J. Phys. Chem. 1993, 97, 1712-1721
1712
Mechanisms of Vectorial Transmembrane Reduction of Viologens across Phosphatidylcholine Bilayer Membranes Eberhardt R. Kuhn and Jama K. Hunt’ Biomolecular Materials Research Loboratory, Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006- 1999 Received: October 19, 1992
Zero-order kinetics were observed for one-electron transmembrane oxidation-reduction in vectorially organized phosphatidylcholine vesicles containing entrapped viologens and chemical reductants in the external aqueous phase. Oxygenation-reduction cycling established that the viologen was retained within the vesicle during this reaction. The rates were independent of the identities of the reactants and concentration of reductant as well as a wide variety of medium conditions. At high salt concentration, however, reduction of N,N’-diheptyl4,4’-bipyridinium ion [(C7)2V2+],the most intensively studied viologen, exhibited anion-specific rate acceleration and autocatalysis, with the relative effectiveness of the anions following approximately the lyotropic series. Addition of valinomycin in K+-containing media or other lipophilic ions also accelerated the reaction rate. Sigmoidal kinetics were observed when valinomycin was present, and following reduction, the viologen was shown to have diffused out of the vesicles. The (C7)2V2+dication was demonstrated to be membrane permeable by 14C-radioisotope-exchangetechniques. These data were interpreted to indicate that net transmembrane oxidation-reduction occurred stepwise by (i) (c7)2v2+ ion diffusion across the bilayer, (ii) reduction in the external aqueous phase, and (iii) uptake of the (C7)2V+ product ion by the vesicles, with the overall reaction being rate limited by movement of charge-compensating aqueous ions. When other membrane-permeable ions were present, their translocation dissipated the membrane potential formed by outward diffusion of entrapped (C7)2V2+ ion, allowing accumulation of the (C7)tV’ radical cation at the external vesicle interface. This accumulation, in turn, gave rise to a (C7)2V+-mediatedtransmembrane redox pathway. The relative contribution of this pathway increased as the reaction proceeded, thereby accounting for the autocatalytic character of the reaction. Photostimulated transmembrane oxidation-reduction of entrapped (c7)2v2+ion by EDTA ion was also observed with several photosensitizers that were confined to the external aqueous phase. These reactions could also be accommodated by the general reaction scheme presented but not by other previously advanced mechanisms.
Introduction Transmembraneoxidation-reduction and transportof lipophilic ions across closed bilayer membranes are complex processes whose mechanisms are only now becoming understood.’-3 Passive diffusion of aqueous ions is often an integral step in these reactions and can become rate limiting under certaincircumstances. These steps are coupled because transmembrane electron transfer or lipophilic ion translocation is electrogenic in the absence of compensating ion movement and leads to development of a membrane electrical potential that opposes further reaction. For small vesicles and related supramolecular assemblies, the potential develops quite rapidly and is large after charge translocation constituting the equivalent of only a few ions per particle has occurred.] Leakage of aqueous ions provides a means for dissipating the potential. This phenomenon has been used to advantage to verify the occurrence of transmembrane electron transfer in vectorially organized vesicle assemblies.’ For example, carrier-mediated inward electron transfer between hydrophilicreductants located in the bulk external aqueous phase and oxidants occluded within the internal aqueous phase was accompanied by acidification of vesicle interior^.^ This is prima facie evidencethat transmembrane charge separationhas O C C U K since ~ ~ proton accumulation against its concentration gradient would occur only if coupled to forces driving the redox reaction, e.g., by membrane polarization with the inner surface negatively charged. Similarly, we have recently shown that transmembrane reduction of dihexadecyl phosphate To whom correspondence should be addressed.
0022-3654/93/2097- 1712$04.OO/O
(DHP) vesicle-bound methylviologen (N,N’-dimethyl-4,4’-bipyridinium, MV*+)by dithionite ion occurs with cotransport of one MV+ radical or other lipophilic cation per electron across the bilayer.5~6 Although redistribution of bound methylviologen clearly establishes that transmembrane redox has occurred, the reaction mechanism is not so evident. In particular, it is difficult to distinguishbetween pathways involving transverse diffusion, e.g., ‘flip-flop” exchange, of bound redox components and pathways involving transmembrane electron transfer. In principle, these mechanisms can be distinguished by quantitative comparison of transmembraneoxidation-reduction and viologen diffusion rates.] We have used this approach to investigate viologen-mediated transmembrane reduction of a series of N-alkyl-N’-methyl-4,4’bipyridinium (C,MV2+)ions across DHP bilayer m e m b r a n ~ . ~ < 8 By comparing for various viologens (i) the extent of interfacial redistribution accompanying the reaction, (ii) relative rates of transmembrane oxidation-reduction and viologen diffusion (measured by I4C-radioisotopic labeling), and (iii) the rateenhancing effects of added lipophilic ions, we have established the existence of two distinct pathways. One pathway, which is predominant for the short-chain analogs (n Ilo), is diffusional in character and was distinguished by an approximate 1:l stoichiometric comigration of the redox-mediating C,MV+ radical cation with the electron. Theother pathway, which is predominant for the long-chain analogs ( n > lo), was expressed only when other lipophilic ions were added to the reaction medium and was distinguished by an absence of net transmembrane movement of the redox-mediatingC,MV+ ion. Since, in this case, the CnMV+ Q 1993 American Chemical Society
