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Electric field modulation of charge transfer processes in reaction centers of photosynthetic bacteria
Volume 116, number 5
17 May 1985
CfiJZMICAL PHYSICS LETTERS
ELECTRIC_ FIELD MODULATION OF CHARGE TRANSFER PROCESSES IN REACl’ION CENTERS OF~PHOTOSYNTHE TIC BACTERIA 2-D:
POKWIC,
G-J. KOVACS,
P.S. VINCET-I-
Xerox Research Cenrre of Cana&, 2660 Speakman Drive, Mississauga, Ontario. Canada WK 2LI
and P-L. DU’ITON Depwtment
of Bipchemistry and Biophysics.
Univgrsity of Pennsylvania, Philadelphia, PA 19104,
UsA
Received 30 August 1984; in final form 25 February 1985
External electric field modulation of charge separation 2nd recombinattonprocesses has been achievedin native photosynthetic reaction centers. Langmuir-Blodgett monolayer films of reaction centers isolated from the photosyntheticbacterium Rhodopseudomonas sphaero~des, have been incorporated into electroded sandwichcell structures allowingfield modulation of
both photoinduced bleaching and time-resolved absorption recovery.
I. Introduction After the isolation of the reaction centers (RCs) of photosynthetic bacteria fllj much effort has been invested [2] in elucidating their structural and kinetic properties. The major driving foice behind these efforts is the desire for detailed understanding of the primary charge separation process, which proceeds with a remarkable quantum efficiency approaching unity. The distances involved&i the ,various charge transfer steps have been estimated byapplying a va$ety of techniques to the n-dtivechromatophori: membranes of the bacteria and to recon&i&ted rriembrane inultilayers of the RCs (see ref. [3] anil citations therein). Efforts to obtain distancesip the profile of the niembr+e have come also from iight$tduced electrical transient measurements on R&J reconstituted into biltiyer membranes and films [4~7]%rid ofi closi-packed monolayers of oriented RCs obtained-byUngmuii_Blodgett (LB) film techniques [8]. It is desirable to be able to apply electric fields to these systems in orde; fopetirb ‘the charge transfer reactions.-Measurements of the-field-depetidence-of these processes are of -great importance for testirig var0 009-2614/85/$03.30 @ Elsevier.Science Publishers B.V. (North-Hol&nd Physi.cs Publishing Division)
ious rmdltiphonon tunnelling theories of electron transfeih photosynthesis [9-l I], and for establishing thereby the fundamental nature of the primary photosynthetic mechanism. What ihe application of the exterilal electric field basically does is to change the relative energy levels between different sites in the charge transfer chain. This approach is completientary to the use of chemical substitution [12] to achieve the same goal. The electric field experiments, however, offer the unique possibility of studying the influence of energy level shiftswithout introducing any structural changes. RC-reconstituted bilayer membranes have proven useful to reveal field modulated elctron transfer between cytochrome c and the RC bacteriochlorophyll dimer [X3], (BC&, a+ also between the RC quinone, Q, and (BChlj2, in RCs that have had the native ubiquinone replaced by anthraquinone [14]. In both these cases the free energy drop and distance were suitable to yield effec’ts with inodest fieids. LB films are a natural vehicle which to study the effects of large exterrial electric fields on the charge transfer processes iu isolated RCs. It has already been .established [8] that the RCs -m these LB films are orierited in a f&on &nilar to that encountered in the 405
Volume 116, number5
17 May
CHEMICAL PHYSICS LETTERS
1985
native membrane except that they are a mixture of two vectorially opposing “up” and “down” states_ Concomitant measurements of electrical and optical activities were possible on these ultrathin films, and the latter showed that a significant fraction (up to 40%) of the RCs retained their photochemical activity_ In the present comrnunation, we give the results of our preliminary experiments on external field modulation of the charge transfer steps in RCs of Rhodopseudomonas sphaeroides which have been formed into LB f&ns. We show that by two quite similar experiments one can observe significant modulation of both the charge separation and recombination processes_ The effects of electric field on the total photoinduced bleaching and on the rate constants of absorption recovery are determined.
that is stable well into the millisecond time scale_ The loss and subsequent recovery of absorption, at 860 nn describes the transfer of an electron from the excited singlet, (BChl);, through Bph to QA and (if present) QI The state, (BChl): Qz has a lifetime in the 100 ms range, while the (BChljl Q;i- state lasts into the second of time. Measurements on finished cells were perforrnei in a reflection geometry, with the probe beam enterin the sample from the quartz slide side and being refl ectc from the top Al electrode, thereby making a double pass through the RC film-The maximum signal obtainer by a saturating bleaching pulse corresponded to a reflectivity change (at an angle of incidence of 45”) at 860 nm of 0_2%, indicating that ~40% of the RCs were photochemically active_ The influence of the elel tric field on the charge transfer kinetics was studied using the two experiments described below.
