9588
J. Phys. Chem. 1994,98, 9588-9593
Transmembrane Electron Transfer Mediated by Diheptylviologen: Disproportionation of Viologen Radical and Viologen-Induced Leakage of External Reductant Leif Hammarsfroin,*Helena Berglund, and Mats Almgren Department of Physical Chemistry, University of Uppsala, Box 532, S-751 21 Uppsala, Sweden Received: January 26, 1994; In Final Form: June 17, 1994@
Transmembrane electron transfer in lecithin vesicles mediated by diheptylviologen was studled by stoppedflow and absorption spectroscopy. The reaction was initiated by addition of dithionite to the bulk phase which reduced viologen distributed over the outer vesicle interface and the bulk. The viologen reduced 2 equiv of ferricyanide in the vesicle interior. The observed reaction was of second order with respect to the viologen mole fraction, with a first half-life of = l s for 1.5 mol % viologen in the outer monolayer of the vesicles. The reaction was found to proceed by transmembrane diffusion of the doubly reduced, uncharged viologen ((c7)2v0) formed by disproportionation of two viologen radicals ((c7)2v+). This lends support to our suggestion that this mechanism is generally at work in microheterogeneous media. Observations on a longer time scale indicate that leakage of dithionite through the membrane may be induced by introduction of .c 10 mol % diheptylviologen in the membrane. Arguments against a high membrane permeability of the dicationic (C7)2V2+ are presented.
Introduction Vesicles have for many years attracted attention as carriers of biomimetic reaction systems, e.g. for charge separation.' However, many features of these surfactant assemblies and their behavior during reactions are still poorly understood, e.g., induced transmembrane leakage of hydrophilic solutes and the mechanisms of transmembranecharge transfer. Viologen (N,N'substituted 4,4'-bipyridine) is one of the most well-known redox mediators in this field and other. In spite of numerous studies, the mechanism of transmembrane electron transfer mediated by viologen remains controversial; diffusion of the reduced viologen r a d i ~ a l , ~ electron -~ tunneling between viologen molecules through the membrane,7-l0 and a combination of those two m e ~ h a n i s m s ~ J have - ' ~ been proposed. We have earlier presented results concerning transmembrane electron transfer in lecithin vesicles mediated by cetylmethylviologen (C16MV).'4-'6 The results showed that electron tunneling between viologen bound at opposite interfaces of the vesicle membrane was not responsible for the observed process. Neither were any of the cationic forms (C16MV2+and C16MV+) found to penetrate the membrane on the experimentaltime scale. Instead we proposed a mechanism (Figure 1) where a ratedetermining disproportionation reaction
-
2C16MV+
kd
+
C16MV2+ C16MVo kc formed a doubly reduced, uncharged viologen (C&lVo) which rapidly (rate constant 21 x lo3 s-l) diffused through the membrane where it reduced 2 equiv of ferricyanide. The CMV2+ formed was reduced again by dithionite which was in excess. The conproportionation(eq 1 from right to left) seemed to be negligible. The use of an electron acceptor other than viologen in the vesicle interior combined with addition of viologen only to the external phase proved to be a fruitful approach. Thus, we were able to exclude transmembrane electron tunneling between viologens, to unambiguously identify and observe the transmembrane reaction step and determine the reaction stoichiometry by direct absorption spectroscopy without subsequent steps of analysis. @
Abstract published in Advance ACS Abstracts, August 15, 1994.
Figure 1. Proposed disproportionationmechanism for transmembrane electron transfer mediated by viologen, as described in the text. The membrane interfaces are indicated by the curved lines. The direction of electron transfer in this figure is inward, i.e., from dithionite in the bulk to ferricyanide in the vesicle interior. The viologen is trapped as the membrane impermeable dication (V2+), inaccessible to dithionite in the bulk on the experimental time scale. The disproportionation reaction (eq 1) is indicated by its rate constant kd.
