photovoltaic effects in biomembranes/spl minus/reverse-engineering naturally occurring molecular...

Felix T. Hong Deportment of Physiology

Wayne State University School of Medicine

Photovoltaic Effects in Biomem branes

Re verse- Engineering Naturally Occurring Moleculur Optoelectronic De vices

ioelectric phenomena are intimately re- B lated to the theory of electricity and electrochemistry. In 1780, Luigi Galvani discovered that electric currents appear when two different types of metals (which generates a Volta potential) are brought into con- tact with a frog muscle. Galvani believed that this “animal electricity” is characteristic of living tissues (which has its physiological origin in the diffusion potential). This sparked a controversy with Allessandro Volta, who believed electricity is of inanimate origin. As subsequently transpired in science history. they both were partly right and partly wrong. The Volta potential and the diffusion potential are now the standard sta- ples in textbooks of electrochemistry and of electrophysiology, respectively. In 1964, Kenneth Brown and Motohiko Murakami [ 11 discovered yet another kind of “animal electricity.” They found that intense illumination of a monkey retina with a photographic flash lamp generated a bioelectric potential with an ultrafast rise-time (i.e., less than one microsecond). This was an unusual finding because it was widely known that bioelectric phenomena originating from ionic diffusion are rela- tively slow in onset (usually with a millisecond response time). This new signal was named the early receptor potential (ERP) in order to be distinguished from a slower signal, the late receptor potential (LRP), which is also known as the a wave of the electroretinogram [2]. It was soon established that the ERP depends on the integrity of rhodopsin, the visual pigment, which is located in the photoreceptor membrane. An intense pursuit of the subject followed in the subsequent decade, but physiologists and biophysicists came up empty-handed because no known physiological functions could be assigned to the early receptor potential. The ERP has since been dismissed as an epi-phenomenon, i.e., an evolutionary ves- tige of no biological importance.

When Ti Tien [3] demonstrated in 1968 a photovoltaic effect in an artificial bilayer lipid membrane (BLM) that contained chlo-

roplast extracts, many investigators thought it was an experimental artifact, presumably caused by a localized temperature jump as a result of dissipation of the absorbed photon energy. It turned out that this photoelectric signal actually is similar to the ERP. The consensus among physiologists is that this type of photosignal is generated by light-induced rapid charge displacement in the pigment molecules residing in the membrane. We shall refer to this class of photosignals as the fast photoelectric voltage/current or the displacement photocurrent.

In this article, we shall present the fundamental principles of the fast photoelectric effect and its applications to molecular electronics research. For two reasons we choose to illustrate the principles with experimental data obtained from reconstituted bacteriorhodopsin membranes. First, bacteriorhodopsin is one of the few biomaterials that have successfully been used as an advanced material for molecular device construction.

February/Marth 1994 IEEE ENGINEERING IN MEDICINE AND BIOLOGY 0739-51 75/94/53.0001994 75

1. Diagram showing the relation of the chromophore and several key amino acid residues of bacteriorhodopsin. The cylindrical shape object inserted in the bilayer lipid membrane (fence-like structure in the diagram) represents bacteriorhodopsin with its seven a-helices, A, B, C, D, E, F, and G. The cytoplasm is a t the top of the diagram. The chromophore is attached to lysine residue 216 via a Schiff's base linkage. Photoi- somerization of the chromophore causes a sudden decrease of thepK, (or rather, increase of thepKb) of the Schiff's base proton binding site. Aspartic acid residue 96 is the immediate proton donor to the Schiff's base proton, and aspartic acid 85 is the immediate proton acceptor. (Reproduced with permission from Ref. [6]).

______ 0.9 ms Ref. 14 1.3 ps 17 ps 0.06 rns

Ref. 15 25 ps 150 ps 2.4 rns 5.8 ms

Ref. 16 4.4 ps 81 1s 2.5 ms 8 rns

Ref. 17' 57 ps 1.06 rns 13 rns

Ref. 18*+ 115ps 4.5 rns

~

~

I 640 ms

Second, bacteriorhodopsin offers an un- precedented opportunity to decipher Na- ture's design principles of photosynthetic and visual apparatus: bacteriorhodopsin re- sembles a visual pigment chemically, but performs the task of photosynthesis.

Ref. 19 2 ms

Halobacterium Halobium: A Salt-Lov- ing Bacterium That Uses a Visual Pig- ment to Car ry Out Photosynthesis

Bacteriorhodopsin (bR) is the photosynthetic pigment of an archaebacterium Haln- bacterium halobium, whose natural habitat

1000 rns

is a salt flat [4,5]. Halobacterium halobizim contains four kinds of pigments in its membrane, all of which have a vitamin A alde- hyde as the chromophore (the light-absorbing element) that is the same as in the visual pigment rhodopsin. The most abundant of the four, bacteriorhodopsin. actually forms a two-dimensional crystal lat- tice of hexagonal cells that constitutes the purple membrane. Like its sister molecule rhodopsin, bR is a single chain polypeptide that transverses the membrane seven times with seven %-helices [6]. Bacteriorhodopsin

14 derived trom Fig. l d in Ref. [31]

76

is essentially a light-driven proton pump; the absorbed photon energy is utilized to transport protons from the intracellular space to the extracellular space and thus is converted to the electrochemical energy of a transmembrane proton (concentration) gradient (Fig. 1). Concurrent with the proton movement, bR undergoes a cyclic photochemical reaction (known as the photocycle) [7] (Fig. 2). A photovoltaic signal can be detected when oriented bR is reconstituted in a bilayer lipid membrane [8]. In fact, when the membrane is illuminated with a light pulse (microsecond or less in duration), the resulting photovoltaic signal is almost indistin- guishable from the ERP [9, 101.

The topic of the fast photoelectric effect in reconstituted bR membranes is controversial [ll-131. While most investigators ad- here to a common practice of decomposing the measured photosignal into several exponential terms, there are not any two groups in agreement with regard to the relaxation time constants (Table 1). The molecular interpretation of the measured signal is dependent on the detail of the photocycle. When the photocycle underwent a major overhaul recently [7], the molecular interpretation had to be revised.

In this article, we shall describe an alter- nate approach that is not dependent on the photocycle, but which constitutes an inter- marriage of electrophysiology and electrochemistry. We believe a more coherent picture will emerge with this approach than was evident with the conventional approach. An additional advantage of our combined electrophysiological and electrochemical approach is its generality. While a specific model must be concocted for each particular

I

2. An improved model of the photocycle. The photointermediates represented by J, K, L, MI, M2, N, and 0 have characteristic absorption maxima and kinetics. Most of the reactions are reversible, but MI + M2 is preferential in the forward direction. (Reproduced with permission from Ref. [7]).

IEEE ENGINEERING IN MEDICINE AND BIOLOGY FebruorylMarch 1994

photosystem, our general model is applicable to the photoelectric effect of all pigment- containing membranes (201.

Two Types of Light-Induced Charge Separation

Photosynthesis, Nature's way of solar energy conversion, involves the conversion of photon energy into chemical energy by first storing the energy in the form of charge separation. That energy will be dissipated when charges recombine. The task of photosynthesis (solar energy conversion) is to convert this temporarily stored energy into a more stable form so that it will not be dissipated immediately as heat. Nature's solution is to store this energy as an electrochemical gradient either across the plasma membrane that forms the boundary of a cell or across the membrane that lines an intracellular or- ganelle (e.g.. the thylakoid membrane of chloroplasts). At first glance, it is similar to mankind's solution of storing electric energy in a capacitor (condenser), but in detail it is much more sophisticated.

Thus, Nature's goal is to separate the charges across the membrane, which is a reasonably good insulator that minimizes excessive (internal) charge recombination. When the charges are allowed to recombine via an outer circuit loop, the dissipated energy can then be used to form energy rich chemical compounds such as ATP (adeno- sine triphosphate) (in all cases of photosynthesis and the purple membrane) or NADPH (in the case of photosynthesis in green plants and cyanobacteria), or used directly to power a bacterial flagella, which a molecular motor. Hence, not only the separated electric charges stored on the membrane condenser are available for ATP synthesis, but also the chemical energy in the from of a transmembrane pH gradient can be tapped for that purpose. Space does not permit a detailed description of various photosynthetic systems. Interested readers are referred to standard reviews in the literature [21-231. A simplified account. with molecular electronics applications in mind, will be given here.

Typically, the thickness of a biological membrane is about 60 A. There is no possi- bility of biological charge separation of that magnitude to be accomplished in a single step. In the bacterial reaction center, charge separation takes place in a relay fashion via a series of electron donors and acceptors. Electrons are transferred over a short distance in each step, but together they manage to span the thickness of the membrane. In each of these steps, the separated charges have the option of either recombining with each other or relaying the separated charge to the next step in the form of coupled consecutive charge transfer reactions (Fig. 3).