2_ Experimental
ing
Inthefirsttypeofexperiment,aftersample-bleach-
The RCs were isolated from the photosynthetic bacterium,Rhodopseudornonas sphaeroides (R26). as described previously 1lSJ. The resulting RCs were a mixture containing predominantly two quinones (QA uartz slides with a sputtered IT0 (iridium andQE)[lSl_Q tin oxide) transparent conductive layer (a 150 run) and a sputtered SiOa blocking layer (=SOO run) were used as substrates for LB film deposition. LB monolayers were deposited using the procedure described ln ref. [S] in a circular Teflon “Fromherz” trough [16] at a constant surface pressure of ~25 mN/m. To allow high electric fields to be applied without cell breakdown, a 1.75 pm polymer blocking layer (Goodyear Pliolite OMS, solvent Exxon Isopar G) was dipcoated [ 171 on top of the RC monolayer. An evaporated opaque Al layer formed the top electrode of the sandwich. Photochemical activity of the RC films was checked at each step of the fabrication process by measuring the absorption at 860 run, thereby monitoring the (BChl)2 photooxidation and reduction_ This was done using a low-intensity probe beam, after bleaching with a 5 ms pulse from the 514.5 M-I line of Ar+ ion laser. (A 750 nm short-wavelength cut-off filter placed in front of the light detector prevented its saturation due to the high intensity of the bleaching light pulse.) Photochemical activity is operationally defined as the ability of the RCs to support flash activated (BChl)Z oxidation 406
at zero field. the electric field was switched on wit in a few miliseconds and the time-resolved absorption recovery was recorded_ This type of measurement gives information about field-modulated recombination processes of already separated electron-hole pairs. The total recording time was 0.5 s and data were take typically by averaging 40 measurements cycles. We shal refer to this type of experiment as absorption recover In the second type of experiment, to be referred to as “quenching of bleaching”, a flash exposure (-5 ps in duration) wx applied to the sample by a Genera Radio xenon flashlamp, equipped with a 600 nm long wavelength cut-off filter. A 5 ms voltage pulse was used to bias the sample during bleaching and was switched off immediately after termination of the light pulse. The measured quantity was AI, which represents the maximum bleaching_ A1, the maximum change of the probe beam intensity, was recorded immediatelyafter termination of the light pulse and turning off of the sample bias. This experiment gives information about the field modulation of the primary charge separafiol; steps. The flash lamp intensity was sufficiently Iow that bleaching was proportional to the total flash energy, and AI was normalized by this energy. In this case, we can define the quenching of bleaching, Qb, due to the electric fieldE, by Q&9
= [UO)
- N(E)1
/AI(o)
(1:
In order to avoid possible charge accumulation in
Volume 116, number 5
CHEMICAL PHYSICS LETTERS
the samples, the usual sequence in time-averaging measurements was as follows: positive field, zero field, negative field, zero field, etc. No significant difference was observed for positive and negative polarities, which confirms the equal up and down populations of the RCs, and the data shown for both experiments are an average over both polarities. The magnitude of the electric field applied to the sample is deduced from the cell thickness determined from a cell capitance measurement, with a dielictric constant of 3 assumed for the composite sample between the conductive eiectrodes.
3. Results and discussion Before discussing our own results, it is pertinent to note that a preliminary report [14] has discussed the effect of transmembrane potentials on charge recombination in RCs incorporated into planar bilayers. No
17 May 1985
effect was found with applied voltages up to 125 mV for RCs containing the native ubiquinone, but an e-fold change in recombination rate was observed for an applied potential of 175 mV for RCs reconstituted with anthraquinone. The applied 125 mV corresponds to a fi.eld of at most 20 V/pm (if one assumes the entire voltage drop is across the 6 nm span of the RCs). We have applied fields an order of magnitude larger, and according to our results, it is not surprising that, in ref. [ 141, no effect was observed in the natural photosynthetic system. Another preliminary report [ 181 has discussed Stark effect absorption changes in the RCs, whereby the electric field alone changes the amplitude of the various absorption bands. These experiments were done by applying electric fields to RCs embedded in polyvinyl alcohol films. In principle this Stark effect could change the amplitude of the flash activated bleaching, and therefore contribute to our measurements of Qb.
0 -1
-2
T ,o P ; h U Z X -
_r
-3 0 -_1 -2 -3 0 -1
-2 -3
Fig. 1. Absorption recovery of RCs as a function of time for various values of the applied electric field. The zero-field reference is given in each case. No detectable shift occurs for fields less than 60 V/urn.
