent cation flow in an artificial two-phase system
Bioelectrochemistry and Bioenergetics, 13 (1984) 171-181 A section of J. Electroanal. Chem., and constituting Vol. 174 (1984)
171
Elsevier Sequoia !%A., Lausanne - Printed in The Netherlands
630-COUPLING BETWEEN A REDOX REACTION, AN INTERFACIAL PROTON FLOW AND A COUNTERCURRENT CATION FLOW IN AN ARTIFICIAL TWO-PHASE SYSTEM *
R. NAUMANN Kiesstr. 43, 6100 Darmstadt
(F.R.G.)
(Revised manuscript received June 10th 1984)
SUMMARY A model reaction for biological proton transfer is constructed in which Fe(II1) salts of different ion exchangers react with hydroquinone in two-phase systems. The dissociation constants of the reacting species are shown to affect the direction of proton flow. The rate of the redox reaction is shown to depend on the electrolyte concentration within the aqueous phase. From this, the interdependence of the three processes electron exchange, interfacial proton flow and countercurrent cation flow can be derived. The same type of redox reaction is used to demonstrate the principle of electron exchange in a two-phase system taking place uncoupled to ion exchange processes. Compared to the single-phase reaction, this is characterized by an appreciably higher reaction rate as well as a shift in equilibrium, although there is no net proton transfer across the interface. Possible consequences for biologically relevant redox-driven ion flows are given.
INTRODUCTION
In the present paper a model reaction will be presented which follows the principles of redox-linked proton flow proposed earlier [l]. Several attempts at constructing artificial two-phase systems with the characteristics of biological membrane transport have been reported [2-41. These were shown to bring about the transport of ions against their electrochemical potential difference, driven by a chemical reaction. Recently, Shinbo et al. [5] described not only a redox reaction driving an uphill ion flow but also the reverse, an ion gradient driving a redox reaction against its free energy difference. These authors, however, only considered transmembrane reactions, i.e. those in which the reacting species are situated on either side of a non-aqueous phase containing a mediator. In contrast to these models, the redox reaction described here is characterized by at least one of the redox couples being within the non-aqueous phase. Therefore, l Presented at the 7th International Symposium on Bioelectrochemistry, Stuttgart (F.R.G.), 18-22 July 1983.
0302-4598/84/!$03.00
0 1984 Elsevier Sequoia S.A.
172
interfacial rather than transmembrane ion flows are coupled to the redox reaction. This was verified experimentally by using ion exchange-type compounds forming Fe(II1) salts that are soluble in the non-aqueous phase and insoluble in water. These were made to react with hydroquinone in a two-phase system containing different quantities of electrolyte. The same type of reaction was used to show experimentally the uncoupling mechanism of a proton-involving redox reaction in a two-phase system. EXPERIMENTAL
Two series of experiments were carried out: (I) 0.5 g A 0.433 cm3 of the strong acid ion exchange resin (5244) Merck S1080 Gl loaded with 1.39 meq Fe3+, the total capacity of the resin being 3.89 meq/g, was suspended in 20 cm3 KNO, solution (0.1 M), thus forming a two-phase (water I resin) system.*The remaining protons as well as the Fe3+ ions within the resin equilibrated with potassium ions in the water phase, leading to an exchange of H+ for K+ ions and of Fe3+ for K+ ions, respectively. The changes in proton and potassium concentrations within the aqueous phase were detected by a Schott (H 61) pH-combination glass electrode and an Ingold (PK 201) potassium-selective electrode, respectively. The Fe3+ concentrations in the aqueous phase prior to the redox reaction were determined voltammetrically using a d.m.e. To start the redox reaction, 200 mg of hydroquinone, dissolved in 1 cm3 of ethanol, was added. The formation of benzoquinone was measured voltammetrically, using a Beckman (188501) rotating gold disc electrode, 3000 r.p.m., and a Metrohm EA 440 double junction reference electrode Ag I AgCl, sat. KC1 IIKNO, (3 M). Oxygen was excluded throughout the experiments by purging with nitrogen. Voltammetric measurements were carried out using a Wenking potential control amplifier (model PCA 72 L) connected to a Wenking voltage scan generator (model VSG 72). Because of the Fe3+-K+ exchange prior to the redox reaction, a homogeneous redox reaction occurred in addition to the two-phase reaction. This was taken into account by performing a second experiment with the aqueous phase only. After equilibration with the resin, this was made to react with hydroquinone after separation of phases. The change in quinone as well as H+ ion concentration subtracted from the respective overall data yielded the data representing the two-phase reaction. Hydroquinone may at least in part be dissolved in the resin phase, while Fe3+ is in part exchanged by K+. Therefore, the overall redox reaction may take place in both phases, according to Scheme 1. H+, K+ and NO; ions redistribute between the phases due to dissociation and exchange equilibria. In every case the proton concentration is expected to increase within the aqueous phase in line with benzoquinone formation and the decrease in potassium concentration. The experimental results are shown in Fig. 1; the H+- and K+-electrodes had been calibrated in concentration units, therefore cu+ and ck+ are given in the graph. In the following, the same kind of experiment was carried out, however, with
173 aqueous phase
resin phase
Fe 3+ Fe 3+ + 1/2 Q H 2 ~ I / 2
.