The occurrence of disproportionationis a simple consequence of having two consecutive, reversible reduction steps. The disproportionation constant Kd is usually evaluated from electrochemical data according to
where A P is the difference in reduction potential between the two steps (At?' = @1/2 - E'112). Even though Kd is very low in water ( 1 0 y 7the disproportionation can be efficient in microheterogenous systems since the different redox forms are solubilized preferentially in different environments, so that separation of the disproportionationproducts is enhanced. This has earlier been claimed to be the case in microemulsions.l8 The efficiency of the processes in eq 1 as determined from redox data is not necessarily the same in microheterogeneous media, as, e.g., vesicles. This is because the various redox forms are distributed differently over the environments in the system. Both the nature of the distribution and the rates of exchange between
0022-365419412098-9588$04.5010 0 1994 American Chemical Society
J. Phys. Chem., Vol. 98, No. 38, 1994 9589
Transmembrane Electron Transfer in Lecithin the different environments have to be considered for each species, as discussed before.15 Apart from the difficulties in describing such a system quantitatively, it might not even be meaningful to describe the conproportionation with a simple second-order rate constant. Apparent values of A F might then lead to erroneous conclusions regarding the efficiency of disproportionation. Recently, the generality of our mechanism was questioned.19 It was proposed that a number of viologens (diheptyl-, dioctyl-, didecyl-, and dibenzylviologen), even in the oxidized form (C,)2V2+, were all intrinsically highly membrane permeable in lecithin vesicles. This would allow for transmembrane electron transfer by cyclic diffusion of (C,)2V+/(C,)2V2+ through the membrane.20 It seemed important to test this proposal. In the present study we found results showing that (C7)2V behaves qualitatively the same as C16MV as a transmembrane redox mediator between dithionite in the bulk phase and ferricyanide in the interior of lecithin vesicle^.'^-'^ Thus, the disproportionation mechanism seems to be valid also for this system. The results further indicate that (c7)2v2+ is membrane impermeable on the experimental time scale but that high mole fractions of viologen induce transmembrane leakage of dithionite. The results and mechanism presented in ref 19 is discussed in view of the present results. In the text the charge of the viologen is indicated only when necessary to distinguish between the different redox states. Further, we have expressed the concentration of viologen in number per vesicle. We believe that this is easier to follow than if mole fractions were used, even if the ratio [viologen]/ [lipid] is the important property in the reactions discussed. This must be remembered when comparison is made with vesicles of different size than the small, unilamellar vesicles used in the present report.
Experimental Section The vesicles were prepared from egg lecithin (first grade, Lipid Products, Nutfuield, England) by sonication and gelexclusion chromatography as described earlier,I4 resulting in small, unilamellar vesicles as confirmed by cryotransmission electron microscopy. The average diameter was ~ 2 2 A, 0 and each vesicle consisted of %3000 lecithin monomers on the average: 2000 in the extemal and 1000 in the internal monolayer.21 The percentage of different hydrocarbon chains in the egg lecithin was, according to the supplier, 16:0, 32.1%; 16:1, 2.1%; 18:0, 11.7%; 18:1,36.2%; 18:2, 12.5%; 20:4, 5.5% (number of carbons:number of double bonds). Diheptylviologen dibromide (Eastman Kodak) was recrystallized from butanol. Dibenzylviologen dichloride (Sigma) and all other chemicals were of highest commercially available purity and were used as received. Absorption spectra were recorded on a Varian CARY 2400 spectrophotometer. The subminute stopped-flow experiments were made using a High-Tech SF-5 1 working anaerobically, with supplied accessories. Data were recorded using a Phillips PM 3350 digital oscilloscope. For the slower stopped-flow experiments represented in Figure 4b a High-Tech SFA-11 Rapid Kinetics Accessory was used with an observation cell fitting in the CARY. This simple stopped-flow equipment did not allow deoxygenation; consequently, a higher concentration of dithionite (oxygen scavenger) was used. These experiments were complemented by experiments where small amounts of concentrated dithionite solution were injected with a syringe directly into a deoxygenized solution in the cuvette of the CARY. In these experiments a low concentration of dithionite could be used (Figure 4a).
u.