Periplasm , Membrane

~

Cytoplasm

H , O

- H,O

Cytoplasm Space

Membrane Phase

Extracellular Space

3. Coupled consecutive charge transfer reactions in the bacterial reaction center (a) and in bacteriorhodopsin (b). (a) The primary electron donor is the "special pair" (SP). BCh, BPh, QA, and QB stand for monomer bacteriochlorophyll, bacteriopheophytin, quinone A, and quinone B, respectively. Cyt b/ci and Cyt c2 are mobile cytochromes in the membrane phase and the aqueous phase, respectively. Cyt is the fixed cytochrome of which four exist but only one is depicted for the sake of simplicity. The solid arrows indicate the direction of forward reactions. Reverse reactions are not shown. Dotted arrows indicate diffusion of mobile charge carriers. The consecutive charge transfer reactions are coupled in the sense that the reactant of a reaction comes from the product of the preceding reaction and the product of the reaction becomes the reactant of the subsequent reaction. Notice that electrons are transported from the periplasmic side to the cytoplasmic side. However, the net result is the transport of equivalent protons in the opposite direction. This is accomplished by the reaction of quinone QB, which binds two electrons and two protons and becomes a neutral car- rier so that an electron and a proton are cotransported from the cytoplasmic side back to the periplasmic side. QB thus converts an electron current to a proton current. (Reproduced with permission from Ref. [24]). (b) The exact proton transport pathway is presently unknown. Only five binding sites are depicted for simplicity. A3 is the Schiff's base proton binding site. I t is understood that the Schiff's base is neutral when unprotonated and is positively charged when protonated. A2 is Asp96 and A4 is Asp85, as shown in Fig. 1. Only A3 -+ A4 is shown to be light-driven. In reality, there are more than one light-driven steps. (Reproduced with permission from Ref. [25])

Februory/Marth 1994 IEEE ENGINEERING IN MEDICINE AND BIOLOGY 77

The competition between these two options is an important factor in determining the conversion efficiency.

These individual charge movements will generate a photoelectric signal. If charge recombination were the only option, there would be no net charge transport and a capacitative signal will be observed (which we shall call the AC photoelectric effect). If charge recombination could be ignored and only continuation of forward charge transfer could be possible, then a net charge transport and a DC signal would be observed (called the DC photoelectric effect). In real life, both AC and DC photoelectric signals are present. The AC photoelectric signals will be the dominant feature when a short light pulse is used to initiate the charge transfer, whereas the DC photoelectric effect will become evident if steady illumination is main- tained to allow a steady state to be established. That the ERP is an AC photoelectric signal was established by an important finding of McGaughy and Hagins in the late 60s [26]. They found that the photocurrent, l( t) , associated with the ERP satisfies the condition of a zero time-integral:

roo

J I ( t ) dt=O 0 [ I 1

This means that, most, if not all, of the charges that have been separated subsequently recombine. It turns out that all fast photoelectric signals are a manifestation of the AC photoelectric effect. We shall hence- forth restrict our discussion to the AC photoelectric effect.

For the sake of understanding fast photoelectric signals, we need only consider two types of charge separations (Fig. 4). De- pending on the environment where charge separation takes place, a charge recombination can follow either simple first order kinetics or second order kinetics. For the initial light-induced charge separation in a chlorophyll-based photosynthetic reaction center and perhaps also in bacteriorhodopsin and in rhodopsin, the separated charges are con- fined to the membrane phase and the recombination is most likely to be a first order process. This is because the space charge concentration inside the membrane is so small and its diffusive mobility is so slow that the separated pair of charges remain correlated all the time. This process generates a transient array of electric dipoles inside the membrane, and is referred to here as the oriented dipole (OD) mechanism [27]. That these transient dipoles are oriented with respect to the membrane is a consequence of the fixed and asymmetric orientation of the photopigments. One further consequence is the generation of an externally measurable

IPT Mechanism

aqueous membrane aqueous

E.

OD Mechanism

aqueous membrane aqueous

n CtT

common irreducible equivalent circuit

I

4. Interfacial proton transfer (IPT) and oriented dipole (OD) mechanisms of light-induced rapid charge separation and recombination. The diagrams are self-explana- tory. The charge density profiles are also shown. The two equivalent circuits are derived from a Gouy-Chapman electrostatic calculation. Cd and C, are the double layer capacitance and the geometric (dielectric) capacitance, respectively. E'p is the photoemf source with an internal resistance Rp. Both equivalent circuits can be reduced to the same irreducible equivalent circuit shown at the bottom. C, is chemical capacitance, which is a composite of Cd and C,, similar to the membrane capacitance Cm (not shown) but of different physical connections. (Reproduced with permission from Ref. [27]).

capacitative current that accompanies the process of charge separation and recombination.

A second type of charge separation occurs at the membrane-water interface. When a proton is taken up by the photopigment or the reaction center complex, its counter-ion must be left behind in the adjacent aqueous phase. The separation of charges across the membrane-water interface leads to polariza- tion of the immediate vicinity of membrane surfaces, known as the diffuse electrical double layers. Because of the abundance of ions in these layers and because of their high diffusive mobility in water, the separated pair of charges become rapidly de-correlated with each other. That is, in the subsequent

charge recombination caused by the reverse charge transfer reaction, the pair of charges that recombine is no longer the same as the pair that separates. Therefore, the recombination will follow either a second order or a pseudo-first order kinetics. This is an important feature that allows the second mechanism, which we shall refer to as the interfacial proton transfer (IPT) mechanism [27], to be differentiated from the oriented dipole mechanism.

Both mechanisms of charge separation described above will satisfy the zero time- integral condition, because the amount of separated charge is exactly equal to the amount of recombined charge. Our next task is to link these two molecular models to

78 IEEE ENGINEERING IN MEDICINE AND BIOLOGY Februory/Morth 1994

macroscopically measured electric signals. Since conventional electrophysiology util- izes equivalent circuits to characterize the bioelectric phenomenon being studied, the task can be accomplished if an equivalent circuit can be derived from either model.

Charge separation will generate an electric field in the vicinity of the membrane, which in turn will affect the distribution of ions in the adjacent aqueous phase. The al- teration of ion distribution in the vicinity of the membrane will generate an additional electric field, which is then superimposed onto the field generated by charge separation. Since all of these processes are time-dependent, the calculation can be quite involved. Fortunately, it turns out that the redistribution of ions in the aqueous phase is infinitely faster than the charge separation and recombination inside the membrane. For all practical purposes, the ion distribution in the diffuse double layers can be regarded as being always in (quasi-)equilibrium. On the other hand, charge redistribution inside the membrane as a result of coulombic interac- tions is infinitely slower than the fast photoelectric effect , and can therefore be completely ignored. This peculiar situation leads to substantial simplification of the problem; the kinetic calculation of charge separation and recombination can be carried out independently of the electrostatic calculation. The latter calculation is prescribed by the standard procedure of the Gouy-Chap- man diffuse double layer theory, a detailed analysis having been published elsewhere

The result of the calculation, re-interpreted in terms of lumped circuit elements, is shown in Fig. 4. The equivalent circuits for the two models are slightly different. Both circuits contain a photocurrent source (photoemf, Ep(f)) , which is located either at the membrane-water interface (IPT mechanism) or inside the membrane (OD mechanism). The injection of photocurrent, i.e, charge separation, leads to charging of three elementary capacitors: a geometric capacitance, Cg, (formed by the membrane dielectric) and two double layer capacitances, c d . The charging patterns matching the space charge density profiles are also shown in the diagram.

Though the equivalent circuit for the IPT mechanism is different from that for the OD mechanism, both are equivalent to the same irreducible equivalent circuit, shown at the bottom of Fig. 4. Consequently, the two mechanisms are electrically indistinguish- able. The differentiation then must rely on chemical kinetic analysis, as indicated above.

In the irreducible equivalent circuit just mentioned, there is a capacitor, C,, which is

[27-291.

Februory/Morth 1994

positioned between the photosignal source, Ep, and the external circuit. In the jargon of electric circuit analysis, the capacitor, C,, is connected in series with the photovoltage source, Ep, and the latter is AC-coupled to the outside world. The presence of this series capacitor was quite puzzling to investigators in the field. because electrophysiologists have become used to the notion of a membrane capacitor by the analogy that the membrane material (phospholipid) serves as the insulator interposed between two plates of a parallel plate condenser. Yet, our analysis insists that Cp is physically distinct from the conventional membrane capacitor, Cm (see Fig. 5) . For this reason, we gave Cp a distinct name, chemical capacitance [30]. Several review papers have been published to ex- plain this sticky point [12,29,31]. Here, we shall only demonstrate the effect of having these two capacitors in the generation of the fast photoelectric signals, and how some otherwise puzzling experimental observa- tions can be readily reconciled.