. ~'
Fe~_
~ ~ "035- "O3 S_
Q + Fe 2+ + H +
+ 1/2 Q H 2 ~
1/2 Q + ~
K +
NO3 , -SO3H¢~_~-SO ~
Fe 2"
+ H+
F e 2 + . ~ " "O3S_ O3S-
NO 3 Q
Scheme
1.
varying K N O 3 concentrations in the aqueous phase. The result of the entire set of experiments, corrected for the homogeneous redox reaction due to K + - F e 3+ exchange is s h o w n in Fig. 2. The rate of the coupled redox and H +-transfer processes increases with increasing K N O 3 concentrations. For a discussion see below. (II) The second series of experiments was designed to demonstrate a redox
~
•°
~
K NO~ ( 0.1 ,e')
i
O ~q
(rain)
-2
-4
®ACK÷
-6
Fig. 1. Reaction of strong acid ion exchanger, Fe(III) form, with hydroquinone; coupling of benzoquinone formation (CQ) to the increase in proton concentration (CH~); and the decrease in potassium concentration (CK+) within the aqueous phase (0:1~M KNO3).
cl
t(min) 1
2
5
4
1
2
a
4
5
6
7
a
9
1c
f (min) 5
Fig. 2. Reaction of strong acid ion exchanger, Fe(M) form, with hydroquinone in KN03 solutions of different concentrations; increase in benzoquinone concentration (cQ) coupled to the increase in proton concentration (cH+ ) in the aqueous phase. The concentrations of KN03 are indicated on the graph.
175
reaction of the same type but without proton transfer coupling. Here, a weak acid ion exchanger, 2-propylpentanoic acid in its Fe(II1) form *, was made to react with hydroquinone (in a 0.1 M solution). Both redox couples were dissolved in the non-aqueous phase of a two-phase system consisting of 20 cm3 pentanol (0.1 M tetrabutylammonium chloride (TBACl)) and 20 cm3 water (0.1 M TBACl). The distribution equilibrium of TBACl was found to have a constant of 1.12. Thus the concentrations of TBACl within the two phases after equilibration will exhibit small deviations from the initial values. The two phases were agitated separately, the upper (pentanol) phase with a rotating gold disc electrode (r.d.e.), 3000 r.p.m., and the aqueous layer with a Metrohm (504) synchronous magnetic stirrer (500 r_p.m.)_ Mass transport between the phases was found to be sufficient for a distribution equilibrium of, for example, 1 X low3 M HCl to be attained within less than 60 s. The distribution-equilibrium constant was found to be 455 for 2-propyl pentanoic acid in the pentanol I aqueous phase system. Fe3+ and Fe*+ were shown to be confined to the pentanol phase during the course of the experiment, with a detection limit of 2 X lo-’ M for Fe3+/Fe2+ within the aqueous phase. The experimental set-up was completed by a double junction reference electrode (Metrohm EA 440) Ag I AgCl, sat. KC1 IIethanol, 0.1 M TBACl, a Pt-auxiliary electrode, both inserted in the pentanol phase in order to determine voltammetrically the quinone concentration together with the r.d.e. For comparison purposes, the
Fig. 3. Two-phase compared to the one-phase reaction hydroquinone. cQ = concentration of benzoquinone.
of 2-propylpentanoic
acid, Fe”’
salt with
* 10 g of 2-propylpentanoic acid mixed with 10 g of pentanol was shaken 5 times with fresh solutions of 15 g of FeCl, in 30 crd of water, washed with water until a pH of 5 was obtained, separated from the water phase, and filtered through H,O-free Na,SO,. 2 cd of this solution was taken per experiment.
176
reaction was carried out in a single phase system, which consisted of the pentanol phase alone (pentanol saturated with water). Figure 3 shows the formation of benzoquinone as a function of time in the two-phase compared to the single-phase system. It is clearly seen that in the two-phase system the rate of the redox reaction is greatly accelerated and the equilibrium is shifted to the right. This is explained by Scheme 2:
aqueous
phase
c-non-aqueous
-
phase
Fe(RCoo)j
+
l/2
QH,
Fe(RCOO)2
+
l/2
Q
K
RWOH
4
Rcoo- +
li+
cw
CL* RCW-
Ii+ c
__\ +
RCC,3H
K RcooE
+
2
a+
cp
w
RCW”
RCOOH
Scheme 2.