0
5
Time (s)
15
10
Time (s) Figure 2. (top) Typical kinetic trace from stopped-flow experiments
where dithionite was added to vesicle solutions containing extemal diheptylviologen and intemal ferricyanide. The curve represents the transmembrane reduction step where the initially formed (C7)2V+on the vesicle exterior disappears as the disproportionation product (C7)2Vo migrates to the vesicle interior and is reoxidized by ferricyanide. The experimental curve and the fitted second-order curve are superimposed. Only the part of the experimental curve over which the fit was made is shown. (bottom) Plot of the residual from the fit to a second-order decay. Conditions: [lecithin] = 1.1 mM, [diheptylviologen] = 7.0 pM (20 per vesicle), [dithionite] = 5 mM, [ferricyanide],, = 0.2 M (70-80 pM overall), 50 mh4 phosphate buffer (pH = 8.0). [KCl],,, = 0.25 M, T = 21 "C. With the exception indicated above, all solutions used in experiments involving redox reactions were deoxygenized by bubbling with nitrogen gas of high purity prior to the experiment. Phosphate buffer (50 mM, pH = 8.0) was used for all solutions. KCl was added to the buffer used in gel-exclusion chromatography to compensate osmotically for entrapped species. The temperature was 21 & 1 "C, except where stated otherwise. Fitting of kinetic curves were performed as an unweighted least-squares fit using a SIMPLEX algorithm. The second-order curves were fitted to the equation Abs = A B/(1 B O ) , where Abs is the absorbance, A and B are the absorbance at infinite time and additional absorbance at time I = 0, respectively, and C is the second-order rate constant (identical to kd in eq 1 according to the mechanism proposed). The inverse of the first half-life ((t1,2)-l = BC) was used in Figure 3 since B and C individually were more sensitive to fitting conditions. The fits were made over > 80% of the total amplitude; a typical example is shown in Figure 2. In second-order kinetics, the last 10% of the amplitude decays over long times. Using longer time scales to include this part of the curve would compress the earlier parts, leading to loss of information. That the experimental curves were truly second order to the end was checked separately.
+
+
Results and Discussion Reduction of Diheptylviologen in Vesicle Solution. It has been shown that the binding of diheptylviologen to lecithin vesicles approximately follows a Langmuir isotherm with a maximum binding capacity of around 220 viologens per extemal vesicle interface19 (2000 lecithin molecules). In the present study, upon addition of an excess of dithionite to a vesicle solution containing diheptylviologen in the extemal phase (the outer vesicular interface and the bulk) the diheptylviologen was immediately reduced to the radical cation ((c7)2v+). The latter
9590 J. Phys. Chem., Vol. 98, No. 38, 1994
Hammarstrom et al.
is probably completely vesicle bound due to its low solubility in water. The rate of viologen reduction was proportional to [ S Z O ~ ~ - ](data ' / ~ not shown), consistent with SOz- being the reducing specie^.^^^^^
s20,2-= 2s0,SO,-
+ (C7)2V2++ OH- -HSO3- + (C7)2V+
(3b) 1
When no secondary electron acceptor was present in the vesicle intenor, the characteristic absorption spectrum from the viologen radical was constant for at least 30 min at the temperature used in this study (21 f 1 "C). At 30 "C, however, the absorption spectrum changed slowly and after 30 min it corresponded to equal amounts of viologen radical and doubly reduced (c7)2v0, as was previously observedlg for this system when diheptylviologen was localized in the vesicle interior only. This extremely slow second reduction step is not connected with transmembrane processes but can be explained by a low driving force (only about half of the viologen was reduced to (C7)2V0 even though dithionite was in excess, thus AGO > 0) and a very low concentration of the actual reductant SOZ- ([S02-l2/ [ S Z O ~ ~= - ] 1.4 x M,22 [S2042-] = 300-400 pM). The extent of reduction to (C7)2Vo is expected to be very sensitive to variation in concentrations of residual oxygen and d i t h i ~ n i t e ~ ~ and will vary for different experimental conditions. We note, however, that all other experiments were performed at 21 f 1 "C where no (c7)2v0 was observed. Transmembrane Electron Transfer Mediated by Diheptylviologen. Dithionite was also added to solutions where the electron acceptor ferricyanide was present in the vesicle interior and diheptylviologen in the external phase only. The behavior was qualitatively the same as when cetylmethylviologen ( C I ~ M V )was used.I4 According to the mechanism schematically presented in Figure 1, the initially reduced viologen radicals ((C7)2V+) formed doubly reduced (c7)2v0 by disproportionation. The hydrophobic (c7)2v0 migrated rapidly to the vesicle interior where it was reoxidized by 2 equiv of intemal ferricyanide. The external (C7)2V2+ formed in the disproportionation reaction was reduced again by dithionite which was in excess. When [Fe(CN)63-] > 2 x [(Cj)2V], no external viologen was left, all viologen being trapped as (C7)2V2+ in the interior, inaccessible to dithionite on all but long time scales (see below). When the vesicles were destroyed, by addition of the surfactant Triton X-100, all viologen was immediately reduced again by the dithionite remaining in the bulk phase. The rate of viologen-mediated transmembrane electron transfer was followed by stopped flow. As the viologen diffused to the interior and was reoxidized by femcyanide, the initially high absorbance of the reduced, external (Cj)2V+ disappeared. The decay of absorbance at 602 nm ((c7)2v+ maximum) versus time yielded traces that fitted well to a second-order decay model (Figure 2). The inverse half-life ( ( t l / z ) - l , see Experimental Section) was mainly proportional to the surface concentration of viologen (Figure 3). This is expected for a reaction that is second order in the surface concentration of viologen radical ([(Cj)zV+]), in which case