Equivalent Circuit Analysis The most important effect due to the

presence of Cp is that the externally observed photosignal will have a different time course of relaxation depending on whether the photosignal is measured under an open circuit or a short circuit condition. An open circuit condition means that the measuring circuit draws negligible current from the membrane and the condition is fulfilled if the so-called access impedance, Re, (the impedance interposing between the measuring device and the membrane system) is much larger than the source impedance (the intrin-

sic impedance of the photosignal source). On the other hand, a short circuit condition means that the measuring device provides a preferential pathway for the photo-induced current to complete a round-trip, and the condition is fulfilled if the access impedance is much smaller than the source impedance. However, a short-circuit measurement of the fast photoelectric signal can be tricky. Be- cause of the series capacitance C,, the source impedance is much reduced at the time scale of its relaxation (MHz); the source impedance may decline from its DC value (approx. R,, typically, lo7 or IO* Q) to its value at 1 MHz (approx. Rp, typically 60 kn) .

Thus, it is possible that an attempted short circuit measurement of the fast photocurrent using current amplifiers designed for low frequency applications (typically with an input impedance of 100 kQ) may turn out to be inadvertently an open circuit measurement. The commonly observed first derivative relationship between the measured photocurrent and the measured photovoltage [32] is tell-tale evidence indicating that the intended short circuit measurements were actually made under near open circuit condition (see p. 222 in Ref. [ZO]). Most of our photocurrent measurements were carried out with an access impedance that is comparable to the source impedance in the MHz range (Re = 40 kn) . We routinely include the finite non-zero access impedance in the analysis.

By allowing the access impedance to vary from zero (ideal short circuit condition) to infinity (ideal open circuit condition), an analysis of the equivalent circuit in Fig. 5 allows us to determine how the waveform of

Measuring Device -

Pigmented Membrane

5. Equivalent circuit representing interaction of the photoemf source, the inert membrane RC network and the measuring device. R,, the access impedance, is the combination of the input impede ance, the electrode impedance and the impedance of the intervening electrolyte solution. (Reproduced with permission from Ref. [201).

IEEE ENGINEERING IN MEDICINE AND BIOLOGY 19

-pass filter with a m e

t nor short circuit, but actually h

TIME (MICROSEC)

0 5 10 15 20 25 30 35 40 45 50 TIME [MICROSEC)

0 5 10 15 20 25 30 35 40 45 5( TIME (MICROSEC1

80 IEEE ENGINEERING IN MEDICINE AND BIOLOGY Februory/Morch 1994

the photosignal would evolve as the passive relaxation time constant, T ~ , is varied [20]. Incidentally, the value of Z~ can be short- ened either by an external short-circuit (reduced Re) or by an internal short-circuit (reduced Rn, as a result of action of some ionophores; typical value of Rm in BLM is IO9 R; for Teflon support films, Rm exceeds 10'6fi]. The result is summarized in Fig. 6.

In the case of an idealized short circuit measurement, the photocurrent surges to a sharp peak upon illumination, representing charge separation. The signal is seen to reverse polarity upon cessation of illumination, which represents the preponderance of charge recombination. The exponential decay follows the simple first order or pseudo- first order charge recombination process. We must point out that the relaxation kinetics are determined solely by the chemical relaxation process of charge recombination. The electric parameters, Rm and C,, of the inert supporting structure (lipid membrane] have no effect. This is of course because of the ideal short circuit condition, which allows the photocurrent to proceed exclusively to the external measuring circuit.

However, this current flow would not be the case if the short circuit condition is less than perfect. A fraction of the photocurrent would then be diverted to partially charge the membrane capacitance. Cm. The relaxation would involve the interaction of two RC circuits, containing Cm and C, , respectively, plus other resistive elements; thus, it will be a bi-exponential process in general. In the extreme case of an idealized open circuit measurement, when all of the photocurrent proceeds to charge C , , the relaxation would be dominated by the RC time constant (RnICtn) (see Sidebar 1).

Component Analysis of the ERP-Like Signal from Reconstituted Bacteri- orhodopsin Membranes

The above described behavior is expected in a conventional device consisting of a photocurrent source that is embedded in an inert supporting thin film structure with its characteristic RC features. Here we must point out that the above model was developed for the idealized case of a single step of charge separation, followed by a simple first order or pseudo-first order process of charge recombination. In real life, bR has a complex photochemical scheme, and the bacterial reaction center has a scheme of multiple steps of charge separation/recombination (Fig. 3). There is no hope of quanti- tatively testing the validity of the equivalent circuit unless we develop methodology to isolate a single step of charge separation that gives rise to a pure photocurrent component.

FebruarylMorth 1994

BRIEF PULSE STIMULATION LONG RECTANGULAR PULSE STIMULATION

-+ I

H, > 0

7,- 7.. > n

e.g., Q ~ -'

6. Relaxation time course of a pulsed light induced photoelectric signal with a single component. Responses to both a brief light pulse and to a long rectangular light pulse are shown, but only the former is discussed in the text. The parameter Tm is the characteristic RC relaxation time of the system which depends on Rm, Re, and Cm shown in Fig. 5. Tmc is the critical value around which the time course is changed from monotonic to biphasic and vice versa (not discussed in the text). See text for further explanation. (Reproduced with permission from Ref. [ZO]).

Through the combination of several membrane reconstitution methods, we were able to make such an isolation. In bR containing membranes, we identified at least three individual components, which we named B1, B2 and B2'. Experimental evidence suggests that the BI component is most likely caused by intramolecular charge separation of the oriented dipole type that accompanies the electronic excitation of the chromophore and its subsequent photoi- someriazation (see below). This component has an unresolved rise-time and has an almost negligible temperature dependence. This signal is analogous to the Rl component of the ERP, which is known to persist at low temperatures. A second component, B2, is analogous to the R2 component of the ERP. Both the B2 and the R2 components are temperature-sensitive and can be reversibly inhibited by low temperatures (OOC). Experimental evidence suggests that B2 and R2 are generated by proton uptake from the cytoplasmic aqueous phase and the subsequent proton release (into the same cytoplasmic aqueous space), i.e., they are generated by means of the interfacial charge (proton) transfer mechanism. A similar third component, B2'. which has no known counterpart in the ERP, is generated by the interfacial proton release to the extracellular space and its subsequent re-uptake [33).

If bR is reconstituted into a genuine BLM. all three major components. B I , B2 and B2', can be observed. If an oriented layer of purple membrane is attached to a thin Teflon film (6.35 pm in thickness) accord-

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ing to a method developed by Trissl and Montal [9] (similar to the Langmuir- Blodgett technique), then the B2' component is suppressed because the extracellular surface of the purple membrane is pressing against the supporting Teflon film; only B 1 and B2 are observable. My colleague, Ting Okajima, further modified the Trissl-Montal method and formed multiple layers of oriented purple membranes on the Teflon film [ I I] . She further allowed the multilayered film to air-dry for at least four days. The resulting thin film assembly, when rehy- drated and mounted in a chamber, exhibits a photosignal that has almost no temperature dependence (see Sidebar 2). This latter signal is consistently in quantitative agreement with the equivalent circuit. For this and other reasons, we identify this signal as the pure B 1 component. This signal can be superimposed, after normalization, with the com- puted signal based on the equivalent circuit. A couple of comments are in order. First, all the input parameters are determined by experimental measurements, and there is no free parameter to adjust. Second, although the signal relaxes in two exponential time constants, deconvolution allows for the re- covery of a single exponential relaxation time constant T ~ , 12.3 k 0.7 p, that is inde- pendent of the access impedance or the Tef- lon film thickness [I I ] .

We have recently developed a method to isolate a pure B2 component [36], but we have not yet carried out a detailed equivalent circuit analysis for this component. How- ever, the behavior of B2 is drastically differ-

81

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IEEE ENGINEERING IN MEDICINE AND BIOLOGY Februory/Morch 1994

-

ent from that of B l . Many experimental parameters, such as pH, ionic strength, and proton/deuterium ion exchange, affect B2 exclusively but not B1 [33]. These clear-cut differences suggest that our signal decomposition is consistent with separation of the underlying molecular processes. These quantitative and semiquantitative results, when taken together, strongly suggest that the B2 component is almost certainly generated by the interfacial charge (proton) transfer mechanism. In this regard, we have previously studied a model membrane system with a pure photosignal component aris- ing from a pulsed light induced interfacial electron transfer under a pseudo-first order regime [28,30]. We have shown that such a signal is indeed in agreement with the equivalent circuit, and the second order rate constant of the reverse interfacial electron transfer (charge recombination) can be determined from such an analysis.