where the weak acid, RCOOH, its charged and uncharged forms are distributed quasi-independently between the phases. This assumption seems justified in view of the excess amount of TBACl present in both phases. The protons formed during the redox reaction recombine with the uncharged form of the weak acid (the pK of propylpentanoic acid was found to be 4.83). The recombination of the protons formed during the redox reaction with anions of the weak acid either in the pentanol or in the water phase leads to a redistribution of charged and uncharged forms of the weak acid, in this case the ion exchanger itself. The result is an acceleration as well as a shift in the equilibrium of the redox reaction, although no net amount of protons traverses the interface. Despite some small oscillations of the pH value, no difference in the proton concentration of the aqueous phase could be detected. DISCUSSION OF mE EXPERIMENTS
The experiments will now be discussed in greater detail and in particular with respect to their relevance to biological membrane processes. Both the proton-coupled and the uncoupled reactions follow the same general equation:
QS
+2FeR
3*Q+2FeR,+2Hr
JrK, Jr& R-+ H+ QH-+ H+ The dissociation constants concerned are: 2-propylpentanoic
(I) acid: K, = 1.48 X 10e5,
177
pK = 4.83; hydroquinone: K, = 4.5 X lo-“, pK = 10.35 [6]; strong acid ion exchanger (sulfonic acid type): K, = lo-‘, pK = 1. Hence in both cases K, > K, B K, and the reaction takes place according to QH, + 2 FeR, ;tQ+2FeR,+2Re+2H+
(2)
where protons are formed during the reaction. This leads to a shift in the equilibrium of the redox reaction in the two-phase system because protons are withdrawn according to the reaction mechanisms given above. In the case of the strong acid ion exchanger, the dissociation equilibrium of the -SO,H group causes free protons to be formed and transported into the aqueous phase. K+ and other ions (NO;) redistribute between the phases in order to satisfy the mass balance of the ion exchanger. [-SO;]
res- 3 [Fe3’] res - 2 [Fe’+] res =
WI res+ W 1res- [NO;]res
(3)
In the more dilute bathing solutions (0.01 and 0.1 M), the transfer of NO; ions into the resin phase does not occur to an appreciable extent because of co-ion exclusion. Therefore, in these cases the exchange of H+ for K+ can be expected to be fully coupled to each other as well as to the redox reaction. This was shown experimentally (Fig. 1). At higher concentrations of the electrolyte, the formation of H+ within the aqueous phase still appears to be coupled to the redox reaction, however with increasing reaction rates. In an attempt to arrive at a quantitative treatment of this phenomenon, the following calculations were carried out. Under the conditions prior to the start of the redox reaction, the mass balance of the resin phase is described by
[-SO;],,,-3[Fe3+l,,,=[K+l,,,+[H+l.,,-[N0,],,,
(4)
while the Donnan potential across the interface is determined by the activities of the ions able to permeate both phases:
=
$$ In
[Fe3+] aq [Fe3+l res
(5)
Concentrations rather than activities are accessible experimentally. However, taking the concentrations in the aqueous phase of the resin, only approximate values of u Donnanmay be obtained and thus with the aid of equation (6) the variations of (Y with ionic strength and with the progress of the redox reaction.
(6) This equation was established by Katchalsky [7], taking into account the influence of the electrostatic energy on the titration curve of a water/resin system (see also Refs. 8-10).
179
The variation in (Y is derived from the experiments with varying electrolyte concentrations. With known concentrations of the ions in the aqueous phase, i.e. Wlaq, K+laq9 WJ,, and P3+la,, the concentration of protons in the resin can be calculated using equations (4) and (5). phase, WI,,,, The tendency of U,,,,,,, to decrease as a function of electrolyte concentration in the aqueous phase may be derived by simply taking the concentration of, for example, K+ ions, calculated according to equation (5), for the computation of u DOlllltlll and Apk+, respectively. This is shown in Table 1, where the activity coefficients are neglected. Therefore Un,,,,, cannot be taken as absolute values but they are adequate for calculating the respective AU,,,,,. They reflect the fact that the starting conditions of the reactions described by Fig. 2A-D are characterized by a change in Api in the positive direction. The addition of hydroquinone starts the redox reaction and makes available a further lot of -SO,H groups resulting in a redistribution of ions such that a new state of equilibrium is established. The rate of both the redox and the ion exchange processes varies with the starting conditions given above. In the following, the influence of various factors on the reaction rate will be discussed. Firstly, the potassium potential within the resin phase increases appreciably as a consequence of the change of the Apx + in the positive direction. The variation in the standard potential of the Fe’+/*+ couple in the resin phase as a function of the potassium potential can be evaluated using the data plotted in Fig. 1. The changes in [K+],, and [H+],, were used to calculate the changes in [K+],,, and [H+],,, by calculating the difference to the initial values. [K+],,, and [K+],, thus obtained were used to calculate U,,,,,. Known concentrations of quinone and hydroquinone within the aqueous phase yielded the potential within the aqueous phase U,,according to: (0.059) log
44 = Uiq,Q,cm +2
IQ1aq[H+I:, tQfbla,
(7)
where Uiq,Q,QH2 = 0.699 V. Then the potential within the resin phase U;,,, was obtained by calculating the difference to the Donnan potential: (8)
u*q - Q30,,,, = IL
Finally, the standard potential of the Fe3+/Fe2+ couple within the resin phase
U" res,Fe3+,Fe~+ was calculated using the equation u,,, = U;s,Fe3+,Fe2++ 0.059 log
[Fe3+l res [Fe*+1 res
(9)
where [Fe*+] res was taken to be proportional to the amount of quinone formed during the reaction. This was done following the curves shown in Fig. 1. From this, values of
180