Vectorial Transmembrane Reduction of Viologens ions were unable to traverse the bilayer on the time scale of the transmembraneredox reaction, the pathway must involve electron exchange between C,MV+ and C,MV2+ ions located in the opposite bilayer leaflet^.^ The structural bases for expression of the separate pathways are not completely understood but may include differing modes of binding of the short-chain and longchain viologens to the vesicle and the greater tendency of the long-chain viologens to aggregate, or form separate domains, within the membrane. The physical properties of C,MV2+ ions bound to DHP ~ e s i c l e s ~are ~ ~consistent -l~ with a model wherein the short-chain viologens bind at the aqueous-rganic interface primarily by electrostatic forces to the DHP phosphate head groups, but the more amphiphilic long-chain congeners intercalate within the DHP surfactant ions, forming part of the bilayer structure. Transmembrane reduction of viologens across phosphatidylcholine (PC) bilayer membranes is distinct from analogous reactions involving DHP vesicles in not requiring that a redoxmediating C,MV2+ be present on the same side of the membrane as the reductantsfor the reaction to occur. One might superficially conclude from this observation that direct electrontransfer across the bilayer from reductants located in the bulk aqueous phase to compartmented viologens is possible. However, we describe in this paper results of experiments based upon approaches developed to study the corresponding reactions in DHP-organized systems which indicate that a more plausible mechanism involves transverse diffusion of the occluded viologen, followed by its reaction with reductant in the same microphase. A strong interdependence between transmembrane redox rates and transmembrane diffusion of aqueous ions was observed, which allowed us to investigate the nature of the coupling of the ion fluxes. As discussed, these results may also bear significantly upon similar reactions reported by others that had been interpreted in terms of rate-limiting transmembrane electron exchange between PCbound redox-mediating amphiphiliccoordinationcomplexes.l3-I5
Experimental Section Materials. Phosphatidylcholine (PC) was extracted from fresh hen egg yolk and purified using chromatographic procedures described in the literature.16 Thin-layer chromatography of the isolated compound on silica with 25: 1 5 4 2chloroform-methanolacetic acid-water gave a single spot at r = 0.24. N,N’-Diheptyl4,4’-bipyridinium (diheptylviologen, (C7)2V’+) dibromide was obtained from Eastman Kodak. Other dialkylviologendibromides used in this study were prepared by refluxing 4,4‘-bipyridine with 4-fold excess of the corresponding alkyl bromide in acetonitrile for 12 h, filtering, and recrystallizing the yellow solids from acetone/methanol,I7 Thin-layer chromatography on silica with 3:3: 1methanol-water-ethylammonium hydrochloride gave single spots. Proton NMR spectra were recorded using a General Electric QE-300 instrument; in all cases, spectral peak positions and relative intensitieswere consistent with the patterns expected from the molecular structures. Radiolabeled diheptylviologen was prepared as follows: 0.85 mg of [I4Cl]heptanol and 7.7 mg of cold heptanol (0.07 mol) were dissolved in 2 mL of ether, and 0.6 mg of PBr3 (0.025 mol) in 0.5 mL of ether was added in 50-pL increments over a period of 1 h while cooling the reaction mixture in an ice bath. Following subsequent addition of 0.1 mL of 0.1 M NaOH to neutralize the phosphoric acid produced and decompose unreacted PBr3, the solvent was evaporated, and 10.9 mg of bipyridine (0.07 mmol) in 2 mL of DMF was added to the oily residue. The mixture was refluxed for 15 h and then cooled to -17 OC to precipitate the product. Thin-layer chromatographygave a single spot migrating at a position identical to the commercial (C7)2V’+ ion. Radiolabeled N-hexyl-N‘-methyl-4,4’-bipyridinium (C6MV2+)ion was prepared by adding 3.3 mL of 14CH31in nitromethane (0.033 mmol, 0.35 mCi) toa 10-fold excess of N-hexyl-4,4’-bipyridinium
The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1713 chloride in 14 mL of acetonitrile contained within a thick-walled glass ampule. The ampule was cooled in liquid N2,sealed under vacuum with a torch, and then heated to 105 OC for 25 h. After cooling, the ampule was opened and 470 mg (3.3 mmol) of cold CH3I was added. The ampulewas resealed and heated for another 24 h. The contents were then recovered, the solvent was removed by rotary evaporation, and the solid was washed with ether and recrystallized from acetone/methanol. Thin-layer chromatography on silica showed a single spot which comigrated with an authentic sample of the C6MV2+ion. The dibromide salts were converted to dichloride salts by precipitation from aqueous solutions with picric acid, redissolving the isolated solid in acetone, and precipitating the viologens by bubbling gaseous HCl through the solution. Dibromides were converted to dinitrates by precipitating B r from aqueous solutionswith a 50%stoichiometric excess of AgNO3 and then removing excess Ag+ by precipitation as the phosphate salt in 0.1 M phosphate buffer, pH 8. 4,4’-Dihexadecylcarbxamido-2,2’bipyridine was synthesized by following procedures described in the literatureI7-l9by KMn04 oxidation of 4,4’-dimethyL2,2’-bipyridine to the corresponding dicarboxylic acid, followed by conversion to the acid chloride with S02Cl and then reaction with excess hexadecylamine. The isolated product was purified by extraction with butanol in a Soxhlet extractor. The derivatizedbipyridine (90 mg, 0.1 mmol) was refluxed with cis-dichlorobis(2,2’-bipyridine)ruthenium(II) (60 mg, 0.1 mmol) under nitrogen for 4 h to form the 4,4’-dihexadecylcarbxamido-2,2’-bipyridinebis( 2,2’-bipyridine)ruthenium(11) (Ru(bpy)z [4,4’-(C I 6CONH)2bpy]2+) complex ion. After cooling, aqueous NaC104was added to initiate precipitation of the perchlorate salt. The brown solid was filtered, dissolved in acetone to remove any unreacted starting material, and then recrystallized from methanol after recovery of the solid. Thinlayer chromatography on silica with 25: 15:4:2 chloroformmethanol-acetic acid-water gave a single spot. The optical absorption spectrum was identical to published spectra. Chromous ion was prepared by anaerobic reduction of Cr(C104)3over zinc amalgam. N-Alkyl-4,4’-bipyridinium chlorides, N-alkyl-N’-methyl-4,4’-bipyridinium dichlorides, and dipyridone were gifts from Dr. David H. Thompson at OGI; (5,10,15,20tetrakis(N-methylpyridinium-4-yl)porphinato)zinc(II) tetrachloride (ZnTMPyPC14)was a gift from Dr. K. Kalyanasundaram, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland. Other materials were reagent grade and used as received.