Demonstration ofthe existence ofthe B2’ component requires the use of a genuine BLM as the supporting matrix for the oriented bR molecules. This can be accomplished by means of either the method developed by Dancshizy and Karvaly [37] or the method developed by Drachev, et al. [38]. However, differentiation between B2 (intracellular proton uptake) and B2’ (extracellular proton release) may be trouble- some because both components are generated by the interfacial proton transfer mechanism, and hence will exhibit the same polarity and perhaps even similar pH dependence. The differentiation between B2 and B2’ can be achieved, however, on the basis of kinetic analysis. Referring to the scheme of coupled interfacial proton transfer reaction, we suspect that the transmembrane coupling may be slower than the interfacial processes of proton binding and release. If so, the two interfacial processes will become essentially chemically decou- pled during the time course of interfacial charge recombination. This consideration is formulated as the concept of local reaction conditions [39]. Interfacial proton transfer processes at each membrane surface will be sensitive to the pH in the adjacent aqueous phase but not to the pH at the opposite side of the membrane. Thus, if the intracellular pH and the extracellular pH are varied independently, B2 and B2‘may be differentiated. In Fig. 7, it is evident that the negative peak of the photosignal can be reversibly inhibited by lowering the intracellular pH, as expected from the known behavior of the B2 component. If, however, the extracellular pH is lowered while the intracellular pH remains low, a negative peak with a faster decay appears instead. We interpret this lat-

Februory/Morth 1994

~~ ~ ~

Cytoplasm Extracellular

SIGNAL 1 PH 7 PH 7

PH 0 PH 7 SIGNAL 2

PH 0 SIGNAL 3

8 a I I I

0 200 400 600 aoo 1001 TIME (MICROSEC)

The “differential” experiment showing the existence of two components of photosignals, due to proton uptake and release, respectively, at the two membrane-water interfaces. See text for explanation. Reproduced with permission from Ref. [33]

ter negative peak as the B2‘ component because of its extracellular origin and because of the pH dependence that is opposite to that of the B2 component: low pH enhances rather than inhibits B2’.

Lessons Learned from the Equivalent Circuit Analysis

The analysis presented above constitutes a major departure from the conventional practice of decomposing a pulsed light induced relaxation signal into a number of exponential components by curve fitting. It is readily seen that the conventional approach is incompatible with both the condition of a zero time-integral and the fundamental interpretation of the fast photoelectric signal as charge separation and re- combinat ion. A single exponent ia l component of the photocurrent can never satisfy Eq. I . meaning that the separated charges never recombine. Therefore, the conventional approach violates the definition of the capacitative (AC) photocurrent that it intends to describe.

Despite the controversial nature of the topic, our line of thinking and our interpretation of the ERP-like photosignals are actually in conformity with conventional practices in electrical engineering. For example, the effect of varying the access impedance is not surpr is ing. Many microelectronic devices are configured as thin films. The performance of a signal generator so configured is expected to be affected by different external loading conditions. Since every signal generator has a finite and non-zero source impedance, the signal waveform ought to vary while the


loading condition changes from an open circuit (no load) condition to a short-circuit (maximum load) condition. This obvious effect, while responsible for the variability of reported kinetic data in bR thin films, has not been generally recognized in the community of bacteriorhodopsin research until fairly recently. Using the Langmuir-Blodgett technique to deposit oriented bR on IT0 (indium tin oxide) electrodes, Ikonen, et al. [40], have correlated the variations of decay time constants of the fast photosignal to the changes of the loading resistance.

As a corollary, variations in the thin film structures are expected to affect the relaxation time course. For example, using Teflon films of different thickness to reconstitute bR. we observed differences in the apparent relaxation time coursec [41]. Yet, when the measured signals were de-convoluted in ac- cordance with our equivalent circuit, the same first order charge recombination time constant was obtained regardless of the thickness of the Teflon film. This result demonstrated that the deconvoluted time constant is more fundamental than the apparent signal relaxation time.

Our results and interpretation also demonstrated that there is no fundamental differ- ence between bioelectric signals generated by bR and inanimate electric signals generated in conventional microelectronic devices: they appear to obey the same physical laws. There is, however, one important dif- ference: it is possible to modulate the bioelectric signal by virtue of its chemistry. In the section below, we shall reinforce this claim by demonstrating that standard chemi-

83

Sidebar 3 ‘Apparent Paradox’

Wld Type bR Thin Film

-20 -I

84

&I -20 0 20 40 60 80 100 120 140 160 180 200

TIME (MICROSEC)

(c)

cal kinetic analysis has a place in the analysis of the AC photoelectric effect.

Resolving the Apparent Lack of pH Dependence of the R2 Component

The peril of interpreting open-circuit photovoltage data without regard to the vari- ation of the access impedance is further ex- emplified by the elusive pH dependence expected of an interfacial proton transfer reaction that exists in bR membranes, but which has not been evident in open-circuit data [9]. This same consideration also ap- plies to rhodopsin.

It has been known for a long time that the R2 component of the ERP appears and decays with the same time course as the forma-

tion and disappearance of metarhodopsin I1 from its precursor, metarhodopsin I 1421. It is also known that this reaction in the rhodopsin photobleaching sequence involves the net uptake of a proton from the aqueous phase [43]. These known facts are actually consistent with the assignment of R2 to an interfacial proton transfer mechanism because the net uptake of one proton from the adjacent aqueous phase takes place during the metarhodopsin I + I1 reaction. Such a mechanism had been considered by Cone [44] and by Ostroy [45] but was later aban- doned presumably because the expected pH dependence of the R2 component could not be demonstrated experimentally. Thus, the conventional wisdom has attributed the ERP


solely to the oriented dipole mechanism. For similar reasons, the ERP-like signal from reconstituted bR membranes has also been attributed to this same mechanism.

This “apparent paradox” can be readily reconciled by means of the present analysis. Referring to Fig. 6, the measured signal relaxation time courses do not reflect the in- trinsic molecular kinetics except under a strictly short circuit condition. Figure 6 also indicates that under open circuit conditions, a single charge recombination process will appear as two exponential decays: the fast decay time constant is about half of the true relaxation time constant, and the slow decay time constant is equal to the RC relaxation time constant [20]. A survey in the literature

February/Morch 1994

reveals that the ERP data were almost exclusively recorded under open circuit conditions, and most reported the relaxation time course in the range from milliseconds to seconds [27]. Apparently, the fast decay constant, which takes place in the microsecond range, was not recorded because of limited time resolution. It is understandable that the slow decay component, which reflects nothing but the RC relaxation of the membrane, does not reveal a pH dependence, as expected by kinetic analysis, except at ex- tremely high or low pH. While Trissl attributed the latter effect to the existence of pKa of an ill-defined molecular process [32], we attributed the effect to an inappropriate procedure of signal decomposition, inherent with the conventional approach (see Side- bar 3).

Concept of Intelligent Materials The “differential” experiment described

in an earlier section was motivated by a consideration of the law of mass action (see Sidebar 4). However, the result of the “differential” experiment runs diametrically op- posi te to the predict ion. A s imple consideration of the law of mass action pre- dicts the following. Prolonged proton pumping by light from the intracellular space to the extracellular space will lead to an increase of the intracellular pH and a decrease of the extracellular pH. The resultant pH changes at both sides of the membrane will enhance the reverse reactions in those do- mains. Thus, the intracellular proton uptake and the extracellular proton release will be retarded. Therefore, the proton pumping will eventually become less efficient; the proton concentration gradient generated by the pump will eventually exert enough “back- pressure” to compromise the pumping ac-

tion. In other words, the increase in intracellular pH is expected to speed up the back reaction (proton re-release) and will some- what diminish the B2 amplitude. and likewise, the decreased extracellular pH is expected to diminish the B2’ amplitude.

The disagreement between prediction and observation forced us to consider another factor in determining the reaction rate, namely, the binding constant of the proton binding site. We thus reached the conclusion that the pKas of the proton binding sites are themselves pH-dependent. In other words, there exists a cooperative interaction among various proton binding sites, some of which may or may not be directly on the proton transport path. By increasing the value of the ?’KO of the intracellular proton binding site as the local pH increases, the equilibrium is shifted to the right in Fig. 3B. Similarly, by decreasing the value of pKO of the extracellular proton binding site as the local pH decreases, the equilibrium is also shifted in favor of the forward proton transfer. This built-in pH-dependentpKa effect has an obvious survival value and thus a behavior that fits the definition of an intelligent materials [46]. According to the Science and Technol- ogy Agency of Japan, an intelligent material is one that has the “ability to respond to environmental conditions intelligently and manifest their functions” [47].