U”res,re3+/re2+at different potassium potentials within the resin phase were obtained, e.g. 0.556 V and 0.621 V at [K+]_ = 0.78 and 0.89 respectively. Again, these values are not absolute values and only serve to show that, in principle, the standard potential of the Fe3+/Fe2+ couple increases in the positive direction with increasing potassium potentials in the resin phase. This clearly would have an effect not only on the equilibrium but also on the rate of the redox reaction, which depends on the rate of diffusion of redox and ionic components. Secondly, [Fe3+lres decreases considerably with increasing concentration of K+ in the bathing solution due to the Fe 3+-K+ exchange prior to the redox reaction. Consequently, the term log([Fe3’],.,,/[Fe2’],,,) increases in the negative direction. This would at least in part counterbalance the effect of increasing standard potentials discussed above. Thirdly, the variation in Ap k+ itself is expected to exert an effect on the rate of ion exchange and-because of the coupling-on the entire process. In the more dilute bathing solutions, the inward potassium flow is forced to occur against a considerable Ap x+ which decreases with increasing KNO, concentration. This, therefore, should accelerate the redox and H+/K+ exchange processes. For the effect of ionic gradients on ion exchange kinetics, see, for example, Ref. 11. At present, it is not clarified which of these factors plays the predominant role on the reaction rate. In every case, the interdependence of the three processes redox reaction, proton and potassium transfer can be demonstrated. A final remark will be devoted to the proton/electron stoichiometry. Not more than about a quarter of the protons expected according to equation (2) were found. This result can only be accounted for by the change in (Ywith the change in pH at the respective Uo,,,. Regarding now the weak acid ion exchanger, this has a double function: first, as the anion of the Fe3+/Fe2+ couple. This makes it a participant of the redox reaction according to equation (2). Second, as an interfacial buffer which facilitates transport of protons between the two phases. Protons, however, are not transported across the interface as charged particles as in experiment 1 but rather as uncharged molecules formed by recombination of the proton with the anion of the uncoupler. Therefore, the proton-involving redox reaction takes place without an accumulation of protons in one of the two phases. This second function of the weak acid ion exchanger is proposed as a general principle of uncouplers of biological proton transfer. Due to their ability to be present in their charged and uncharged forms in the water as well as in the membrane phase, they are considered to prevent the formation of ion gradients while the redox reaction itself goes on unimpeded or even accelerated compared to a two-phase redox reaction coupled to ion transfer as shown in experiment 1. This idea gains support from the work of Terada and Van Dam [12]. These authors proposed a shuttling mechanism of the uncoupler supported later by Hitchens and Kell [13]. From the viewpoint of the present author, the uncoupler shuttles between phases in order to inhibit redox coupled ion exchange processes.
181
CONCLUDING
REMARKS
In the present paper, a model reaction was presented according to which a proton-involving redox reaction causes protons to flow across the interface of a two-phase system. Proton transfer occurs due to the non-vanishing proton balance of the redox couples comprising the overall redox reaction. This was proposed earlier [l] to be the reason for interfacial proton transfer in biological systems. In order to obey the electroneutrality condition, the interfacial proton flow is accompanied mainly by a countercurrent cation flow. Hence the chemical reaction is coupled to an ion exchange process by virtue of the mass balance of the resin or, in other words, by the electroneutrality condition. This was proposed to be the reason for redox induced ion exchange in mitochondria [l]. These conclusions apply to biological membrane transport only if the model reactions described here have something in common with biological systems. The significance of ion exchange resins as models for biological membrane systems was investigated earlier by Katchalsky [7] and emphasized only recently by De Korbsy [14]. Fe(II1) bound to a fixed charge network can be regarded as a redox center. But these are not the main points this paper is dealing with. The goal was to show a two-phase system exhibiting peculiarities of biological ion-transport and yet consisting of two compartments only. Hence attention is focussed on interfacial rather than transmembrane ion transfer processes. ACKNOWLEDGEMENTS
The author is extremely grateful to Prof. I.R. Miller, The Weizmann Institute of Science, Rehovot, Israel, for his constructive criticism and his important suggestions regarding this article. Further, I wish to express my gratitude to Prof. H. Wendt, Technische Hochschule Darmstadt, and to Dr. W. Pusch, Max Planck Institut fur Biophysik, Frankfurt/M., for very profound discussions on the present paper. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14
R. Naumamr, Bioelectrochem. Bioenerg., 9 (1982) 663, D.K. Schiffer, A. Hcchhauser, D.F. Evans and E.L. Cussler, Nature (London), 250 (1974) 484. T. Shinbo, K. Kurihara, Y. Kobatake and N. Kamo, Nature (London), 270 (1977) 277. J.J. Grimaldi and J.M. Lehn, J. Am. Chem. Sot., 101 (1979) 1333. T. Shinbo, M. Sugiura, N. Kamo and Y. Kobatake, J. Membr. Sci., 9 (1981) 1. J.C.D. Hodgman in Handbook of Chemistry and Physics, R.C. Weast (Editor), 46th ed., CRC Press, Cleveland, OH, 1965. A. Katchalsky, Progr. Biophys. Biophys. Chem., 4 (1954) 1. A. Katchalsky and S. Lifson, J. Polym. Sci., 11 (1953) 409. A. Katchahky and I. Michaeli, J. Polym. Sci., 15 (1955) 69. I. Michaeli and A. Katchahky, J. Polym. Sci., 22 (1957) 683. F. Helfferich, Ionenaustauscher, Vol. 1, Verlag Chemie, Wiesbaden, 1959, p. 239. H. Terada and K. van Dam, Biochim. Biophys. Acta, 387 (1975) 507. G.D. Hitchens and D.B. Kell, B&hem. Z., 212 (1983) 25. F.D. De Koriisy, Bioelectrochem. Bioenerg., 9 (1982) 391.