407
Volume 116, number5
However the magnitudes of these effects are very small in comparison with our measured Qb and can be neglected . With regard to our own results, we will first discuss field induced changes in the kinetics of the charge recombination between (33Chl)‘-t and QT in the RCs. Fig. f shows the logarithm o iLreflectivity variarion versus time for different fiefds applied to the sample. The zero-field data can be well fitted with two exponentials having time constants of 70 and 360 ms. The first time constant is in good agreement with the values(60-100 ms) normally observed in absorption recovery experiments on RCs containing a single quinone (i.e. QA)_ The second, however, is about three times shorter than usually observed (=I s) in RCs with two quinones (i.e. QA and QB) in which the charge recombination occurs from Qg . We do not understand the reason for this discrepancy, but it is presumably due to subtle structural changes or distortions, which occur on formation of the RCs into a densely packed monoplayer, It is obvious that application of the electric field dramatically changes the recombination kinetics in photoactivated RCs. We would expect that the recombination process can either be hastened or hindered, depending on the RC orientation with respect to the external field. The two time constants cbserved at zero field should therefore split into four, and in principle it should be possible to deconvolute them from the decay curves. Fig. 1 shows that the curves genera&j shift increasingly upward showing decreasing slope at longer times when compared with the zero-field data. This can be understood in the framework of rate constant splitting:. At longer times, the time evolution of the decay will be governed by the slow rate constants, leading to a decreased rate as observed. Attempts to deduce the field dependence of the decay rate constants by assuming arbitrary rate constant splitting were not successful; the number of fitting parameters, four independent rate constants in this case, was simply too large. While excellent fits to the experimental data could be obtaining using this procedure, ;t did not yield meaningful variation of the rate constants with electric field_ In order to limit the number of fitting parameters and to impart order to their extracted values, we have assumed that, in the first approximation, the field-induced splitting of rate constants is symmetric. Mathematically this can be expressed in the following fashion. If the time vari408
17 May 1985
CHEMICALPHYSICSLETTERS ation of the bleaching at zero field is A.$f, E = 0) = 234,
exp(-kit),
where the Ai are the light intensity amplitudes associated with the rate constants, kj, then the field induced symetric splitting (by Lyci) of rate constants will lead to the following time dependence A.I(t,E)
= C
+ exp[-(ki
iAi{exp[-(kf -
ak,)t]).
+ Aki)t] (3)
One might suspect that the inability to extract ameaningful progression of rate constants from the experimental data without the assumption of symmetric splitting, may be due to the fact that RCs are not up and down oriented, but rather possess a dispersion in their orientation. However, the orientation of LB f%-~-~s of RCs has been confirmed by linear dichroism measurements [8]. In any event an assumption of dispersed orientation would introduce additional fitting parameters, further compounding the problem of extraction of rate constants; the immediate problem is in the prolificacy rather than the paucity of fitting parameters. If the RCs do assume a dispersion in orientation, then the rate constants determined (under the assumption of symmetric splitting) may be interpreted as averages of the resulting distributions of rate constants. In fig_ 2 the field dependence of the rate constants, deduced by fitting eq. (3) to the experimental data, is given. The amplitudes, A, deduced by fitting eq. (2) to the zero-field data are retained in fitting eq. (3)_ While this procedure cannot be regarded as an exact deconvolution, it can at least give an idea about the magnitude of the field-induced changes. For the range of fields applied Cup to 200 V/m), the rate constants appear to change by about an order of magnitude. These changes are caused by shifts in the energy levels of QA and QE relative to (SChl]~, on application of the uniform electric field. The shift is given by --de& where d is the dipole moment of the electron-hole pair and E is the electric field, If it is assumed that the RC cylindrical axis lies along the electric field direction, a projected separation distance of +1.5 nm for the pair is reasonable for purposes of estimation. From this separation distance one estimates a substantial change in energy separation of 20.3 V for the range of fields
Volume 116, number 5
17 May 1985
CHEMICAL PHYSICS LETPERS
-a
.
oooo
k,-Ak, .
00. 000%~ k2+Ak2
0
0
ka-Ak2 00
t 1.0
0,
E2 (V/pm)
‘0
Ooo
0
L
o-4o>
F%. 3. Quenching of bleaching the applied electric field.
2
as a function
of the square of
300 E (V/pm)
Fig_2. Field dependence of recombination rate constants assuming symmetricrate constant splitting (see eq. (3% deduced
applied. This change will in general be somewhat different for QA and QB and will depend on their precise vectorial separation from (BChl)$. It would appear that an alternative approach to simplifying the-analysis would be to use singlequinone RCs with single decay kinetics. However, while singlequinone RCs have been prepared in solution, an equilibrium population of zero-, one- and two-quinone RCs can developed with time, at a rate depending on the storage conditions. This process appears to be accelerated in RCs cast into a solid-state film, and the use of singlequinone RCs did little, if anything, to improve the situation. Films prepared from singlequinone RCs showed almost identical decay kinetics to those pr& pared from twoquinone RCs. Alternatively we attempted to block QA to QB electron transfer by treating the RCs with ortho-phenanthroline [ 191. Again the resulting films showed basically identical decay kinetics to those of films not treated by the electron transfer inhibitor, irrespective of whether the treatment occurred before or after film deposition, or both. It therefore appears that LB films of RCs are deposited into a unique configuration which is not easy to change.
In fig. 3 the dependence of electric-field-induced quenching of bleaching as a function of the square of the applied electric field is given. This data representation gives a straight line, which is easily explained in terms of equal populations of up and down oriented RCs in the LB films. We now introduce t!re yield, ?iCT, of the chargeseparated state induced by bleaching ilfumination. The quantity AI(E), averaged over positive and negative fields, is proportional to $ [r7CT(E) + qc~( - E)]. Consequently for the quenchmg of bkaching, averaged over positive and negative field directions, we can writer Q,(E)
= 1 - 4 fr’Ic#)
+ “?c-+-E)]
/r&o)-
(4)
Quenching of bleaching is therefore a measure of the field-induced change in the yield of the charge separated state. This change is caused by the change in energy level separation between the intermediate bacteriopheophytin state, (BPh)-, relative to (BChl); and to Q-. From relative distance measurements [7] the changes are expected to be somewhat smaller, but of the same order,as the changes estimated above between the levels, Q- and (BChl)$ .