Reverse Engineering the Bacterial Re- action Center and Bacteriorhodopsin

Some investigators believed that the pre- requisite for bR to work as a solar energy converter was to have at least one step of proton transfer that was irreversible. But most charge transfer reactions are accompa- nied by reverse reactions. In fact. the existence of reverse reactions does not prevent

solar energy conversion from proceeding. A simple kinetic analysis indicates that the minimum requirement for coupled consecutive charge transfer reactions along a transmembrane pathway to work as a solar energy converter is that one of the forward charge transfer steps be driven by light. Experimen- tal proof can be provided by a simple model membrane that consists of a membrane- bound and mobile electron donor in the membrane phase, and a transmembrane re- dox gradient formed by an asymmetric distribution of water-soluble electron acceptor, potassium ferricyanide, and electron donor, potassium ferrocyanide. Light causes electrons to be ejected from the membrane bound pigment, magnesium porphyrin, to the aqueous electron acceptor. Continuous illumination allows electrons to be transported from the donor-rich side of the aqueous phase to the acceptor-rich side. We have shown that coupling of the two interfacial electron transfer reactions is mediated by the transmembrane diffusion of the mobile pigment and its oxidized species, magnesium porphyrin monocation [28]. However. the prominent reverse electron transfer reaction does compromise the efficiency of such an electron pump.

Thus, minimizing the impact of charge recombination is one of the primary goals in the research of artificial solar energy conversion. The pH-dependentp& of bR is merely one of Nature’s ingenious designs for this purpose. It is instructive to examine how the photosynthetic reaction center of Rhodop- seudomonas viridis manages to minimize the reverse charge transfer (charge recombination).

Superficially, there is very little in common between the bacterial reaction center and bacteriorhodopsin. The structures and

plication of the Law ansfer Reaction

ent revealed just the oppo

Februory/Morth 1994 IEEE ENGINEERING I N MEDICINE AND BIOLOGY 85

100

50

- E 3 0 1- U

-50

-1 00

0

-1 00 Y - E 3 (Jj -200 a

-300

100

- E

2- 2 50

0

N ii reaction coordinate

(4

reaction coordinate

(b)

reaction coordinate

(c)

8. Enthalpy (a), entropy (b), and free energy (c) changes relative to bacteriorhodopsin during the photocycle. The greatest free energy and entropy changes occur at thc transition from M I to M?. (Reproduced with permission from Ref. [7]).

the chemical constituents are quite different. However, similarities begin to emerge if one examines the two photosynthetic apparatus at the level of reaction schemes. As shown

in Fig. 3, both schemes can be characterized as coupled consecutive charge transfer reactions. In bR, the transmembrane proton pathway arises from a series of consecutive

proton binding sites provided by amino acid side chains, such as carboxylic groups of aspartic acid or glutamic acid, or quartemary ammonium groups of arginine or lysine. In the case of the bacterial reaction center, it is electrons that are being transported across the membrane; the various prosthetic groups form an electron path known as the electron transport chain. While light-induced charge separation in the form of electron-hole pairs transports electrons from the periplasmic (extracellular) side to the intracellular side of the membrane, the net result is a transport of protons in the opposite direction.

The bacterial reaction center as an intelligent supramolecular structure is best illus- trated by Kuhn’s analysis [48]. He found the spatial positioning of prosthetic groups within the reaction center to be optimal for an efficient forward electron transfer and for a diminished reverse electron transfer. Spe- cifically, the goal is to separate the electron from the hole as fast and as far away as possible. Since electron tunneling can take place for only a limited distance (about 10- 20 A), the supramolecular structure evolves to include three highly conjugated molecules (two bacteriochlorophyll and one bacteriopheophytin molecules). One way to enhance the forward reaction and to retard the reverse reaction i s to increase the differ- ence of free energy, i.e., to make AG more negative. However, by doing so, each and every step of electron transfers would incur an unacceptable amount of energy loss. Na- ture’s compromise is to make most of these steps highly reversible, i.e., negative but small AG. It is only after the electron has been separated from the primary donor, the “special pair,” by about 30 8, that a decrease of energy level of 0.4 eV, via vibronic relaxation of quinone A-, is allowed to take place so as to minimize charge recombination. An additional factor further retards charge recombination: the “special pair,” the monomer bacteriochlorophyll, and the bacteriopheophytin are arranged in a curved, “banana shaped” extended n-electron system. As a consequence, charge recombination by direct electron tunneling is forced to go through the o-portion of the protein moi- ety (L subunit). The alternative route of charge recombination, via the extended 7 ~ - system, must now face an “uphill” reaction due to the 0.4 eV energy loss during forward reaction, mentioned above. A comparison with the thermodynamic data recently reported by V6ro and Lanyi [7] reveals a striking resemblance at the energetic level. As shown in Fig. 8, most of the transition between various photointermediates incurs little loss of energy, with the exception of the Mi to M2 reaction, which i s the highly exo-

86 IEEE ENGINEERING I N MEDKINE AND BIOLOGY Februory/Morrh 1994

thermic. Thus, Nature seems to use the same strategy but different physical constructs to achieve the same objective.

Reverse Engineering Visual Rhodopsin and Bacteriorhodopsin

The switching step of the M I to M2 reaction in bR is reminiscent of the metarhodopsin I to metarhodopsin I1 reaction in the photobleaching sequence of visual rhodopsin. The similarity is more than superficial. Figure 8 reveals that the change of free energy during the Mi to M2 reaction is pre- dominantly entropic. In other words, there is a significant conformational change. As for visual rhodopsin, the metarhodopsin I to metarhodopsin I1 reaction also involves a dramatic conformational change, causing several sulfhydryl (SH) groups to be exposed to the aqueous phase and the net binding of one proton per rhodopsin molecule [43]. It is now well understood that the metarhodopsin I to metarhodopsin I1 reaction is a crucial step in visual transduction: metarhodopsin binds and act ivates transducin (a G-protein) 1491.

Activation of transducin is the initiation of the so-called cyclic GMP cascade, which sequentially leads to activation of phos- phodiesterase and to hydrolysis of cyclic GMP 1501. Cyclic GMP is the ligand of the

February/Marth 1994

sodium ion channel that keeps it open. Therefore. hydrolysis of cyclic GMP leads to closure of sodium ion channels and diminution of the massive sodium ion dark current that enters the outer segment in the absence of light [51]. Diminution of this dark current constitutes the “excitation” of the photoreceptor and the late receptor potential. Between the absorption of a photon by rhodopsin and the onset of the late receptor potential, there is a 100,000 fold increase of energy, and a time interval of 1.7 ms. In other words, the ERP and the LRP take place at the opposite ends of this time interval. In addition, the ERP is a linear function of the stimulating light, whereas the LRP has a highly nonlinear light dependence. One wonders whether there is any mechanistic linkage between the two electric signals.

Recalling that the R2 component of the ERP appears at the same time as metarhodopsin 11, and that the metarhodopsin I binds a proton to become metarhodopsin 11, we have hypothesized that R2 is generated by an interfacial proton transfer mechanism and we have predicted the appearance of a positive surface potential when metarhodopsin I1 forms [27]. This prediction was verified experimentally by Cafiso and Hubbell 1521, who used a spin probe to detect the surface potential.


As revealed by a simple Gouy-Chapman analysis, the appearance of a surface potential generates at the same time an intense but localized electric field (see Sidebar 5) . The transmembrane potential profile indicates that the surface potential must drop to zero in the remote region of the bulk aqueous phase. But the remote region is only at a distance of the Debye length away from the membrane surface (no more than 1 A in a physiological solution). Thus, a modest surface potential of SO mV is translated into an interfacial electric field of S X 1 O5 kV/m. For comparison, a transmembrane diffusion potential of 60 mV generates an intramem- brane electric field of only lo4 kV/m.

Furthermore, this interfacial electric field is switched on with a rise-time limited only by the rise-time of the surface potential because the ionic cloud in the diffuse double layer relaxes in about 0.5 ns in 0.1 M NaCl or about 0.05 ns in 1 M NaC1. Thus for all practical purposes, the interfacial electric field rises with the formation of metarhodopsin I1 or the appearance of R2. An intense but localized electric field provides a good switching mechanism for visual transduction. However, in order for a surface potential based switching mechanism to work for visual transduction, the proton binding must take place at the cytoplasmic side, i.e.. at the

a i

I I U

Electrolyte Bilayer

(a1

Electrolyte

Time

(b)

9. (a) An experimental prototype showing photogating of an ionic current in an artificial BLM. The BLM contains magnesium octaethylporphyrin, which is lipid-soluble (3.6 rnM). The aqueous phases contain the electron acceptor, anthraquinonesulfonate, in equal concentrations on both sides (0.1 mM). Tetra- phenyl borate ions, B-, are partitioned into the membrane at the region of polar head groups of the lipid, the so-called boundary region. Photoactivation of rnagne- sium octaethylporphprin generates two symmetrical positive surface potentials (or rather, boundary potentials), which increases the surface concentration of B-. (b) The photocurrent induced by photoexcitation from a laser pulse delivered at the time indicated by the arrows. The upper and lower traces represent ionic currents when a +40 mV and a -40 mV potential are applied across the membrane, respectively. (Reproduced with permission from Ref. [57]).