171
Elsevier Sequoia !%A., Lausanne - Printed in The Netherlands
630-COUPLING BETWEEN A REDOX REACTION, AN INTERFACIAL PROTON FLOW AND A COUNTERCURRENT CATION FLOW IN AN ARTIFICIAL TWO-PHASE SYSTEM *
R. NAUMANN Kiesstr. 43, 6100 Darmstadt
(F.R.G.)
(Revised manuscript received June 10th 1984)
SUMMARY A model reaction for biological proton transfer is constructed in which Fe(II1) salts of different ion exchangers react with hydroquinone in two-phase systems. The dissociation constants of the reacting species are shown to affect the direction of proton flow. The rate of the redox reaction is shown to depend on the electrolyte concentration within the aqueous phase. From this, the interdependence of the three processes electron exchange, interfacial proton flow and countercurrent cation flow can be derived. The same type of redox reaction is used to demonstrate the principle of electron exchange in a two-phase system taking place uncoupled to ion exchange processes. Compared to the single-phase reaction, this is characterized by an appreciably higher reaction rate as well as a shift in equilibrium, although there is no net proton transfer across the interface. Possible consequences for biologically relevant redox-driven ion flows are given.
INTRODUCTION
In the present paper a model reaction will be presented which follows the principles of redox-linked proton flow proposed earlier [l]. Several attempts at constructing artificial two-phase systems with the characteristics of biological membrane transport have been reported [2-41. These were shown to bring about the transport of ions against their electrochemical potential difference, driven by a chemical reaction. Recently, Shinbo et al. [5] described not only a redox reaction driving an uphill ion flow but also the reverse, an ion gradient driving a redox reaction against its free energy difference. These authors, however, only considered transmembrane reactions, i.e. those in which the reacting species are situated on either side of a non-aqueous phase containing a mediator. In contrast to these models, the redox reaction described here is characterized by at least one of the redox couples being within the non-aqueous phase. Therefore, l Presented at the 7th International Symposium on Bioelectrochemistry, Stuttgart (F.R.G.), 18-22 July 1983.
0302-4598/84/!$03.00
0 1984 Elsevier Sequoia S.A.
172
interfacial rather than transmembrane ion flows are coupled to the redox reaction. This was verified experimentally by using ion exchange-type compounds forming Fe(II1) salts that are soluble in the non-aqueous phase and insoluble in water. These were made to react with hydroquinone in a two-phase system containing different quantities of electrolyte. The same type of reaction was used to show experimentally the uncoupling mechanism of a proton-involving redox reaction in a two-phase system. EXPERIMENTAL
Two series of experiments were carried out: (I) 0.5 g A 0.433 cm3 of the strong acid ion exchange resin (5244) Merck S1080 Gl loaded with 1.39 meq Fe3+, the total capacity of the resin being 3.89 meq/g, was suspended in 20 cm3 KNO, solution (0.1 M), thus forming a two-phase (water I resin) system.*The remaining protons as well as the Fe3+ ions within the resin equilibrated with potassium ions in the water phase, leading to an exchange of H+ for K+ ions and of Fe3+ for K+ ions, respectively. The changes in proton and potassium concentrations within the aqueous phase were detected by a Schott (H 61) pH-combination glass electrode and an Ingold (PK 201) potassium-selective electrode, respectively. The Fe3+ concentrations in the aqueous phase prior to the redox reaction were determined voltammetrically using a d.m.e. To start the redox reaction, 200 mg of hydroquinone, dissolved in 1 cm3 of ethanol, was added. The formation of benzoquinone was measured voltammetrically, using a Beckman (188501) rotating gold disc electrode, 3000 r.p.m., and a Metrohm EA 440 double junction reference electrode Ag I AgCl, sat. KC1 IIKNO, (3 M). Oxygen was excluded throughout the experiments by purging with nitrogen. Voltammetric measurements were carried out using a Wenking potential control amplifier (model PCA 72 L) connected to a Wenking voltage scan generator (model VSG 72). Because of the Fe3+-K+ exchange prior to the redox reaction, a homogeneous redox reaction occurred in addition to the two-phase reaction. This was taken into account by performing a second experiment with the aqueous phase only. After equilibration with the resin, this was made to react with hydroquinone after separation of phases. The change in quinone as well as H+ ion concentration subtracted from the respective overall data yielded the data representing the two-phase reaction. Hydroquinone may at least in part be dissolved in the resin phase, while Fe3+ is in part exchanged by K+. Therefore, the overall redox reaction may take place in both phases, according to Scheme 1. H+, K+ and NO; ions redistribute between the phases due to dissociation and exchange equilibria. In every case the proton concentration is expected to increase within the aqueous phase in line with benzoquinone formation and the decrease in potassium concentration. The experimental results are shown in Fig. 1; the H+- and K+-electrodes had been calibrated in concentration units, therefore cu+ and ck+ are given in the graph. In the following, the same kind of experiment was carried out, however, with
173 aqueous phase
resin phase
Fe 3+ Fe 3+ + 1/2 Q H 2 ~ I / 2
.