Methods Veclicle Preparation. Vesicles were prepared from egg phosphatidylcholine by either ultrasonic dispersal or high-pressure extrusion. In either case, a chloroform solution containing 100 mg/mL PC was first rotary evaporated to dryness in a roundbottomed flask, coating the glass surface with a film of the amphiphile. In the first method, 10 mM phosphate buffer, pH 8.0, was added, and the lecithin was dispersed by two 10-min sonications at 40% power using the standard horn and tip of a Heat Systems Model W-225 sonicator; the solution was cooled in an ice bath during sonication. The resulting translucent solutions were then filtered through 0.22-pm pore size Millipore HAWP filters and centrifuged for 90 min at 100 OOOgand 15 “C in a Beckman LS-65 ultracentrifuge using a TY65 fixed-angle rotor. The supernatant, which containssmall unilamellarvesicles of about 15-nm radius and is free of larger aggregatesIz0was taken for experimentation. To prepare vesicles by extrusion,21 the turbid phosphate suspensions were first subjected to five repetitive freeze-thaw cycles to completely hydrate the phospholipid. The mixtures were then repeatedly extruded (10-15 times) using N2at 600 psi through track-etched Nuclaopore filters. By varying the filter pore size, unilamellar vesicles could be prepared with narrow size distributions around various average radii from 15 to 50 nm.Z1 Dimyristoylphosphatidylcholine
1714 The Journal of Physical Chemistry, Vol. 97, No. 8,1993
(DMPC), dipalmitoylphosphatidylcholine (DPPC), and dihexadecyl phosphate (DHP) vesicles were prepared by sonication without cooling or by extrusion at temperatures greater than 10 deg above the surfactant gel-liquid crystalline phase transitions. Viologens (or other ions) were entrapped within the vesicles by forming the vesicles in viologen-containing buffer and then removing the remaining external ions by passing the suspensions down buffer-equilibrated Bio-Rad AGSOW-Xt? or Chelex 100 cation-exchange columns in their Na+ or K+ form. Prolonged sonication of PC suspensions containing (c7)2v2+gave rise to formation of minor species whose fluorescence properties resembled pyridones.17J2 These impurities appeared to be noninterfering, since direct addition of up to 20 pM dipyridone to viologen-containing liposomes formed by extrusion did not alter the subsequentkinetics of one-electronreduction by S Z O Padded to the bulk aqueous phase. However, viologen-containing egg PC vesicles that were formed by sonication of material that contained residual CHC13 showed after sonication a pronounced lag in the onset of viologen radical formation following S ~ 0 4 ~ addition. Ultraviolet spectroscopic changes indicated that oxidation of the reactant had occurred over this time period. These effects, which were eliminated by rigorous removal of CHCl3, could possibly be attributed to ultrasonic formation of dichlorocarbenes or similar species, which readily add to lipid double bonds to form S2042--reactive dichlorocyclopropanes. The synthetic lipids, DPPC and DMPC, whose alkyl chains contain no unsaturated carbon bonds, did not produce any redox-active contaminants upon sonication in the presence of CHCl3. With the precaution noted above, vesicle suspensions prepared by the sonication or extrusion methods gave equivalent results. Most of the data described herein were taken on liposomes prepared by extrusion. In several experiments,the K+-specificionophore23valinomycin was added to preformed vesicles as a methanolic solution (1-5% v/v). The background leak rates of both entrapped potassium ion, measured with an Orion Model 93-19 potassium ion-specific electrode, and entrapped (C7)2V2+,measured by reduction to (C7)2V+.withexternally added S Z O ~(described ~below), were unaffected by this amount of methanol, indicating that it did not alter the intrinsic impermeability of the bilayer toward ions. Similarly, valinomycin addition did not facilitate leakage of entrapped I4C-radiolabeledsucrose, indicating that its incorporation did not disrupt the membrane. For these experiments, vesicle samples were passed through Sephadex G-50 dextran sieving gels to remove external sucrose,and the scintillationcounts in the recovered vesicle fractions were determined as described below. Ion Adsorption to PC Vesicles. The extent of vesicle binding by various reaction components was determined by equilibrium dialysis using 10 mL Bel-Art Products cells, which have very favorable membrane surface-to-sample volume ratios. The two half-cells were separated by a No. H40299 cellulose membrane possessing a nominal molecularweight cutoff of 6000 amu. Control experiments in which the diffusible ion was added to one compartment and the other compartment contained an equal volume of buffer established that equilibrium was achieved when the concentrations of diffusible ions became equal in the two compartments. These studies also established that equilibration required a minimum time of 10 h under gentle agitation. In some instances, the dialysis membrane also bound weakly the dialkylviologens. The measured binding interactions to PC vesicles were corrected for this nonspecific loss, which in all instances was less than 10% of the total added viologen. In individual binding experiments, viologens or other lipophilic ions of interest were added to either compartment, i.e., containing the PC vesicles or just buffer. The same equilibrium distributions were obtained regardless of the original location of the viologen. Ion concentrations in the two compartments were generally