where kd is the second-order rate constant in eq 1 and [(C7)2V+]t=~is the surface concentration of viologen radical when all viologen has been reduced but before the transmembrane electron transfer has started. This rate dependence is consistent with a rate-determining disproportionation step.
0
10
1
20 30 viologen/vesicle
I
I
40
50
Figure 3. Rate of transmembrane electron transfer (inverse of the first half-life) mediated by diheptylviologenversus the number of viologens per vesicle. The points are averages over 5-8 individual runs using the same vesicle preparation. The line is a least-squares linear fit to the data points with an intercept of 0.16 s-I. The number of lecithin molecules per vesicle is e2000 in the outer monolayer and olO00 in the inner. Thus, 20 viologens per vesicle in the outer monolayer correspond to [viologen]/[lipid],,, = 0.010. Conditions: as in Figure 2, except [diheptylviologen] = 4.0-10.0 pM, [lecithin] = 0.50-5.0
m.
Thus, we conclude that the general behavior is the same as for the previously studied system with C16MV, and it can be explained by the disproportionation mechanism, as proposed earlier'4-16 (Figure 1). If not circumstantial, the indication of a slightly positive intercept could be due to a minor fraction of (c7)2v0 formed directly by dithionite, also at 21 "C. This would allow for a parallel reaction path that would be of first order with respect to [(C7)2V+]. Since dithionite was in excess, the reaction could in that case, as well as when (C7)2Vo was formed by disproportionation, be driven to completion. If the reaction would proceed through parallel second- and pseudo-first-order reaction paths, the resulting half-life would, at times t -= (k;)-l, be described byz4
where kz' = ~ ~ [ S O Zand - ] k2 is the second-order rate constant for reduction of (C7)2V+ by dithionite. However, in that case 2k2' should have been in the order of the intercept in Figure 3 ( ~ 0 . 1s-'), which is not consistent with the slow reduction observed at 30 "C (see above). Also, the kinetic traces would have been noticeably deflected from the second-order shape if the contribution from this pseudo-first-orderprocess would have been significant. We conclude, therefore, that the dependence of the rate of transmembrane electron transfer on the surface concentration of viologen is best described by eq 4a. The rate of transmembrane electron transfer was 4-5 times lower with (C7)2V than with C&lV, which indicates a lower yield of disproportionation products upon encounter, or a lower encounter rate. Transmembrane Leaking of Dithionite. After all viologen had been trapped in the vesicle interior in the experiments above, there still remained external dithionite and intemal femcyanide. A slow further reduction of the latter could be followed as a bleaching of its absorbance at 420 nm (Figure 4). When viologen was present at concentrations up to about 60 per vesicle, the rate of this slow reduction was the same as for vesicles without viologen ( ~ x5 lo-* M s-I, [lecithin] = 3.0 mh4). Since no transmembrane diffusion of ferricyanide was observed for at least several hours,14 it seems that dithionite slowly permeated the membrane and reduction took place in the vesicle interior. When the concentration of viologen was increased even further, however, the reduction of femcyanide suddenly became more rapid (Figure 4). The lowest number of viologen
Transmembrane Electron Transfer in Lecithin
0
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200
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J. Phys. Chem., Vol. 98, No. 38, I994 9591
800
Time (s) 0
4
-2 v
-10
-20
* e
-30 -40
%
-60
-50 -70
-80
'
0
I
I
I
1
I
100
200
300
400
500
230
250
270 290 310 Wavelength (nm)
330
350
Figure 5. Absorption spectra normalized at 260 nm. Solid line: (benzyl)zVz+(no vesicles); broken line: (C7)2VZf (no vesicles); circles: external phase of a vesicle solution separated from the vesicles by gel exclusion chromatography. The vesicles were prepared with (C7)2Vz+in the interior and an equimolar amount of (benzyl)2V2+was added to the external phase 20 min before the chromatographic procedure was initiated. A slight light-scatteringcomponent is obvious in the external phase which raises the curve at shorter wavelengths, resulting in a slight error in the normalization (the maximum is blue shifted compared to both (C7)2V2+and (benzyl)2V2+).Otherwise the overlap with the spectrum for (benzyl)2V2+is complete, as is most evident at 1 2 300 nm where light-scattering is negligible.