. ~'
Fe~_
~ ~ "035- "O3 S_
Q + Fe 2+ + H +
+ 1/2 Q H 2 ~
1/2 Q + ~
K +
NO3 , -SO3H¢~_~-SO ~
Fe 2"
+ H+
F e 2 + . ~ " "O3S_ O3S-
NO 3 Q
Scheme
1.
varying K N O 3 concentrations in the aqueous phase. The result of the entire set of experiments, corrected for the homogeneous redox reaction due to K + - F e 3+ exchange is s h o w n in Fig. 2. The rate of the coupled redox and H +-transfer processes increases with increasing K N O 3 concentrations. For a discussion see below. (II) The second series of experiments was designed to demonstrate a redox
~
•°
~
K NO~ ( 0.1 ,e')
i
O ~q
(rain)
-2
-4
®ACK÷
-6
Fig. 1. Reaction of strong acid ion exchanger, Fe(III) form, with hydroquinone; coupling of benzoquinone formation (CQ) to the increase in proton concentration (CH~); and the decrease in potassium concentration (CK+) within the aqueous phase (0:1~M KNO3).
cl
t(min) 1
2
5
4
1
2
a
4
5
6
7
a
9
1c
f (min) 5
Fig. 2. Reaction of strong acid ion exchanger, Fe(M) form, with hydroquinone in KN03 solutions of different concentrations; increase in benzoquinone concentration (cQ) coupled to the increase in proton concentration (cH+ ) in the aqueous phase. The concentrations of KN03 are indicated on the graph.
175
reaction of the same type but without proton transfer coupling. Here, a weak acid ion exchanger, 2-propylpentanoic acid in its Fe(II1) form *, was made to react with hydroquinone (in a 0.1 M solution). Both redox couples were dissolved in the non-aqueous phase of a two-phase system consisting of 20 cm3 pentanol (0.1 M tetrabutylammonium chloride (TBACl)) and 20 cm3 water (0.1 M TBACl). The distribution equilibrium of TBACl was found to have a constant of 1.12. Thus the concentrations of TBACl within the two phases after equilibration will exhibit small deviations from the initial values. The two phases were agitated separately, the upper (pentanol) phase with a rotating gold disc electrode (r.d.e.), 3000 r.p.m., and the aqueous layer with a Metrohm (504) synchronous magnetic stirrer (500 r_p.m.)_ Mass transport between the phases was found to be sufficient for a distribution equilibrium of, for example, 1 X low3 M HCl to be attained within less than 60 s. The distribution-equilibrium constant was found to be 455 for 2-propyl pentanoic acid in the pentanol I aqueous phase system. Fe3+ and Fe*+ were shown to be confined to the pentanol phase during the course of the experiment, with a detection limit of 2 X lo-’ M for Fe3+/Fe2+ within the aqueous phase. The experimental set-up was completed by a double junction reference electrode (Metrohm EA 440) Ag I AgCl, sat. KC1 IIethanol, 0.1 M TBACl, a Pt-auxiliary electrode, both inserted in the pentanol phase in order to determine voltammetrically the quinone concentration together with the r.d.e. For comparison purposes, the
Fig. 3. Two-phase compared to the one-phase reaction hydroquinone. cQ = concentration of benzoquinone.
of 2-propylpentanoic
acid, Fe”’
salt with
* 10 g of 2-propylpentanoic acid mixed with 10 g of pentanol was shaken 5 times with fresh solutions of 15 g of FeCl, in 30 crd of water, washed with water until a pH of 5 was obtained, separated from the water phase, and filtered through H,O-free Na,SO,. 2 cd of this solution was taken per experiment.
176
reaction was carried out in a single phase system, which consisted of the pentanol phase alone (pentanol saturated with water). Figure 3 shows the formation of benzoquinone as a function of time in the two-phase compared to the single-phase system. It is clearly seen that in the two-phase system the rate of the redox reaction is greatly accelerated and the equilibrium is shifted to the right. This is explained by Scheme 2:
aqueous
phase
c-non-aqueous
-
phase
Fe(RCoo)j
+
l/2
QH,
Fe(RCOO)2
+
l/2
Q
K
RWOH
4
Rcoo- +
li+
cw
CL* RCW-
Ii+ c
__\ +
RCC,3H
K RcooE
+
2
a+
cp
w
RCW”
RCOOH
Scheme 2.