Kuhn and Hurst determined by optical absorption spectroscopy using published extinction coefficients. The viologen dications are colorless with a strong UV band at -260 nm, but the corresponding radical cations are intensely absorbing blue or purple species, depending upon their states of a g g r e g a t i ~ n . ~ ~At- *low ~ concentrations, where light scattering by the vesicles interfered significantly with measurements in the ultraviolet region, the viologens were reduced to their radical cation forms with S20d2-ionand their concentrations determined from the visible spectra. For these experiments, the lipid concentration was 4 mM and the concentration of viologens was varied from 10 pM to 1 mM. The medium was 0.1 M sodium phosphate, pH 8.0; this high ionic strength minimized osmotic effects that would perturb the intercompartmental viologen distribution. Photochemistry. Photochemical systems consisted of PC vesicles containing entrapped viologens and a photoredox-active chromophore in the bulk medium. Chromophores used in this study included Ru(bpy)3Z+(bpy = 2,2/-bipyridine), the amphiphilic analog Ru( bpy) 2 [4,4'- (C 16CONH)zbpy] 2+, ZnTMPyP+, and (5,10,15,20-tetrakis(4-sulfonatophenyl)porphinato)zinc(II) (ZnTPPS") ions. The electron donor, ethylenediaminetetraacetic acid, was added to the aqueous medium for studies involving net viologen reduction. Two-milliliter samples within an optical cuvette were deoxygenated by bubbling with NZ and immersed in a constant temperature bath. Thesamples were illuminated with filtered light from a 100-Whigh-pressure xenon lamp (Oriel Model C-60-80). The incident light beam was passed through a 10-cm-path length water cell and then Corning 3-75 UV cutoff and CS-470 blue-green filters prior to impinging upon the sample, thus limiting the spectral irradiance range to 400600 nm. By Reinecke salt actinometry,28 the incident light intensity was determined to be 8.7(*1.3) X einstein s-I. The temperature inside the cuvette was monitored in some runs by introducing a copper/constantan thermocouple; by this method, it was determined that the temperature of the illuminated sample did not vary by more than 0.5 OC over the course of a 1-h experiment. The solution within the cuvette was continuously stirred by means of a magnetic stirrer bar and submersiblewaterdriven magnetic stirrer. The course of photochemical reactions was followed by periodically removing the cuvette from the light beam and recording the absorption spectrum of the sample on a Hewlett-Packard 8452A diode array spectrophotometer. Chemical D ~ M ~ c sRates . of leakage of internally localized viologen dications from PC vesicles were determined by adding S 2 0 d 2 - to deoxygenated suspensions of the viologen-containing vesicles at various times following their preparation and measuring the extent of viologen radical formation by adsorption spectrophotometry. Similarly, net transmembrane reduction of entrapped viologens was monitored by recording the time course of viologen radical formation in septa-stoppered optical cellsto which dithionite had been added using a gas-tight hypodermic syringe. Spectral changes were measured using either the diode array spectrophotometer or, where precise temperature control was required, a Perkin-Elmer Lambda 9 spectrophotometer. With the latter instrument, reaction rates were generally monitored at 604 nm, and temperature was maintained constant to fO.l OC using a thermostat4 cell holder/bath assembly. Reactions that were too rapid to measure accurately by these methods, Le., transmembrane reduction in the presence of ionophores or viologen-mediated transmembrane reduction, were measured using a Gibson-type stopped-flow instrument which was specifically designed for handling anaerobic solutions.I7J9 Voltage-time waveformswere recorded on a Nicolet 4094B digital oscilloscope equipped with a 4568 plug-in interfaced to a DEC Pro-350 computer. Data were analyzed using either an a d a p tation30of the program CURFIT by Be~ington,~' based upon an
The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1715
Vectorial Transmembrane Reduction of Viologens algorithm by Marquardt, which fits the experiment traces after conversion to absorbance to a series of first-order processes, or a general simulation program for which measured absorbances were compared to calculated data for any given mechanism consisting of a series of steps with any specified reaction order.30 The programs were run using the RT-11 operating system with a sample size of 794-3968 data points taken from the individual kinetic waveforms. Goodness of fit was evaluated by comparing the random residual of the best-fit calculated curves to the actual data. Tnnswmbmne Viologen Exchange. The exchange rates of (C7)2V2+ and C6MV2+ ions were estimated by preparing PC vesicles containing 14C-radiolabeledviologens and measuring the rate of loss of radioactivity from the particles following addition of nonradioactive viologen to the external medium. For these studies, the vesicles were formed in the presence of the l4C-1abeled viologen and external viologen was removed by ion-exchange chromatography. The concentration of entrapped viologen was determined spectrophotometrically. An equal amount of unlabelcdviologenwas then added to theextemal medium. At various subsequent times, portions of this sample were passed through Bio-Rad Chelex-100 resin, and radioactivity in the vesiclecontaining fraction was measured with a Beckman LS-3 150T scintillation counter using Beckman Ready Solv GP scintillation cocktail. To increase theseparation speed and minimizedilution effects, dry resin was placed in a fritted funnel atop a bell jar. The sample was then pulled through the resin under vacuum from a water aspirator into the scintillation vial. The minimum sampling time by these procedures was about 20 s.
--5
-3
-4
log [ ( C 7 ) 2 V 2 + I b u f f ( M I
Figure 1. Langmuir adsorption isotherm for (C7)2V2+ion binding to PC vesicles. Conditions: 4 mM egg phosphatidylcholine inO.10 M potassium phosphate, pH 7.5, at 22 OC. Squares are data points for individual runs; the solid line is the curve calculated from eq 2 with pmax= 220, K = 2.5 x 103 M-1.