C-terminus side of rhodopsin, where binding and activation of transducin takes place. The test of this crucial criterion was provided by the work of Ostrovsky and his colleagues [54].

Using a pH indicator dye and using an affinity chromatographic method to prepare two kinds of rhodopsin containing phospholipid vesicles: one with the correct orientation of rhodopsin and the other with the

inverted orientation, Shevchenko. et al. [53, 541 found that the proton binding during metarhodopsin I to metarhodopsin I1 reaction takes place at the cytoplasmic side. Fur- thermore. there is no proton release at the N-terminus side of rhodopsin.

The finding of Ostrovsky's group is consistent with a surface-potential-based switching mechanism for two reasons. First, the positive surface potential appears in the

88 IEEE ENGINEERING IN MEDICINE AND BIOLOGY

right place and at the right time. Second, the fact that the interfacial proton transfer occurs at both membrane surfaces in bR but only at the cytoplasmic surface in rhodopsin is consistent with their respective roles of light energy converter and light signal transduction. Cytoplasmic proton uptake and extracellular proton release are two obligatory steps in proton translocation. In contrast, the presence of a proton uptake mechanism without a proton release mechanism on the opposite side of the visual membrane pre- cludes this membrane's possible function as a proton pump, and therefore, as a light energy converter. But Nature never meant to design the visual membrane as a light energy converter, anyway. With the built-in ampli- fication mechanism outlined above, the photon acts merely as a trigger to unleash the energy previously stored as a transmembrane sodium ion gradient. As explained above, a surface potential-based trigger mechanism suits the purpose well. In addition, a surface potential-based de-activation mechanism may also be involved in visual transduction. This mechanism may be im- plemented by light-induced phosphorylation of rhodopsin at its cytoplasmic domain. The phosphorylation results in the appearance of a striking negative surface potential, because there are nine amino acids residues (threonine or serine) that are photophospho- rylated [55] . We further point out that there are no comparable events in bR.

A surface-potential based mechanism is presently speculative, but the work of Drain, et al. [56, 571 demonstrated that such a mechanism is experimentally realistic. These investigators studied the ionic conduction across an artificial lipid bilayer membrane when lipid-soluble tetraphenyl- borate ions are under the influence of an applied transmembrane potential (Fig. 9). The ionic conductivity is, in part, dependent on the (local) surface concentration of tetraphenyl borate ions. A switching mechanism similar to which we proposed for rhodopsin was set-upin themembrane by light-induced electron transfer from the membrane-bound magnesium porphyrin to an aqueous electron acceptor that resides in the two aqueous phases in equal concentrations. A positive surface potential appears at each membrane surface upon illumination with visible light, but no photovoltaic effect can be detected because of the cancellation of two equal interfacial electron transfers with opposite polarities. The appearance of this positive surface potential coincides with the sudden increase of ionic conductivity, as monitored by the transmembrane current carried by tetraphenyl borate ions. If the lipid-soluble ion that carries the transmembrane current were positively charged, the light-induced

Februory/Morrh 1994

positive surface potential would lower its surface concentration. and consequently would cause a sudden reduction of ionic conductivity. This latter prediction was indeed verified by a similar experiment with tetraphenylphosphonium ions replacing tetraphenyl borate ions.

Bacteriorhodopsin as an Advanced Bioelectronic Material

Bacteriorhodopsin is an excellent advanced material for molecular electronics. Most prototype bR-based bioelectronic devices exploit the rapid rise-time of this material’s AC photoelectric signal. Simmeth and Rayfield [58] have demonstrated the rise-time of the B 1 component to be less than 5 ps. This measurement is still limited by instrumentation available for electrical re- cording and the true rise-time could be much faster. Thus, if an ultrafast response time of a bR-based device is important, it would be better to utilize its photochromic effect. because the time resolution for detection of optical signals is presently much better than for detection of electric signals [59]. There are two interesting applications of the AC photoelectric effect of bR that are not pri- marily based on fast response time.

Miyasaka, et al. [60] constructed amulti- pixel photosensor, as shown in Fig. 10. Each pixel consists of an oriented bR film attached to a transparent metal electrode and a counter electrode. The photocurrent output of each pixel is connected to a processing circuit made of conventional microelectronic components. A typical photocurrent response to the onset and the termination of continuous illumination. which is characteristic of an AC photoelectric signal, is shown in Fig. 10. Because the sensor re- sponds only to a change in the level of illumination, the multi-pixel sensor works as a motion detector.

Another example of devices based on the AC photoelectric effect of bR is provided by a cellular automaton network made of patches of bR, similar to the previous example [61]. However, this latter example uses a phenomenon known in the rhodopsin literature as the photoreversal potential [44]. Essentially, when a rhodopsin sample is pre- pared by a previous illumination so that most molecules are in the metarhodopsin state, a subsequent illumination. using light absorb- able by metarhodopsins, converts it back to rhodopsin, and the accompanying ERP has an opposite polarity. Likewise, illumination of a sample of bR enriched with the M state by a prior illumination generates an AC photoelectric signal with an opposite polarity. By manipulating a network of such patches, a rudimentary cellular automaton could be constructed.

Februory/Marth 1994

An interesting device which combines both the photochromic and AC photoelectric effects has been proposed by Birge [62]. Essentially. the local electric field generated by the B 1 component is utilized to manipu- late an optical dynamic random access mem- ory (RAM) made of bR.

Role of Protein Engineering If the photochromic effect of bR can be

manipulated by genetic engineering to fine- tune its performance 1631, can the photoelectric signal be similarly manipulated‘? To answer this question, we used a recently developed Halobacterium hulobium ex- pression system to produce bR mutants [64]. Shown in Fig. 1 1 are the AC photoelectric signals generated in Trissl-Montal films pre- pared from four different mutants (each with a single point mutation) 16.51. All four mutants exhibit distinct pH-dependent patterns of the AC photoelectric signal. Such point mutations have a dramatic effect on the B2 component. Mutant D2 12N is of particular interest to us from the component analysis point of view. because the B2 component is completely absent in the range of pH from about 6 to 1 I . This latter result lends addi-

10. A motion detector constructed with bacteriorhodopsin. (Reproduced with permission from Ref. [60]). (a) Cross section of a photocell shows that bacteriorhodopsin is immobilized on a transparent electrode; (1) SnO? transparent conductive layer; (2) purple membrane LB film (typically six to 10 layers); (3) aqueous electrolyte gel laxer (200 pm thick); (4) Au layer (< 1000 A) as counterelectrode; (5) Teflon ring spacer; (6) glass substrate. (b) An IT0 (indium tin oxide) electrode patterned with 64 pixels is used for image-sensing. PiFels of IT0 (2.5 mm by 2.5 mm, 1000 A thick transparent layer) are two dimensionally arrayed on a glass plate; each pixel has a separate wire leading to the four edge terminals along the sides for interfacing with an amplifier circuit. (c) The photocurrent generated in each pixel by a long rectangular light pulse of about 200 ms is shown along with the light pulse. The positive spike of the “on” response and the negative spike of the “off” response are characteristic of a high-pass filter to a long rectangular pulse of applied current (see also Fig. 6). The high-pass filter is formed by the irreducible equivalent circuit shown in Fig. 4. Here, R, must be set to infinity because of the lack of an electrodic reaction that converts a proton-mediated current to an electron-mediated current.

IEEE ENGINEERING IN MEDICINE AND RIDLOGY

tional credence to our component analysis. because one component is missing but the other is essentially intact [66]. The conventional approach based on exponential analysis gives results that are more complicated and which have no clear-cut effect on the individual components [67,68].