where the weak acid, RCOOH, its charged and uncharged forms are distributed quasi-independently between the phases. This assumption seems justified in view of the excess amount of TBACl present in both phases. The protons formed during the redox reaction recombine with the uncharged form of the weak acid (the pK of propylpentanoic acid was found to be 4.83). The recombination of the protons formed during the redox reaction with anions of the weak acid either in the pentanol or in the water phase leads to a redistribution of charged and uncharged forms of the weak acid, in this case the ion exchanger itself. The result is an acceleration as well as a shift in the equilibrium of the redox reaction, although no net amount of protons traverses the interface. Despite some small oscillations of the pH value, no difference in the proton concentration of the aqueous phase could be detected. DISCUSSION OF mE EXPERIMENTS
The experiments will now be discussed in greater detail and in particular with respect to their relevance to biological membrane processes. Both the proton-coupled and the uncoupled reactions follow the same general equation:
QS
+2FeR
3*Q+2FeR,+2Hr
JrK, Jr& R-+ H+ QH-+ H+ The dissociation constants concerned are: 2-propylpentanoic
(I) acid: K, = 1.48 X 10e5,
177
pK = 4.83; hydroquinone: K, = 4.5 X lo-“, pK = 10.35 [6]; strong acid ion exchanger (sulfonic acid type): K, = lo-‘, pK = 1. Hence in both cases K, > K, B K, and the reaction takes place according to QH, + 2 FeR, ;tQ+2FeR,+2Re+2H+
(2)
where protons are formed during the reaction. This leads to a shift in the equilibrium of the redox reaction in the two-phase system because protons are withdrawn according to the reaction mechanisms given above. In the case of the strong acid ion exchanger, the dissociation equilibrium of the -SO,H group causes free protons to be formed and transported into the aqueous phase. K+ and other ions (NO;) redistribute between the phases in order to satisfy the mass balance of the ion exchanger. [-SO;]
res- 3 [Fe3’] res - 2 [Fe’+] res =
WI res+ W 1res- [NO;]res
(3)
In the more dilute bathing solutions (0.01 and 0.1 M), the transfer of NO; ions into the resin phase does not occur to an appreciable extent because of co-ion exclusion. Therefore, in these cases the exchange of H+ for K+ can be expected to be fully coupled to each other as well as to the redox reaction. This was shown experimentally (Fig. 1). At higher concentrations of the electrolyte, the formation of H+ within the aqueous phase still appears to be coupled to the redox reaction, however with increasing reaction rates. In an attempt to arrive at a quantitative treatment of this phenomenon, the following calculations were carried out. Under the conditions prior to the start of the redox reaction, the mass balance of the resin phase is described by
[-SO;],,,-3[Fe3+l,,,=[K+l,,,+[H+l.,,-[N0,],,,
(4)
while the Donnan potential across the interface is determined by the activities of the ions able to permeate both phases:
=
$$ In
[Fe3+] aq [Fe3+l res
(5)
Concentrations rather than activities are accessible experimentally. However, taking the concentrations in the aqueous phase of the resin, only approximate values of u Donnanmay be obtained and thus with the aid of equation (6) the variations of (Y with ionic strength and with the progress of the redox reaction.
(6) This equation was established by Katchalsky [7], taking into account the influence of the electrostatic energy on the titration curve of a water/resin system (see also Refs. 8-10).
179
The variation in (Y is derived from the experiments with varying electrolyte concentrations. With known concentrations of the ions in the aqueous phase, i.e. Wlaq, K+laq9 WJ,, and P3+la,, the concentration of protons in the resin can be calculated using equations (4) and (5). phase, WI,,,, The tendency of U,,,,,,, to decrease as a function of electrolyte concentration in the aqueous phase may be derived by simply taking the concentration of, for example, K+ ions, calculated according to equation (5), for the computation of u DOlllltlll and Apk+, respectively. This is shown in Table 1, where the activity coefficients are neglected. Therefore Un,,,,, cannot be taken as absolute values but they are adequate for calculating the respective AU,,,,,. They reflect the fact that the starting conditions of the reactions described by Fig. 2A-D are characterized by a change in Api in the positive direction. The addition of hydroquinone starts the redox reaction and makes available a further lot of -SO,H groups resulting in a redistribution of ions such that a new state of equilibrium is established. The rate of both the redox and the ion exchange processes varies with the starting conditions given above. In the following, the influence of various factors on the reaction rate will be discussed. Firstly, the potassium potential within the resin phase increases appreciably as a consequence of the change of the Apx + in the positive direction. The variation in the standard potential of the Fe’+/*+ couple in the resin phase as a function of the potassium potential can be evaluated using the data plotted in Fig. 1. The changes in [K+],, and [H+],, were used to calculate the changes in [K+],,, and [H+],,, by calculating the difference to the initial values. [K+],,, and [K+],, thus obtained were used to calculate U,,,,,. Known concentrations of quinone and hydroquinone within the aqueous phase yielded the potential within the aqueous phase U,,according to: (0.059) log
44 = Uiq,Q,cm +2
IQ1aq[H+I:, tQfbla,
(7)
where Uiq,Q,QH2 = 0.699 V. Then the potential within the resin phase U;,,, was obtained by calculating the difference to the Donnan potential: (8)
u*q - Q30,,,, = IL
Finally, the standard potential of the Fe3+/Fe2+ couple within the resin phase
U" res,Fe3+,Fe~+ was calculated using the equation u,,, = U;s,Fe3+,Fe2++ 0.059 log
[Fe3+l res [Fe*+1 res
(9)
where [Fe*+] res was taken to be proportional to the amount of quinone formed during the reaction. This was done following the curves shown in Fig. 1. From this, values of
180