TABLE I: Ion Binding to PC Liposomes' CnMV2+'
(C,)2V2+
n
F
n
F
1 6 8 12 14 16
4 X 10-2 S-I. The capacity for valinomycin to promote transmembrane diffusion of K+ under our experimental conditions was gauged by measuring rates of release from K+-loaded PC vesicles with an ion-specific electrode in the bulk medium. In a typical experiment, about 100 pM K+ total analate concentration was entrapped by forming PC vesicles in 20 mM potassium phosphate, the external K+ was exchanged for Na+ by column chromatography, and reaction was initiated by adding 10 pM valinomycin as a methanolic solution. The external K+ ion concentration rapidly rose from 10 pM to 90 pM, with a rate constant of k H 0.1 s-I estimated from the initial slope of the electrode response. Photochemical Rerctions. No net reaction occurred upon prolonged illumination of PC vesicles containing entrapped (C7)2V2+ ion in the presence of the chromophores R~(bpy)3~+, R~(bpy)~[4,4'-(C&ONH)~bpy]~+, ZnTMPyP+,or ZnTPPS4-. Passive leakratesof the (C7)*V2+ions in bothilluminatedsamples and those kept in the dark were the same as background rates measured in the absence of the dyes. Configurations in which the dyes were either added to suspensions of preformed vesicles or incorporated into both aqueous phases gave equivalent results. These studies are of interest in relation to the phenomenon of photostimulated diffusion, which occurs in DHP vesicles and entails enhanced transmembrane diffusionof entrapped viologens upon illuminationof m~mbrane-bounddyes.~* Theresultsindicate that these effects do not occur in these systems. When EDTA was added to the bulk medium, illumination in all cases gave net formation of the blue (c7)2v+ radical cation. This reaction was studied most thoroughly using the Ru(bpy)32+ ion as photosensitizer. Following a brief induction period, viologen radical was formed at a steady rate until the (C7)2V2+ ion was entirely consumed (Figure 7). The reaction rate increased with temperature; apparent activationparameters based upon maximal rates estimated from plots of (c7)2v+ yields vs illumination time were = 18 kcal/mol and A S = -32 eu. The same reactivity was observed for vesicle suspensions containing R ~ ( b p y ) 3 ~ion + in both inner and outer aqueous phases as when Ru(bpy)32+ was added to just the external medium. The intravesicular location of (C7)2V+followingphotoreductionwas probed by thesequential oxygenation-dithionite reduction techniques described in the
Viologen Binding. Binding of the (C7)2V2+ion to PC vesicles was adequately fitted to a Langmuir-type adsorption isotherm, which gave a reasonable value for the association constant but a maximal binding number p (,, = 190-240) that was only about 10% of the value expected for monolayer coverage of the outer membrane surface. This difference may reflect antimperative effects originating in a developing repulsive surface potential, which increases as the number of bound amphiphilic cations increases. This effect can be treated approximately using a Stern adsorption model. However, distinguishing quantitatively between these models requires precise data taken over a wide range of coverage;40because our data do not allow this distinction to be made, we have presented only the simpler Langmuir model. Adsorption appears to be driven by hydrophobic forces arising from interaction of the viologen alkyl chains with the hydrocarbon layer of the vesicle membrane. Under comparable medium conditions, the extent of binding increasedwith increasingnumber of carbon atoms for series of viologens containing both one and two alkyl chains of varying length (Table I). Also, for a given carbon number (n), the binding of (C,)2V2+ ions was more extensive than for the corresponding C,MV2+ ions. In contrast, the electrostatic forces between the viologen dications and the PC membrane appear to be weakly repulsive. Binding was not detected for methylviologen or other aqueous polyvalent cations, but the ZnTPPS4-anion did bind appreciably, even at micromolar concentrationlevels (Table I). Thisapparent differencein binding affinities between large cations and anions was surprising because the surfactant headgroups are thought to orient at the aqueoushydrocarbon interface with the choline segments normal to the interface or even projecting inward somewhat toward the hydrocarbon p h a ~ e . ~ Thus, ' - ~ ~ although formally electroneutral, the zwitterionic headgroups may present to the aqueous phase an asymmetric interface composed of an outer negatively charged and an inner positively charged layer. However, the choline segment, being tethered to a phospholipid 'anchor", might more easily respond to an approaching negative charge and reorient toward the aqueous phase. Mechanisms of Transmembrane Oxidation-Reduction. a. Without Added Membrane-Permeable Ions. When lipophilic ions or ion carriers were absent, transmembrane reduction of entrapped (c7)2v2+by reductants located in the bulk solvent occurred with retention of the (c7)2v+ product ion within the vesicle. One might be tempted to conclude from this observation that direct transfer had occurred across the bilayer membrane. However, the 14C-radioisotope-exchange studies clearly demonstrated that (C7)2V2+ is membrane permeable. Consequently, the initially entrapped viologen musr redistribute across the bilayer, reaching equilibrium when the concentration gradient is
Vectorial Transmembrane Reduction of Viologens balanced by the developingtransmembrane potential (A$). This potential, which arises as a consequenceof uncompensated charge movement, is given by
where F is Faraday's constant, n is the ionic charge, and the subscripts i and o refer to equilibrium concentrations of the ions in the inner and outer aqueous phases, respecti~ely.~~ Typically, equilibrium requires outward diffusion of only a few percent of the entrapped ion.' For example, addition of 100 pM MV+ ion to a suspension of dihexadecylphosphate (DHP) vesicles resulted in -8% uptake of the radical cation, giving a transmembrane diffusion potential of A$ N 4 0 mV.8 In the present case, the transmembrane potential opposed net diffusion of (C7)2V2+ions down their concentration gradient, so that the bulkof the viologen was retained within thevesicles. Thevery slow leakrates measured under the prevalent medium conditions confirm that the permeabilities of all other ions were much less than for (C7)2V2+; leakage was undoubtedly caused by membrane-depolarizing electrolyte diffusion. Given these microphase properties, a plausible alternative mechanism is that S2O42- ion reduced only the externally localized subpopulation of (c7)2v2+ions, which were then taken up by the vesicles in response to the transmembrane potential, which is oriented negatively inward (Figure 5a). The net reaction is the same as direct transmembrane reduction of the viologen but avoids the conceptually unappealing proposal that electron transfer can somehow occur over 3 W O A, a distance approximating the membrane bilayer width.' In the diffusional mechanism, uptake of the (C7)2V+ion causes partial collapse of A$, allowing further outward diffusionof entrapped (c7)2v2+ion, which alsoundergoes reduction. The entire process is electrogenic because it involves outward diffusion of a dication and inward diffusion of a monocation. Therefore, the net reaction will proceed only until the transmembrane potential is balanced by the thermodynamic driving force of the chemical reaction.23 Further reduction at this point must await transmembranediffusion of other ions which reduce the electrical potential; the rate correspondingly becomes limited by theseionfluxes(Figure5a). In thismode1,it isassumed that the (C7)2V+ radical cation is membrane permeable. The high permeability of the (C7)2V2+ion precluded testing this point by I4C-isotope exchange methods. However, transmembrane diffusion of viologen radical cations has been found to exceed that of the corresponding dications in other viologen-containing vesicle systems.8.44.45 Several other pieces of evidence support our assertion that measured viologen rates were limited by electrolyte movement in these reactions. First, the approximate zero-order character of the kinetic waveforms (Figure 2) and insensitivity of rates to the identity and concentration of reductants (Figure 3) indicate that the rate-limiting step was not electron transfer. Second, the dramatic increase in rates observed upon deliberate introduction of leaks in the form of lipophilic ions or ionophores indicates that the reaction was limited by a transmembrane potential. Third, the appearance of anion-specific rate enhancement at high salt concentrations suggests that, at least under these conditions, transmembrane diffusion of the electrolyte anion had become rate limiting. In this regard, the relative rate-enhancing effects of the anions followed approximately the lyotropic series, which is a measure of their relative hydration energies.46 If bilayer penetration required desolvation, ion permeabilities would vary inversely with hydration energies, as observed. This order also corresponds to the relative binding affinities of the anions for the surfactant choline headgroups, as measured by NMR spectroscopy.43947Since ion fluxes increaseproportionately with interfacial ion concentrations, the relative rates should also follow the same order as the ion-binding constants. Both effects could contribute to the observed specificities.