4. Conclusions The application of the electric field clearly slows down the net recombination reaction (see fig_ 2) This 409
Volume
116.
number 5
can easily be understood qualitatively, at least for our case of equal up and down populations of RCs, by the idea of rate constant splitting. The RCS whose recombination kinetics have been slowed down dominate the time-resolved absorption recovery at long times and effectively balance the accelerated kinetics of the opposed RCs at short times Our results on the quenching of bleaching show that the electric field decreases the yield (averaged over positive and negative field directions) of the charge transfer state, up to x 13% at the highest fields of 200 V/p. If one assumes that the yield of one of the two opposing RC populations saturates at unity for large “forward” fields, then the yield of the opposing RC population is decreased by =26%. However, even this effect is relatively mild and is in sharp contrast to the very strong field dependence of the barrier generation yield in organic semiconductors in general, where orders of magnitude change would be produced for the same span of the applied electric fields [20]. Nature has apparantly found a way to construct high quantum efficiency carrier generating systems, remarkably insensitive to external field bias. As discussed, the time dependence of absorption recovery under the influence of external -electric fields should enable the electric field dependence of the recombination reaction; this would be of great importance for testing various multiphonon tunnelling theories of electron transfer in photosynthesis [9-l 11. Our present deconvolution scheme is only approximate, and we hope to accomplish more accurate deconvolution in our future work. In this respect one desirable approachwould be to produce LB films showing only single decay kinetics_ In addition, the production of mono-oriented (or Ieast partially oriented) LB frtms or RCs would enable experimental determination ofthe electric field directions with respect to the RC axis which speed up or slow down the reco_mbination reaction, and also the field directions which increase or decrease the yield. Surface potential or electro-absorption measurements on oriented LB films of RCs may answer the previousiy raised question [2 I] regarding the existence of permanently built-in charges and electric fieIds in RCs, wbichmay be of crucial importance for the efficient functioning of the photosynthetic process
410
17 May 1985
CHEMICAL PHYSICS LETTERS Acknowledgement
We would like to thank Dr. D.K. Murti for preparation of the sputter-coated substrates. This work has been funded in part by a grant to PLD from the Department of Energy (USA), grant number DE/ AC02/80/ER 10590.10590.
References 111 D-W. Reed and R-K. Clayton, Biochem. Biophys. Res. Pl r31 [41 151 [61 f71 I81
Comm- 30 (1968) 471. R.K_ Clayton and W-R. Sistrom, eds, The photosynthetic bacteria (Plenum Press, New York, 1978). JX. Blasie, J.M. Pachence, A. Tavormina, P-L. Dutton, J. Stamatoff, P. Eisenberger and G. Brown, BiochimBiophys. Acta 679 (1982) 188. M. Schoenfeld, M_ MontaJ and G. Feher, Proc. Natl. Acad. Sci_ US 76 (1979) 6351, NX. Packham, P-L. Dutton and P. Mueller, Biophys, J. 37 (1982) 465_ HJ_ Appel, M. Snozzi and R_ Bachofen, Biochim. Biophys. Acta 724 (1983) 258. H.-W_Trisd, Proc. Natl. Acad. Sci. US 80 (1983) 7173. D-MM.Tiede, P. Mueller and P.L. Dutton, Biocbim. Biophys. Acta. 681 (1982) 191.
191 M. Redi and J-J. Hopfield. J. Chum. Phys. 72 (1980) 6651. [lOI J. Jortner, J. Am. Chem. Sec. 102 (1980) 6676. 1111 S. Rachkovsky and H. Scher. Biochim. Biophys. Acta 681 (1982)
152.
1121 M.R. Gunner, DMM. Tiede, R-C. Prince and P.L. Dutton,
1131 I141 [I51
in: Function of quinones in energy conserving systems, ed. B .L.Trumpower (Acsdemic Press, New York, 1982) pp- 265-269. N. Packham, P. Mueller and P.L. Dutton. Biophys. J., submitted for publication. A. Gopher, Y. Blat& M-Y. Okamura, G. Feher and M. Mental, Biophys. J. 41 (1983) 121a. RX. Clayton and R-T. Wang, Methods Enzyrnol. 23 (1971) 696;
M.Y. Okamura, L.A. Steiner and G. Feher, Biochemistry 13 (1974) 1394. tJ61 P. Fromhera, Rev. Sci. In&r. 46 (1975) 1380. 1171 C-C. Yang, J-Y. Josefowicz and L. Atexandru, Thin Solid Films 74 (1980) 117. II81 D. DeLeeuv, M. MaRey, G. Buttermann. M-Y. Okamma and G. Feher, Biophys. J. 37 (1982) llla. 1191 H. Arata and W-W. Parson, Biochim- Biophys. Acta 638 (1981) 201. 1201Z_D- Popovic, Chem Phys. 86 (1984) 311, and references therein. 1211 2-D. Popovic, J. Chem. Phys. 77 (1982).498.