Time (s)
Figure 4. Reduction of intemal femcyanide by external dithionite at
different concentrations of (c7)2v2+ in the vesicle interior (see text). The reduction of femcyanide was followed as the decrease in its absorbance at 420 nm. Zero time was set to be when all viologen initially on the vesicle exterior had migrated to the vesicle interior by the disproportionation mechanism. Only the further reduction of femcyanide is shown, which presumably proceeded by transmembrane leakage of dithionite and subsequent reaction in the vesicle interior. The concentration of viologen (no per vesicle) is given in the figures. As can be seen, this leakage was drastically increased over a narrow interval of viologen concentrations. (a, top) Dithionite was syringetransferred to a deoxygenated solution in the cuvette. A small amount of gas bubbles, produced by bubbling with nitrogen gas, rose slowly to the surface and was responsible for the slight decrease in absorbance seen as a curvature in the early part of the curve (total contribution 95% dibenzylviologen; i.e., no observable transmembrane exchange of viologen had taken place (Figure 5 ) . If both viologen dications were membrane permeable, they would redistribute to equal concentrations on both sides in the present experiment, without need for additional charge compensating transmembrane diffusion of other ions. Thus, from this simple experiment we conclude that at least one of the dicationic viologens ((C7)2V2+or (benzyl)2V2+) is membrane impermeable on the experimental time scale. Instead, the increased transmembrane reduction observed with higher concentrations of viologen may be due to viologeninduced leakage of dithionite through the membrane. Dithionite
9592 J. Phys. Chem., Vol. 98, No. 38, 1994
leakage is consistent with the reported zeroth-order shape of the kinetic traces from transmembrane viologen reduction, but the observed rate would be expected to increase when the concentration of dithionite is increased 10 times (even if [SOz-] is increased by only a factor of 3). This was not observed,19 however, and when dithionite was replaced by Crz+, the rate of reduction was reported to be the same. If, however, leakage of the reductant was limited by charge compensating ion diffusion/ leakage, this could possibly explain the observed independence of reductant identity and concentration. Membrane Permeability of (c7)2v2+. The conclusions drawn above are very different from those of Kuhn and Hurst.lg We have to consider their results in some detail, therefore. The key feature of the mechanism proposed by Kuhn and Hurst is the assumption that all redox forms of the viologen are intrinsically highly membrane permeable. When no external viologen is added, the reduction of internal viologen by external dithionite is supposed to proceed by the diffusion of (Cn)2V2+ to the exterior, and back diffusion of reduced (c7)2v+. The rate of this process is suggested to be limited by the inevitable transmembrane diffusion of charge compensating ions. In qualitative accord with this, the addition of valinomycin/K+ to ensure a facile passage of K+ through the membrane reduced the time for complete reduction of internal viologen from 10 min to less than 1 min at a valinomycin mole fraction of 0.1 in the membrane. In separate experiments, without dithionite, it was found that whereas permeation of (C7)2V2+ through the membrane occurred with a half-life of 4.3 h, the exchange of radioactivity labeled (C7)2V2+ in the interior for unlabeled (c7)2v2+ in the external phase seemed to occur with a half-life