The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1719 In the kinetic models presented, both passive diffusion of entrapped (C7)2V2+ion from the PC vesicle and transmembrane reduction were limited by charge-compensating ion diffusion, yet the transmembrane redox reactions were much faster than viologen leakage. This difference can be rationalized in terms of the much greater transmembrane potential associated with the chemical reaction. An estimate of this potential (AV) can be made from the kinetic waveforms. Because both (C7)2V2+ and (C7)2V+ ions are membrane permeable, A V will be rapidly established following addition of reductant to the external medium. The extent of (C7)2V2+ reduction required to generate this potential can be estimated by extrapolating to zero time the zeroorder kinetic trace that monitors the subsequent ion diffusionlimited phase of the reaction. For example, in Figure 2, this value is about 10%of the total (c7)2v2+ion present, as determined by the ordinate intercept. From this value, estimates of the (c7)2v2+/+ reduction potential based upon thermodynamic midpoint potentials measured for CloMV2+/+in PC vesiclecontaining media,]' the S03-/S2042- standard and assuming -90% purity of the reductant, AE was calculated from the Nernst equation to be greater than 290 mV under our experimental conditions. Since LLT A$' and simple diffusion potentials are typically A$ I60 mV, it is apparent that the transmembrane potential undergoes substantial increase upon addition of the reductant (Le., AV >> A$). Diffusion of aqueous ions across bilayer membranes is currently described in terms of two alternative theoretical models.49 In the 'hole-fluctuation" theory, transversediffusionis viewed as occurring through aqueous pores which form transiently as the membrane ruptures and reseals. In this model, the work required to form the hole depends upon the square of the applied voltage. Consequently, the permeability coefficient (P) of the ion is given by the form P = PO exp(a(A$)2/kZ'), where POis the intrinsic permeability. In continuum theories, the membrane is viewed as an energetic barrier which the ion must surmount; assuming a symmetric barrier, P = PO exp(aA$/ZkZ'). Thus, in both theories, ion diffusion rates are exponentially dependent upon, and therefore extremely sensitive to, the magnitude of the prevailing transmembrane potential. The independence of (c7)2v2+reduction rates upon reductant concentrations and relatively low activation energies for these reactions suggest that continuum theories are more appropriate. Specifically, if pore formation allowed transmembrane diffusion of &Os2- ion, its flux should have been dependent upon the magnitude of its concentration gradient, but the reaction rate remained invariant upon changing the external Sz042- ion concentration over a 10-fold range. Also, activation enthalpies for pore formation in lecithin bilayers are projected to be A H N 30 kcal/mol,50 rather than the measured value of AFP N 20 kcal/mol for transmembrane oxidation-reduction. One aspect of these reactions that is not immediately interpretable in terms of the kinetic model is the increase in zero-order rates with increasing concentrations of entrapped (c7)2v2+ions (Figure 3). One possibility is that increasing the mole fraction of bound viologen disrupts the hydrocarbon side-chain packing, promoting ion leakage. Alternatively, anion diffusion might involve ion pairing with theviologens, although to account for the zero-order character over the entire reaction course, the dications and cation radicals would have to be equally effective as anion carriers. b. Including Membrane-Permeable Ions. Adding lipophilic ions or ionophores to the reaction medium profoundly affected the kinetics of (C7)2V2+reduction including (to varying degrees depending upon the addend identity) enhancing reaction rates, introducing autocatalytic character (Figure 4), and promoting release of the (C7)2V+radical cation from the vesicle interior. All of these effects can be understood in terms of the ability of the additional component to collapse the rate-inhibiting transmem-
1720 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993
brane potential. The largest effects were seen with K+valinomycin,which was the system most extensively studied. The kinetic waveforms could be reproduced by the mechanism given in Figure 5b, the key feature of which is countertransport of K+ for (c7)2v+. Progressive accumulation of the radical cation at the external interface ensues, allowing expressing of a (c7)2v+mediated transmembrane reduction step (k4in Figure 5b). The relative contribution of this step to the overall reaction increases with time, accounting for the autocatalytic character. The rate constant for S 2 0 4 2 - reduction of (C7)2Vz+determined by stopped-flow spectrophotometry was used for calculations simulating the kinetic traces, and it was also assumed that valinomycin-facilitatedtransmembrane diffusion of K+(k3) was more rapid than diffusion of (C,)2Vz+and (c7)2v+ ions (kland k ~respectively). , Adequate fits were obtained using a bimolecular rate constant for MV+-mediated transmembrane reduction of (C7)2V2+ (k4) that was very nearly the same as the directly measured constant. The calculated curves were very insensitive to themagnitudeof kz,which was taken tobethedirectly measured value for (C7)2V2+reduction by S02-ion in PC vesicle-containing media. They were fairly sensitive to the value chosen for k4,with perceptible changes in the maximal slope accompanying 2-fold changes in k4. Both the curve shape and maximal slope were extremely sensitive to the values taken for the slow kl and k5 steps. As anticipated, apparent rate constants for (C7)zV+ transmembrane diffusion were about 102-foldgreater than for the (c7)2v2+dication (kl)(Table 11). These constants increased with the amount of added valinomycin, and kl was considerably s-l) measured under less than the lower limit ( k l > 4 X conditions where the membrane was not polarized. Both these results suggest that A+‘ was not completely collapsed by K+valinomycin during the courseof the