17 May 1985
CfiJZMICAL PHYSICS LETTERS
ELECTRIC_ FIELD MODULATION OF CHARGE TRANSFER PROCESSES IN REACl’ION CENTERS OF~PHOTOSYNTHE TIC BACTERIA 2-D:
POKWIC,
G-J. KOVACS,
P.S. VINCET-I-
Xerox Research Cenrre of Cana&, 2660 Speakman Drive, Mississauga, Ontario. Canada WK 2LI
and P-L. DU’ITON Depwtment
of Bipchemistry and Biophysics.
Univgrsity of Pennsylvania, Philadelphia, PA 19104,
UsA
Received 30 August 1984; in final form 25 February 1985
External electric field modulation of charge separation 2nd recombinattonprocesses has been achievedin native photosynthetic reaction centers. Langmuir-Blodgett monolayer films of reaction centers isolated from the photosyntheticbacterium Rhodopseudomonas sphaero~des, have been incorporated into electroded sandwichcell structures allowingfield modulation of
both photoinduced bleaching and time-resolved absorption recovery.
I. Introduction After the isolation of the reaction centers (RCs) of photosynthetic bacteria fllj much effort has been invested [2] in elucidating their structural and kinetic properties. The major driving foice behind these efforts is the desire for detailed understanding of the primary charge separation process, which proceeds with a remarkable quantum efficiency approaching unity. The distances involved&i the ,various charge transfer steps have been estimated byapplying a va$ety of techniques to the n-dtivechromatophori: membranes of the bacteria and to recon&i&ted rriembrane inultilayers of the RCs (see ref. [3] anil citations therein). Efforts to obtain distancesip the profile of the niembr+e have come also from iight$tduced electrical transient measurements on R&J reconstituted into biltiyer membranes and films [4~7]%rid ofi closi-packed monolayers of oriented RCs obtained-byUngmuii_Blodgett (LB) film techniques [8]. It is desirable to be able to apply electric fields to these systems in orde; fopetirb ‘the charge transfer reactions.-Measurements of the-field-depetidence-of these processes are of -great importance for testirig var0 009-2614/85/$03.30 @ Elsevier.Science Publishers B.V. (North-Hol&nd Physi.cs Publishing Division)
ious rmdltiphonon tunnelling theories of electron transfeih photosynthesis [9-l I], and for establishing thereby the fundamental nature of the primary photosynthetic mechanism. What ihe application of the exterilal electric field basically does is to change the relative energy levels between different sites in the charge transfer chain. This approach is completientary to the use of chemical substitution [12] to achieve the same goal. The electric field experiments, however, offer the unique possibility of studying the influence of energy level shiftswithout introducing any structural changes. RC-reconstituted bilayer membranes have proven useful to reveal field modulated elctron transfer between cytochrome c and the RC bacteriochlorophyll dimer [X3], (BC&, a+ also between the RC quinone, Q, and (BChlj2, in RCs that have had the native ubiquinone replaced by anthraquinone [14]. In both these cases the free energy drop and distance were suitable to yield effec’ts with inodest fieids. LB films are a natural vehicle which to study the effects of large exterrial electric fields on the charge transfer processes iu isolated RCs. It has already been .established [8] that the RCs -m these LB films are orierited in a f&on &nilar to that encountered in the 405
Volume 116, number5
17 May
CHEMICAL PHYSICS LETTERS
1985
native membrane except that they are a mixture of two vectorially opposing “up” and “down” states_ Concomitant measurements of electrical and optical activities were possible on these ultrathin films, and the latter showed that a significant fraction (up to 40%) of the RCs retained their photochemical activity_ In the present comrnunation, we give the results of our preliminary experiments on external field modulation of the charge transfer steps in RCs of Rhodopseudomonas sphaeroides which have been formed into LB f&ns. We show that by two quite similar experiments one can observe significant modulation of both the charge separation and recombination processes_ The effects of electric field on the total photoinduced bleaching and on the rate constants of absorption recovery are determined.
that is stable well into the millisecond time scale_ The loss and subsequent recovery of absorption, at 860 nn describes the transfer of an electron from the excited singlet, (BChl);, through Bph to QA and (if present) QI The state, (BChl): Qz has a lifetime in the 100 ms range, while the (BChljl Q;i- state lasts into the second of time. Measurements on finished cells were perforrnei in a reflection geometry, with the probe beam enterin the sample from the quartz slide side and being refl ectc from the top Al electrode, thereby making a double pass through the RC film-The maximum signal obtainer by a saturating bleaching pulse corresponded to a reflectivity change (at an angle of incidence of 45”) at 860 nm of 0_2%, indicating that ~40% of the RCs were photochemically active_ The influence of the elel tric field on the charge transfer kinetics was studied using the two experiments described below.