U”res,re3+/re2+at different potassium potentials within the resin phase were obtained, e.g. 0.556 V and 0.621 V at [K+]_ = 0.78 and 0.89 respectively. Again, these values are not absolute values and only serve to show that, in principle, the standard potential of the Fe3+/Fe2+ couple increases in the positive direction with increasing potassium potentials in the resin phase. This clearly would have an effect not only on the equilibrium but also on the rate of the redox reaction, which depends on the rate of diffusion of redox and ionic components. Secondly, [Fe3+lres decreases considerably with increasing concentration of K+ in the bathing solution due to the Fe 3+-K+ exchange prior to the redox reaction. Consequently, the term log([Fe3’],.,,/[Fe2’],,,) increases in the negative direction. This would at least in part counterbalance the effect of increasing standard potentials discussed above. Thirdly, the variation in Ap k+ itself is expected to exert an effect on the rate of ion exchange and-because of the coupling-on the entire process. In the more dilute bathing solutions, the inward potassium flow is forced to occur against a considerable Ap x+ which decreases with increasing KNO, concentration. This, therefore, should accelerate the redox and H+/K+ exchange processes. For the effect of ionic gradients on ion exchange kinetics, see, for example, Ref. 11. At present, it is not clarified which of these factors plays the predominant role on the reaction rate. In every case, the interdependence of the three processes redox reaction, proton and potassium transfer can be demonstrated. A final remark will be devoted to the proton/electron stoichiometry. Not more than about a quarter of the protons expected according to equation (2) were found. This result can only be accounted for by the change in (Ywith the change in pH at the respective Uo,,,. Regarding now the weak acid ion exchanger, this has a double function: first, as the anion of the Fe3+/Fe2+ couple. This makes it a participant of the redox reaction according to equation (2). Second, as an interfacial buffer which facilitates transport of protons between the two phases. Protons, however, are not transported across the interface as charged particles as in experiment 1 but rather as uncharged molecules formed by recombination of the proton with the anion of the uncoupler. Therefore, the proton-involving redox reaction takes place without an accumulation of protons in one of the two phases. This second function of the weak acid ion exchanger is proposed as a general principle of uncouplers of biological proton transfer. Due to their ability to be present in their charged and uncharged forms in the water as well as in the membrane phase, they are considered to prevent the formation of ion gradients while the redox reaction itself goes on unimpeded or even accelerated compared to a two-phase redox reaction coupled to ion transfer as shown in experiment 1. This idea gains support from the work of Terada and Van Dam [12]. These authors proposed a shuttling mechanism of the uncoupler supported later by Hitchens and Kell [13]. From the viewpoint of the present author, the uncoupler shuttles between phases in order to inhibit redox coupled ion exchange processes.
181
CONCLUDING
REMARKS
In the present paper, a model reaction was presented according to which a proton-involving redox reaction causes protons to flow across the interface of a two-phase system. Proton transfer occurs due to the non-vanishing proton balance of the redox couples comprising the overall redox reaction. This was proposed earlier [l] to be the reason for interfacial proton transfer in biological systems. In order to obey the electroneutrality condition, the interfacial proton flow is accompanied mainly by a countercurrent cation flow. Hence the chemical reaction is coupled to an ion exchange process by virtue of the mass balance of the resin or, in other words, by the electroneutrality condition. This was proposed to be the reason for redox induced ion exchange in mitochondria [l]. These conclusions apply to biological membrane transport only if the model reactions described here have something in common with biological systems. The significance of ion exchange resins as models for biological membrane systems was investigated earlier by Katchalsky [7] and emphasized only recently by De Korbsy [14]. Fe(II1) bound to a fixed charge network can be regarded as a redox center. But these are not the main points this paper is dealing with. The goal was to show a two-phase system exhibiting peculiarities of biological ion-transport and yet consisting of two compartments only. Hence attention is focussed on interfacial rather than transmembrane ion transfer processes. ACKNOWLEDGEMENTS
The author is extremely grateful to Prof. I.R. Miller, The Weizmann Institute of Science, Rehovot, Israel, for his constructive criticism and his important suggestions regarding this article. Further, I wish to express my gratitude to Prof. H. Wendt, Technische Hochschule Darmstadt, and to Dr. W. Pusch, Max Planck Institut fur Biophysik, Frankfurt/M., for very profound discussions on the present paper. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14
R. Naumamr, Bioelectrochem. Bioenerg., 9 (1982) 663, D.K. Schiffer, A. Hcchhauser, D.F. Evans and E.L. Cussler, Nature (London), 250 (1974) 484. T. Shinbo, K. Kurihara, Y. Kobatake and N. Kamo, Nature (London), 270 (1977) 277. J.J. Grimaldi and J.M. Lehn, J. Am. Chem. Sot., 101 (1979) 1333. T. Shinbo, M. Sugiura, N. Kamo and Y. Kobatake, J. Membr. Sci., 9 (1981) 1. J.C.D. Hodgman in Handbook of Chemistry and Physics, R.C. Weast (Editor), 46th ed., CRC Press, Cleveland, OH, 1965. A. Katchalsky, Progr. Biophys. Biophys. Chem., 4 (1954) 1. A. Katchalsky and S. Lifson, J. Polym. Sci., 11 (1953) 409. A. Katchahky and I. Michaeli, J. Polym. Sci., 15 (1955) 69. I. Michaeli and A. Katchahky, J. Polym. Sci., 22 (1957) 683. F. Helfferich, Ionenaustauscher, Vol. 1, Verlag Chemie, Wiesbaden, 1959, p. 239. H. Terada and K. van Dam, Biochim. Biophys. Acta, 387 (1975) 507. G.D. Hitchens and D.B. Kell, B&hem. Z., 212 (1983) 25. F.D. De Koriisy, Bioelectrochem. Bioenerg., 9 (1982) 391.