oxidation-reduction reaction. However, control experiments established that as little as onetenth of the amount of valinomycin added for the redox studies caused rapid release of K+ from the vesicles. The basis for this discrepancy is not known. The progressively slower reduction rataobserved forthelonger-chainanalogs (C8)2Vz+and(Cl0)2V2+ are consistent with viologen diffusion being at least partially rate limiting under these conditions. The sequence of steps given in Figure Sb represents a minimal reaction scheme and is probably not unique. Hammarstr6m and co-workers have presented kinetic evidence that a major pathway for the k4 step in PC vesicles containing bound C16MV2+ion is rate-limitingClaMV+disproportionation,forming CI6MVowhich traverses the bilayer as an electroneutral spe~ies.3~Under conditions approximating those of our study, they noted that this reaction was more complex and could not be fitted to a simple rate law. Our stopped-flow studies were made with equimolar concentrations of internal (c7)2vz+and external (c7)2v+ ions, conditions which do not allow a distinction to be made between (C7)2V0as the redox carrier and other pathways involving (C7)2V+ However, the (c7)2v+1° standard reduction potential in PC vesicles is approximately = -0.74 V (SCE),” very nearly equal to the S03-/S2042-potential. Thus, under the conditions of our experiments, (c7)2v0can accumulate (cf. Figure 2”). On the other hand, the high intrinsic permeability of the (C7)2V+ ion, as implied by the rapid transmembrane diffusion of the (C7)2V2+ion (Figure 6), does not allow alternative pathways involving redox mediation by the radical cation to be excluded from consideration. Comprrirrolls with Other Vesicular Assemblies. On the basis of their kinetic analysis of one reaction, Hammarstrijm and coworkers have suggested that the neutral diradical, Le., VO, is the charge carrier in all viologen-mediated transmembrane redox reactions. Otherwise, “it is...easy to end up in a patchwork of different rate-limiting steps for different circumstances where sometimesdiffusion of unidentified ions and sometimes diffusion or electron transfer between viologens determines the rate of
Kuhn and Hurst transmembrane red~ction’.~’In the present example, it is clear that the transmembrane redox step is not rate limiting in the absence of added lipophilic ions. In general, the pathways available will be determined by the structural characteristics of the membrane and intrinsic permeabilities of the bound dopant ions, both of which canvary widely. For example, transmembrane reduction of analogous ions entrapped within DHP vesicles does not occur7 because the CnMV2+ ions cannot diffuse across this bilayer membrane.8 As discussed in the Introduction, diffusion of the corresponding C,MV+ radical cations is dependent upon the length of the N-alkyl chain? so that different pathways are expressed for the short-chain and long-chain congener^.^ Similar structural dependencies appear to exist in PC vesicles. Hammerstr6m and co-workers report that transmembrane reduction of occluded C16MVZ+ by S Z O ~ion ~ -does not occur unless the viologen is also present at the outer implying that the CI6MVZ+dication is not membrane permeable. In contrast, we show in this work that (c7)2vz+ion rapidly penetrates the bilayer. Finally, standard reduction potentials are 100-200 mV more negative for viologens bound to DHP vesicles than PC vesicles.Il The extent of neutral diradical formation is therefore for less when theviologensarebound toDHP membranes. Aquantitative assessment based upon these potentials and transmembrane diffusion constants8indicated that pathways involving C,MVO or C,MV+ diffusion across DHP bilayer membrane were equally plau~ible.~ Another case in point is the apparent transmembrane reduction of entrapped (C7)2VZ+ion by photosensitizers located in the bulk aqueous phase demonstrated in this work. The spatial orientation of these assemblies is similar to those reported previously where vectorial transmembrane reduction involved the Ru(bpy) [4,4’c&ONH)~bpy]~+ ion bound tobothinterfacesof PCvesicles.I3J5 In those studies, photoexcitation of the ruthenium coordination complex gave net one-electron reduction of (c7)2vz+ion located in the bulk aqueous phase by EDTA located in the vesicleinterior. Rates of (C,)zV+ radical cation formation followed reaction profiles very similar to those shown in Figure 7; the mechanism proposed involved oxidative quenching of the photoexcited sensitizer by (c7)2v2+ ion located on the same side of the membrane, followed sequentially by transmembrane electron transfer from a ruthenium(I1) ion located at theopposite interface and then reduction of the newly formed ruthenium(II1) ion by EDTA on the same side of the membrane. The transmembrane charge-transferstep proposed thereforeinvolved electron exchange between ruthenium(I1) and ruthenium(II1) ions bound at the oppositevesicle interfaces. However,our observationsthat similar reactions occur when the photosensitizer is localized to only one interface or is not membrane bound at all, but confined to the aqueous phase that (nominally) does not contain the viologen (Figure 7), suggest that an alternative mechanism must be operative. Specifically, the results are interpretable in terms of a mechanism analogous to the one given in Figure 5a, in which only the small subpopulation of (c7)2v2+ residing in the same aqueous phase as the sensitizer undergoes photoreduction and are subsequently taken up by the vesicles in response to the electrical polarization of the membrane. The key observation here is that the (C7)2V2+ion is permeable to the PC membrane; therefore, although apparently compartmented, it distributes across the bilayer according to the thermodynamic constraints of the system. In summary, transmembrane redox processes are diverse, and reducing their molecular mechanismswill ultimately require full dynamical characterization of the components of each organized assembly. Acknowledgment. Funding for this research was provided by the Office of Basic Energy Sciences, U.S. Department of Energy, under Grant DE-FG 87ER 13664.