2_ Experimental
ing
Inthefirsttypeofexperiment,aftersample-bleach-
The RCs were isolated from the photosynthetic bacterium,Rhodopseudornonas sphaeroides (R26). as described previously 1lSJ. The resulting RCs were a mixture containing predominantly two quinones (QA uartz slides with a sputtered IT0 (iridium andQE)[lSl_Q tin oxide) transparent conductive layer (a 150 run) and a sputtered SiOa blocking layer (=SOO run) were used as substrates for LB film deposition. LB monolayers were deposited using the procedure described ln ref. [S] in a circular Teflon “Fromherz” trough [16] at a constant surface pressure of ~25 mN/m. To allow high electric fields to be applied without cell breakdown, a 1.75 pm polymer blocking layer (Goodyear Pliolite OMS, solvent Exxon Isopar G) was dipcoated [ 171 on top of the RC monolayer. An evaporated opaque Al layer formed the top electrode of the sandwich. Photochemical activity of the RC films was checked at each step of the fabrication process by measuring the absorption at 860 run, thereby monitoring the (BChl)2 photooxidation and reduction_ This was done using a low-intensity probe beam, after bleaching with a 5 ms pulse from the 514.5 M-I line of Ar+ ion laser. (A 750 nm short-wavelength cut-off filter placed in front of the light detector prevented its saturation due to the high intensity of the bleaching light pulse.) Photochemical activity is operationally defined as the ability of the RCs to support flash activated (BChl)Z oxidation 406
at zero field. the electric field was switched on wit in a few miliseconds and the time-resolved absorption recovery was recorded_ This type of measurement gives information about field-modulated recombination processes of already separated electron-hole pairs. The total recording time was 0.5 s and data were take typically by averaging 40 measurements cycles. We shal refer to this type of experiment as absorption recover In the second type of experiment, to be referred to as “quenching of bleaching”, a flash exposure (-5 ps in duration) wx applied to the sample by a Genera Radio xenon flashlamp, equipped with a 600 nm long wavelength cut-off filter. A 5 ms voltage pulse was used to bias the sample during bleaching and was switched off immediately after termination of the light pulse. The measured quantity was AI, which represents the maximum bleaching_ A1, the maximum change of the probe beam intensity, was recorded immediatelyafter termination of the light pulse and turning off of the sample bias. This experiment gives information about the field modulation of the primary charge separafiol; steps. The flash lamp intensity was sufficiently Iow that bleaching was proportional to the total flash energy, and AI was normalized by this energy. In this case, we can define the quenching of bleaching, Qb, due to the electric fieldE, by Q&9
= [UO)
- N(E)1
/AI(o)
(1:
In order to avoid possible charge accumulation in
Volume 116, number 5
CHEMICAL PHYSICS LETTERS
the samples, the usual sequence in time-averaging measurements was as follows: positive field, zero field, negative field, zero field, etc. No significant difference was observed for positive and negative polarities, which confirms the equal up and down populations of the RCs, and the data shown for both experiments are an average over both polarities. The magnitude of the electric field applied to the sample is deduced from the cell thickness determined from a cell capitance measurement, with a dielictric constant of 3 assumed for the composite sample between the conductive eiectrodes.
3. Results and discussion Before discussing our own results, it is pertinent to note that a preliminary report [14] has discussed the effect of transmembrane potentials on charge recombination in RCs incorporated into planar bilayers. No
17 May 1985
effect was found with applied voltages up to 125 mV for RCs containing the native ubiquinone, but an e-fold change in recombination rate was observed for an applied potential of 175 mV for RCs reconstituted with anthraquinone. The applied 125 mV corresponds to a fi.eld of at most 20 V/pm (if one assumes the entire voltage drop is across the 6 nm span of the RCs). We have applied fields an order of magnitude larger, and according to our results, it is not surprising that, in ref. [ 141, no effect was observed in the natural photosynthetic system. Another preliminary report [ 181 has discussed Stark effect absorption changes in the RCs, whereby the electric field alone changes the amplitude of the various absorption bands. These experiments were done by applying electric fields to RCs embedded in polyvinyl alcohol films. In principle this Stark effect could change the amplitude of the flash activated bleaching, and therefore contribute to our measurements of Qb.
0 -1
-2
T ,o P ; h U Z X -
_r
-3 0 -_1 -2 -3 0 -1
-2 -3
Fig. 1. Absorption recovery of RCs as a function of time for various values of the applied electric field. The zero-field reference is given in each case. No detectable shift occurs for fields less than 60 V/urn.