1 2 3 4

On Light Off

(4

8 9

P I , , , , , , , , , , -20

0 20 40 60 80 100 120 140 160 180 200

TIME (MICROSEC)

(4

20 r I I // pH3.5

-20' " " " " 1 '

0 20 40 60 80 100 120 140 160 180 200

TIME (MICROSEC)

-20 0 20 40 60 80 100 120 140 160 180 200

TIME (MICROSEC)

(4

20 E- 4

4 10

6 0

z F

[r 3

h- pH 0.7

0 20 40 60 80 100 120 140 160 180 200

TIME (MICROSEC)

(d)

11. pH dependence of photocurrents from mutant bacteriorhodopsin (a) D212N (aspartate to asparagine mutation at residue 212); (b) D115N; (c) D96N; (d) D85N. The inset in (a) shows superposition of records at pH 5.4,6.0,7.7,9.4, and 9.9, after normalization of the peak amplitudes. (Reproduced with permission from Ref. [65]).

Interface Problem Some investigators, who make bR de-

vices by attaching an oriented BR film to a transparent metal electrode, often wondered why the DC photocurrent could not be detected. In contrast, the DC photovoltage photocurrent is routinely observed in a reconstituted bR-BLM exposed to two aqueous phases. The answer lies in the conduction mechanism of the DC photocurrent in bR. Unlike the bacterial reaction center, the bR photocurrent is carried exclusively by protons. Yet the metal electrode demands the conversion from a current carried by protons to one carried by electrons in the conduction band. Such a conversion mechanism (an electrodic reaction) is lacking in most bR-based devices. This is why the record shown in Fig. 10 exhibits only the AC

photoelectric signal. In contrast, Greenbaum [69] succeeded in interfacing the photocurrent from the photosynthetic reaction center of a blue green alga by precipitating colloid platinum onto the reaction center. He was able to record a DC photocurrent from such a preparation.

We have previously proposed a possible solution to converting the DC protonic current in bR into a conventional current carried by electrons, a method based on reverse engineering of the photosynthetic reaction center and mitochondria [70]. We point out that these organelles utilize a quinone or a quinone-like compound to convert a transmembrane electron movement into a transmembrane proton movement. We need only engineer this type of molecule (known as a quinoid compound) and make it suitable for

the reverse reactions, helping to convert a proton-mediated current to an electron-mediated current (Cf Fig. 3A).

Physiological Significance of the RI Component of the ERP and Its Analog Signal in Bacteriorhodopsin

So far, we have not commented on the physiological significance of the R1 component of the ERP nor on the B1 component of its bR counterpart. Does the B 1 component represent a proton movement? If it does, then, as Keszthelyi [71] pointed out, the proton moves backward after photon absorption and the forward motion occurs later. This consideration led Keszthelyi to propose a slingshot model. Photon absorption causes the proton to move backward

90 IEEE ENGINEERING IN MEDICINE AND BIOLOGY February/Morth 1994

first. The uncompensated negative charge drags the positive charge (proton), which passes by in moving to the surface. This interpretation of the B 1 component in terms of proton movements, however, is not supported by our experimental analysis. While the time course of the B2 component is significantly, but reversibly, affected by replacing D20 for water with no discernible delay, there is no detectable effect on the B 1 component after an overnight exchange of DzO and water: the charge recombination time for B1 remains unchanged (within 5 percent) [ 111. Based on a theoretical calculation, Birge 1621 is in favor of the interpretation of B 1 as a light-induced electric dipole moment accompanying the photoisomeriza- tion of the chromophore.

While the problem is not completely set- tled, the following interpretation seems rea- sonable and plausible. Since proton movement is considerably slower than electron movement, Nature’s strategy is to convert the photon energy into electrostatic energy, temporarily, as charge separation manifested by the B 1 component [72]. This electrostatic energy is then utilized subsequently to drive conformational changes that eventually switch the molecule from the MI to the M2 state of bR, or from metarhodopsin I to metarhodopsin 11. In light of this new interpretation, the slingshot model is still correct if it is interpreted metaphorically rather than literally.

Concluding Remarks The photovoltaic effect is a prominent

physical event exhibited by several photobiological membranes. Although these membranes have diverse physiological functions, the photovoltaic effect is the manifestation of their common but fundamental characteristic, namely light-induced rapid charge separation. The effect is macroscopically observable because the photopigments in these membranes maintain a specific orientation in the biological membranes. For example, the C- t e rminus in both bacteriorhodopsin and rhodopsin is located intracellularly. The specific orientation is also evident from the functional point of view; bacteriorhodopsin and the bacterial reaction center perform vectorial transport of protons, resulting in alkalinization of the cytosol. The net proton transport results in a DC photovoltage or a DC photocurrent that has a distinct polarity consistent with the direction of proton transport. These charge transport processes are not accomplished in a single step, because the membrane thickness exceeds the limit of distance covered by electron tunneling. Therefore, charge transport is divided into several steps, each cov- ering a short distance, characterized by a

general scheme of coupled consecutive charge transfer reactions. These charge transfer reactions are inherently reversible, and a fraction of separated charges recombine, resulting in no net charge transport. But this charge separation and recombination can also be detected macroscopically as a transient capacitative photovoltage or photocurrent (AC photoelectric effect).

The AC photoelectric effect is a controversial subject. The conventional approach is difficult with regard to molecular interpretation. The present article describes an alternative approach to signal decomposition, starting from a simple but physically realistic molecular model. By combining electrochemistry and electrophysiology in the analysis, we are able to derive an equivalent circuit that links the experimentally measured macroscopic signal with the underlying molecular kinetics. In our analysis, electron transfer and proton transfer are treated as mechanistically equivalent, and the equivalent circuit model is generally applicable to different types of vectorial charge transfer in membranes or in supported thin films with embedded pigments [20]. Thus, the interfacial proton transfer mechanism can be gen- eralized to the interfacial charge transfer mechanism, and is applicable to interfacial electron transfer [28] and to interfacial chloride ion transfer [73]. Our approach offers a coherent and consistent interpretation that is not dependent upon any particular photochemical schemes. In contrast, the conventional approach requires that a specific model be concocted for each particular experimental system, and that the model be modified whenever the photochemical scheme is refined and modified.

The fundamental principle of the AC photoelectric effect was developed with an artificial membrane that contains magnesium porphyrin. Nonetheless, we chose to illustrate the principle and the methodology with bR thin films, because bR shares parts of the main features of both the visual membrane and the photosynthetic membrane. This duality allows us to explore Nature’s secret design principles by means of reverse engineering. Specifically, bacteriorhodopsin, as a stripped down version of photosynthesis, provides a clearer view of what features are essential, and what others are secondary, for a photon energy converter. This insight is not always feasible with the more complicated chlorophyll-based photosynthetic reaction center. By analyzing the energetics, we witness astonishing similarity at a more fundamental level, which is not obvious from a superficial comparison based on structural information alone.

The striking similarity of the AC photoelectric signal from bR and from rhodopsin

also provides insights about the functional differences between a photon energy converter and a photon signal transducer. We propose a surface potential-based trigger mechanism for visual transduction. While the proposed mechanism remains to be tested experimentally, the principle actually worked with an artificially constructed system lacking any structural similarity. The idea of extracting a biological principle and implementing the design with an artificial system by means of either biomolecules or synthetic molecules is the essence of biomimetic science.

The astonishing design features in biom- achinery are thought to be products of trials and errors by Nature through billions of years of evolution. This thinking inspires both the emergence of biomimetic science and the concept of intelligent materials. Our survey of various photobiological systems reveals that Nature may design an intelligent molecule, such as bR, or an intelligent supramolecular structure, such as the bacterial reaction center, with different structural lay- outs but with the same physical principles. Comparison of the ERP and the ERP-like signal in bR also suggests that Nature designs molecular functionality in modular forms, and that the same molecular functionality, such as proton binding, may be configured in similar molecules for different purposes.

In this article, we chose to illustrate the principle underlying the AC photoelectric effect with experimental data obtained from various types of bR thin films. The unusual attention bestowed upon bR is justified by the following consideration. Bacteriorho- dopsin possesses exceptional stability [74, 751 that is suitable for device construction, and was the building block used in a number of successful prototypes. While skeptics may have a legitimate doubt whether bR- based devices will ever become commer- c ia l ly viable , bR related molecular electronics research provides an excellent proving ground to solve various technical problems that may be transferred to other types of molecular devices.

With regard to the use of biomolecules as advanced materials, we have previously pointed out that materials intelligence must be evaluated along with the intended function of the molecular device. What is considered intelligent in its native function may not be sufficiently intelligent for the specific application because Nature may have opti- mized the biomolecule on the basis of a different set of criteria. Thus, modifications of the molecular structure are often required for the intended applications. Fortunately, the advent of modern recombinant DNA technology makes it possible to conduct ar-

Februory/Morch 1994 IEEE ENGINEERING IN MEDICINE AND BIOLOGY 91

tificial breeding of intelligent biomolecules by forcibly providing the assembly code to bacteria, and thus greatly shortening the time required for fine-tuning the molecular design.