Vectorial Transmembrane Reduction of Viologens
References rad Notes (1) Hunt. J. K. Kineiics and Caralysis in Microhererogeneous Sysrems; Surfactant ScienceSeries Vol. 38;Marcel Dekker: New York, 1991;pp 183226. (2) Lymar, S.V.; Parmon, V. N.; Zamaraev, K. I. Phoroinduced Elecrron Transfer III; Topics in Current Chemistry Vol. 159;Springer-Verlag: Berlin, 1991;pp 1-66. (3) Robinson, J. N.; Cole-Hamilton, D. J. Chem. Soc. Rev. 1991,20,49. (4) Runquist, J. A,; Loach, P. A. Biochim. Biophys. Acta 1981,637,231, (S) Pattenon, B. C.; Thompson, D. H.; Hurst, J. K. J. Am. Chem. SOC. 1988, 110, 3656. (6) Patterson. B. C.; Hurst. J. K. J . Chem. Soc., Chem. Commun. 1990, I 137. . (7) Patterson, B. C.; Hurst, J. K. J. Phys. Chem., in press. ( 8 ) Lymar, S.V.; Hunt, J. K. J . Am. Chem. Soc. 1992. 114,9498. (9) Humphry-Baker, R.; Thompson, D. H.; Lei, Y.; Hope, M. J.; Hurst, J. K. Lungmuir 1991, 7,2592. (IO) Colaneri. M.J.; Kevan, L.; Thompson, D. H.; Hurst, J. K. J. Phys. Chem. 1987. 91, 4072. (11) Lei, Y.; Hurst, J. K. J . Phys. Chem. 1991, 95,7918. (12) Thompson. D.H. P.; Barrette, W.C., Jr.;Hurst, J. K. J. Am. Chem. Soc. 1987. 109. 2003. (13) Ford, W. E.;Otvos, J. W.; Calvin, M. Proc. Natl. Acad. Sci. U.S.A. 1979. -..., 76. -, 3590. - .-. (14) Laane, C.;Ford, W. E.;Otvos, J. W.; Calvin, M. Proc. Nail. Acad. Sei. U.S.A. 1981. 78. 2017. (15) Mettee. H. D.;Ford, W. E.;Sakai, T.; Calvin, M. Phorochem. Phorobiol. 1984, 39, 679. (16) Singleton, W. S.;Gray, M. S.;Brown, M. L.; White, J. L. J. Am. Oil Chem. Soc. 1965,42,53. (17) Ford, W. E.;Calvin, M. Chem. Phys. Len. 1980, 76, 105. (18) Sprintschnik, G.; Sprintschnik, H. W.; Kirsch, P. K.; Whitten, D. G. J . Am. Chem. Soc. 1977,99,4947. (19) Laukanis. A.; Lay, P. A.; Mau, A. W.-H.; Sargeson. A. M.; Sasse, W. H. F. Ausi. J. Chem. 1986,39, 1053. (20) Barenholz. Y.; Gibbes, D.; Litman, 8. J.; Goll, J.; Thompson, T. E.; Carlson, F. D.Biochemisrry 1977, 16, 2806. (21) Mayer, L. D.;Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acra 1986,858,161. (22) Mau, A. W.-H.; Overbeck, J. M.; M e r , J. W.; Sasse, W. H. F.J . Chem. Soc., Faraday Trans. 2 1986,82,869.
The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1721 (23)See, e.g.: Nicholls, D. G. Bioenergetics; Academic Press: New York, 1982. Harold, F. M. The Vital Force: A Siudy in Biocnergeiics; W. H. Freeman: New York, 1986. (24)Watanabe, T.; Honda, K. J . Phys. Chem. 1982,86,2617. (25)Poizat, 0.; Sourisseau, C.; Giannotti, C. Chem. Phys. h i t . 1985, 122, 129. (26) Bockman, T.M.; Kochi, J. K. J. Org. Chem. 1990,55, 4127. (27) Geuder, W.; Hiinig, S.;Suchy, A. Tetrahedron 1986, 42, 1665. (28) Wegner, E.E.;Adamson, A. W. J. Am. Chem. Soc. 1966,84394. (29)Norton, K. A.; Hurst, J. K. J . Am. Chem. SOC.1978,100, 7237. (30)Patterson, B. C. Ph.D. Dissertation, Oregon Graduate Institute of Science & Technology, 1990. (3 1) Bevington,P. R. Data Reduction and Error Analysisfor the Physical Sciences; McGraw-Hill: New York, 1969. (32) Huang, C. Biochemisrry 1969,8,344. (33) Tsukuhara, K.; Wilkins, R. G. J. Am. Chem.Soc. 1985,107,2639. (34) Zamaraev, K. I.; Lymar, S.V.; Khramov, M. I.; Parmon, V. N. Pure Appl. Chem. 1988,60,1039. (35) Fendler, J. H. Membrane Mimeric Chemistry; Wiley-Interscience: New York, 1982. (36) See, e.g.: Clement, N. R.; Gould, J. M. Biochemisrry 1981,20,I544 and references therein. (37) Hammarstram, L.; Almgren, M.; Norrby, T. J . Phys. Chem. 1992, 96,5017. (38) Lee, L. Y.-C.; Hurst, J. K.; Politi, M.; Kurihara, K.; Fendler, J. H. J . Am. Chem. SOC.1983, 105,370. (39)Kalyanasundaram, K.; GrHtzel, M. Helu. Chim. Acra 1980,63,478. (40) McLaughlin, S.Curr. Top. Membr. Transp. 1977,9, 71. (41) Dill, K. A.; Stigter, D. Biochemisrry 1988,27, 3446. (42) Wiener, M. C.; White, S.H. Biophys. J. 1992,61,434. (43) Seelig, J.; Macdonald, P. M.; Scherer, P. G. Biochemistry 1987,26, 7535. (44) Tabushi, I.; Kugimiya, S.J . Am. Chem. Soc. 1985,107, 1858. (45) Tabushi, I.; Kugimiya, S. Tetrahedron Lett. 1984,25, 3723. (46) See, e.&: Jencks, W. P. Caralysis in Chemisrry and Enzymology; McGraw-Hill: New York, 1969. (47) Macdonald, P. M.; Seelig, J. Biochemistry 1988,27,6769. (48) Mayhew, S.G. Eur. J. Biochem. 1978,85, 535. (49) Bender, C. J. Chem. SOC.Reo. 1988, 17,317. (50) Hamilton, R.T.; Kaler, E. W. J . Phys. Chem. 1990,94,2560.