407
Volume 116, number5
However the magnitudes of these effects are very small in comparison with our measured Qb and can be neglected . With regard to our own results, we will first discuss field induced changes in the kinetics of the charge recombination between (33Chl)‘-t and QT in the RCs. Fig. f shows the logarithm o iLreflectivity variarion versus time for different fiefds applied to the sample. The zero-field data can be well fitted with two exponentials having time constants of 70 and 360 ms. The first time constant is in good agreement with the values(60-100 ms) normally observed in absorption recovery experiments on RCs containing a single quinone (i.e. QA)_ The second, however, is about three times shorter than usually observed (=I s) in RCs with two quinones (i.e. QA and QB) in which the charge recombination occurs from Qg . We do not understand the reason for this discrepancy, but it is presumably due to subtle structural changes or distortions, which occur on formation of the RCs into a densely packed monoplayer, It is obvious that application of the electric field dramatically changes the recombination kinetics in photoactivated RCs. We would expect that the recombination process can either be hastened or hindered, depending on the RC orientation with respect to the external field. The two time constants cbserved at zero field should therefore split into four, and in principle it should be possible to deconvolute them from the decay curves. Fig. 1 shows that the curves genera&j shift increasingly upward showing decreasing slope at longer times when compared with the zero-field data. This can be understood in the framework of rate constant splitting:. At longer times, the time evolution of the decay will be governed by the slow rate constants, leading to a decreased rate as observed. Attempts to deduce the field dependence of the decay rate constants by assuming arbitrary rate constant splitting were not successful; the number of fitting parameters, four independent rate constants in this case, was simply too large. While excellent fits to the experimental data could be obtaining using this procedure, ;t did not yield meaningful variation of the rate constants with electric field_ In order to limit the number of fitting parameters and to impart order to their extracted values, we have assumed that, in the first approximation, the field-induced splitting of rate constants is symmetric. Mathematically this can be expressed in the following fashion. If the time vari408
17 May 1985
CHEMICALPHYSICSLETTERS ation of the bleaching at zero field is A.$f, E = 0) = 234,
exp(-kit),
where the Ai are the light intensity amplitudes associated with the rate constants, kj, then the field induced symetric splitting (by Lyci) of rate constants will lead to the following time dependence A.I(t,E)
= C
+ exp[-(ki
iAi{exp[-(kf -
ak,)t]).
+ Aki)t] (3)
One might suspect that the inability to extract ameaningful progression of rate constants from the experimental data without the assumption of symmetric splitting, may be due to the fact that RCs are not up and down oriented, but rather possess a dispersion in their orientation. However, the orientation of LB f%-~-~s of RCs has been confirmed by linear dichroism measurements [8]. In any event an assumption of dispersed orientation would introduce additional fitting parameters, further compounding the problem of extraction of rate constants; the immediate problem is in the prolificacy rather than the paucity of fitting parameters. If the RCs do assume a dispersion in orientation, then the rate constants determined (under the assumption of symmetric splitting) may be interpreted as averages of the resulting distributions of rate constants. In fig_ 2 the field dependence of the rate constants, deduced by fitting eq. (3) to the experimental data, is given. The amplitudes, A, deduced by fitting eq. (2) to the zero-field data are retained in fitting eq. (3)_ While this procedure cannot be regarded as an exact deconvolution, it can at least give an idea about the magnitude of the field-induced changes. For the range of fields applied Cup to 200 V/m), the rate constants appear to change by about an order of magnitude. These changes are caused by shifts in the energy levels of QA and QE relative to (SChl]~, on application of the uniform electric field. The shift is given by --de& where d is the dipole moment of the electron-hole pair and E is the electric field, If it is assumed that the RC cylindrical axis lies along the electric field direction, a projected separation distance of +1.5 nm for the pair is reasonable for purposes of estimation. From this separation distance one estimates a substantial change in energy separation of 20.3 V for the range of fields
Volume 116, number 5
17 May 1985
CHEMICAL PHYSICS LETPERS
-a
.
oooo
k,-Ak, .
00. 000%~ k2+Ak2
0
0
ka-Ak2 00
t 1.0
0,
E2 (V/pm)
‘0
Ooo
0
L
o-4o>
F%. 3. Quenching of bleaching the applied electric field.
2
as a function
of the square of
300 E (V/pm)
Fig_2. Field dependence of recombination rate constants assuming symmetricrate constant splitting (see eq. (3% deduced
applied. This change will in general be somewhat different for QA and QB and will depend on their precise vectorial separation from (BChl)$. It would appear that an alternative approach to simplifying the-analysis would be to use singlequinone RCs with single decay kinetics. However, while singlequinone RCs have been prepared in solution, an equilibrium population of zero-, one- and two-quinone RCs can developed with time, at a rate depending on the storage conditions. This process appears to be accelerated in RCs cast into a solid-state film, and the use of singlequinone RCs did little, if anything, to improve the situation. Films prepared from singlequinone RCs showed almost identical decay kinetics to those pr& pared from twoquinone RCs. Alternatively we attempted to block QA to QB electron transfer by treating the RCs with ortho-phenanthroline [ 191. Again the resulting films showed basically identical decay kinetics to those of films not treated by the electron transfer inhibitor, irrespective of whether the treatment occurred before or after film deposition, or both. It therefore appears that LB films of RCs are deposited into a unique configuration which is not easy to change.
In fig. 3 the dependence of electric-field-induced quenching of bleaching as a function of the square of the applied electric field is given. This data representation gives a straight line, which is easily explained in terms of equal populations of up and down oriented RCs in the LB films. We now introduce t!re yield, ?iCT, of the chargeseparated state induced by bleaching ilfumination. The quantity AI(E), averaged over positive and negative fields, is proportional to $ [r7CT(E) + qc~( - E)]. Consequently for the quenchmg of bkaching, averaged over positive and negative field directions, we can writer Q,(E)
= 1 - 4 fr’Ic#)
+ “?c-+-E)]
/r&o)-
(4)
Quenching of bleaching is therefore a measure of the field-induced change in the yield of the charge separated state. This change is caused by the change in energy level separation between the intermediate bacteriopheophytin state, (BPh)-, relative to (BChl); and to Q-. From relative distance measurements [7] the changes are expected to be somewhat smaller, but of the same order,as the changes estimated above between the levels, Q- and (BChl)$ .
4. Conclusions The application of the electric field clearly slows down the net recombination reaction (see fig_ 2) This 409
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can easily be understood qualitatively, at least for our case of equal up and down populations of RCs, by the idea of rate constant splitting. The RCS whose recombination kinetics have been slowed down dominate the time-resolved absorption recovery at long times and effectively balance the accelerated kinetics of the opposed RCs at short times Our results on the quenching of bleaching show that the electric field decreases the yield (averaged over positive and negative field directions) of the charge transfer state, up to x 13% at the highest fields of 200 V/p. If one assumes that the yield of one of the two opposing RC populations saturates at unity for large “forward” fields, then the yield of the opposing RC population is decreased by =26%. However, even this effect is relatively mild and is in sharp contrast to the very strong field dependence of the barrier generation yield in organic semiconductors in general, where orders of magnitude change would be produced for the same span of the applied electric fields [20]. Nature has apparantly found a way to construct high quantum efficiency carrier generating systems, remarkably insensitive to external field bias. As discussed, the time dependence of absorption recovery under the influence of external -electric fields should enable the electric field dependence of the recombination reaction; this would be of great importance for testing various multiphonon tunnelling theories of electron transfer in photosynthesis [9-l 11. Our present deconvolution scheme is only approximate, and we hope to accomplish more accurate deconvolution in our future work. In this respect one desirable approachwould be to produce LB films showing only single decay kinetics_ In addition, the production of mono-oriented (or Ieast partially oriented) LB frtms or RCs would enable experimental determination ofthe electric field directions with respect to the RC axis which speed up or slow down the reco_mbination reaction, and also the field directions which increase or decrease the yield. Surface potential or electro-absorption measurements on oriented LB films of RCs may answer the previousiy raised question [2 I] regarding the existence of permanently built-in charges and electric fieIds in RCs, wbichmay be of crucial importance for the efficient functioning of the photosynthetic process
410
17 May 1985
CHEMICAL PHYSICS LETTERS Acknowledgement
We would like to thank Dr. D.K. Murti for preparation of the sputter-coated substrates. This work has been funded in part by a grant to PLD from the Department of Energy (USA), grant number DE/ AC02/80/ER 10590.10590.
References 111 D-W. Reed and R-K. Clayton, Biochem. Biophys. Res. Pl r31 [41 151 [61 f71 I81
Comm- 30 (1968) 471. R.K_ Clayton and W-R. Sistrom, eds, The photosynthetic bacteria (Plenum Press, New York, 1978). JX. Blasie, J.M. Pachence, A. Tavormina, P-L. Dutton, J. Stamatoff, P. Eisenberger and G. Brown, BiochimBiophys. Acta 679 (1982) 188. M. Schoenfeld, M_ MontaJ and G. Feher, Proc. Natl. Acad. Sci_ US 76 (1979) 6351, NX. Packham, P-L. Dutton and P. Mueller, Biophys, J. 37 (1982) 465_ HJ_ Appel, M. Snozzi and R_ Bachofen, Biochim. Biophys. Acta 724 (1983) 258. H.-W_Trisd, Proc. Natl. Acad. Sci. US 80 (1983) 7173. D-MM.Tiede, P. Mueller and P.L. Dutton, Biocbim. Biophys. Acta. 681 (1982) 191.
191 M. Redi and J-J. Hopfield. J. Chum. Phys. 72 (1980) 6651. [lOI J. Jortner, J. Am. Chem. Sec. 102 (1980) 6676. 1111 S. Rachkovsky and H. Scher. Biochim. Biophys. Acta 681 (1982)
152.
1121 M.R. Gunner, DMM. Tiede, R-C. Prince and P.L. Dutton,
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in: Function of quinones in energy conserving systems, ed. B .L.Trumpower (Acsdemic Press, New York, 1982) pp- 265-269. N. Packham, P. Mueller and P.L. Dutton. Biophys. J., submitted for publication. A. Gopher, Y. Blat& M-Y. Okamura, G. Feher and M. Mental, Biophys. J. 41 (1983) 121a. RX. Clayton and R-T. Wang, Methods Enzyrnol. 23 (1971) 696;
M.Y. Okamura, L.A. Steiner and G. Feher, Biochemistry 13 (1974) 1394. tJ61 P. Fromhera, Rev. Sci. In&r. 46 (1975) 1380. 1171 C-C. Yang, J-Y. Josefowicz and L. Atexandru, Thin Solid Films 74 (1980) 117. II81 D. DeLeeuv, M. MaRey, G. Buttermann. M-Y. Okamma and G. Feher, Biophys. J. 37 (1982) llla. 1191 H. Arata and W-W. Parson, Biochim- Biophys. Acta 638 (1981) 201. 1201Z_D- Popovic, Chem Phys. 86 (1984) 311, and references therein. 1211 2-D. Popovic, J. Chem. Phys. 77 (1982).498.