Acknowledgements The author thanks Richard Needleman,

Michelle Petrak, and Klaus-Peter Zauner for reading the manuscripts. The author ac- knowledges the contribution of his collabo- rators: Janos Lanyi, Lowell McCoy, Mauricio Montal. Richard Needleman. The cited experimental work of Man Chang, Al- bert Duschl, Filbert Hong, Sherie Michaile. Baofu Ni, and Ting Okajima are also ac- knowledged. The experimental work was in part supported by a contract from the Office of Naval Research (N00014-87-K-0047) and a contract from the Naval Surface War- fare Center (N60921-9 1 -M-G76 1 ).

Felix T . Hong was born in Chonghua, Taiwan. He received his medical training at the National T a i w a n U n i v e r s i t y School of Medicine, Taipei, Taiwan (M.D., 1966). He received a Ph.D. in Biophysics un-

der the supervision of Professor David Mauzerall from the Rockefeller Univer- sity, New York, NY (1973).

From June 1973 through July 1977, he was Assistant Professor at the Rockefeller University. From August 1977 through Au- gust 198 1, he was Associate Professor in the Department of Physiology. Wayne State University School of Medicine, Detroit. He has been Professor of Physiology there since September 1981.

His research interest centers around membrane biophysics and photobiology. He has proposed, in his early work, diamagnetic anisotropy as the mechanism for the mag- neto-orientation effect of isolated rod outer segments and a unicellular alga Chlorella. Subsequently, he concentrated his research effort on the development of a comprehen- sive mathematical and molecular model and methodology for investigating the photoelectric effect in a model system of artificial black lipid membranes that contains magnesium porphyrin.

Since he joined Wayne State University, he turned his research effort to the light-induced molecular processes in bacteriorhodopsin, and other related biopigments. He also extended his research interest to include molecular electronics, biomolecular computing and artificial solar energy conversion.

He established the Molecular Electronics Track for the IEEE Engineering in Medicine

and Biology Society in 1988, and served as the Track Chair for four consecutive terms until he stepped down in 1992. He has also organized molecular electronics symposia for the Fine Particle Society (1988), and the Bioelectrochemical Society (1992).

He currently serves on the editorial board of the following journals: Advanced Materi- als for Optics and Electronics, Applied Bio- chemistry and Biotechnology, Bioelectrochemistry and Bioenergetics, and the Newsletter of the International Society for Molecular Electronics and BioCom- puting.

He was the recipient of the Victor K. LaMer Award (American Chemical Society Division of Colloid and Surface Science: 1976).

His address for correspondence is: Department of Physiology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201. Fax: (313) 577-5494; e-mail: [email protected]

References I . Brown KT, Murakami M: A new receptor potential of the monkey retina with no detectable latency. Nature (London) 201 :626-628, 1964. 2. Brown KT: The electroretinogram: its components and their origins. Vision Res 8:633-677, 1968. 3. Tien, HT: Light-induced phenomena in black lipid membranes constituted from photosynthetic pigments. Nuture (London) 219272.274. 1968. 4. Stoeckenius W: The purple membrane of salt- loving bacteria. Sci Am 234(6):38-46, 1976. 5 , Stoeckenius W: The rhodopsin-like pigments of halobacteria: light-energy and signal transducers in an archaebacterium. Trends Bio- chem Sri 10:483-486. 1985. 6. Henderson R, Baldwin JM, Ceska TA, Zem- lin F, Beckmann E, Downing KH: Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:899-929, 1990. 7. Varo G, Lanyi JK: Thermodynamics and energy coupling in the bacteriorhodospin photocycle. Biochenzisfry (Wash.) 30:5016-5022. 1991. 8. Drachev LA, Jasaitis AA, Kaulen AD, Kon- drashin AA, Liberman EA, et al: Direct measurement of electric current generation by cytochrome oxidase, H+-ATPase and bacteriorhodopsin. Nature (London) 249:321-324, 1974. 9. Trissl H-W, Montal M: Electrical demonstration of rapid light-induced conformational changes in bacteriorhodopsin. Nature (London) 26665.5-6.57. 1977. 10. Hong FT. Montal M: Bacteriorhodopsin in model membranes: a new component of the displacement photocurrent in the microsecond time scale. Biophp J 251465472. 1979. 1 I . Okajima TL, Hong FT: Kinetic analysis of displacement photocurrents elicited in two types of bacteriorhodopsin model membranes. Biophys J 50:901-912, 1986. 12. Hong FT, Okajima TL: Electrical double layers in pigment-containing biomembranes. In:

Blank M (Ed): Electrical Double Layers in Biol- ogy, Plenum Press, New York, pp 129.147. 1986. 13. Trissl H-W: Photoelectric measurements of purple membranes. Photochem P hotohiol5 1 :793- 818, 1990. 14. Fahr A, Lauger P, Bamberg E: Photocurrent kinetics of purple-membrane sheets bound to pla- nar bilayer membranes. .I Membrane Biol 60:s 1- 62, 1981. 15. Der A, Hargittai P, Simon J: Time-resolved photoelectric and absorption signals from oriented purple membranes immobilized in gel. J Biochem Biophys Mrtkods 10:29.5-300. 1985. 16. Keszthelyi L, Ormos P: Electric signals associated with the photocycle of bacteriorhodopsin. FEBS Lett 109:189-193, 1980. 17. Rayfield GW: Events in proton pumping by bacteriorhodopsin. Biophys J 41:109-117, 1983. 18. Trissl H-W: Charge displacements in purple membranes adsorbed to a heptane/water interface: evidence for a primary charge separation in bac- teriorhodpsin. Biochim Bioph)>.s Acta 737327- 331, 1983. 19. Drachev LA, Kaulen AD, Khitrina LV, Skulachev VP: Fast stages of photoelectric processes in biological membranes, 1. bacteriorhodopsin. Eur J Biochem 117:461-470, 1981. 20. Hong FT: Displacement photocurrents in pigment-containing biomembranes: artificial and natural systems. In: Blank M (Ed), Bioelectro- clzemisrry: Ions, Surfacus, Memhrunes, ACS Ad- vances in Chemistry Ser No 188, American Chemical Society, Washington, DC. pp 21 1-237. 1980. 21. Andreasson L-E, Vannglrd T: Electron transport in photosystems I and 11. Annu Rev P /ant P hysiol and P /ant Mol B i d 39379-4 1 1, 1988. 22. Barber J: Photosynthetic electron transport in relation to thylakoid membrane composition and organization. P lant Cell Enitiron 6:3 I 1-322,1983. 23. Deisenhofer J, Michel H: The photosynthetic reaction center from the purple bacterium Rho- dopseudomonas viridis. Science ( W a s h . ) 24.5:1463-1473. 1989. 24. Hong FT: Retinal proteins in photovoltaic devices. In: Birge RR (Ed): Molecular and Bio- molecular Electronics, ACS Adv Chem Ser No 240, American Chemical Society. Washington. DC, in press. 2.5. Hong FT: Bacteriorhodopsin as an intelligent material. In: Wang C-Y, Chen C-T. Cheng C-K. Huang, Y-Y, Lin, F-H (Eds): Biomedical Engi- neering in the 21st Century. Center for Biomedical Engineering, National Taiwan University College of Medicine, Taipei, pp 85-9.5, 1990. 26. Hagins WA, McGaughy RE: Molecular and thermal origins of fast photoelectric effects in the squid retina. Science (Wash.) 157:813-816, 1967. 27. Hong FT: Mechanisms of generation of the early receptor potential revisited. Binelectrochem Bioenerg 5:425-455. 1978. 28. Hong FT: Charge transfer across pigmented bilayer lipid membrane and its interfaces. Photo- chem Photobiol24: 155-189, 1976. 29. Hong FT: Interfacial photochemistry of retinal proteins. Prog Surface Sri, in press. 30. Hong FT, Mauzerall D: Interfacial photore- actions and chemical capacitance in lipid bilayers. Pi-oc Nut AcadSci USA 71:1.564-1568. 1974.

91 IEEE ENGINEERING I N MEDICINE AND BIOLOGY Februory/Morth 1994

mailto:[email protected]

3 1. Hong FT: Electrochemical procewes in men- branes that contain bacteriorhodopsin. In: Blank M, Vodyanoy I (Eds): Binmemhrane Elecrro- chemistry, ACS Adv Chem Ser No 235. American Chemical Society, Washington. DC, in press. 32. Trissl H-W: Light-induced conformational changes in cattle rhodopsin as probed by measurements of the interface potential. Photoc,hem Pho-

33. Hong FT, Okajima TL: Rapid light-induced charge displacements in bacteriorhodopsin membranes: an electrochemical and electrophysiological study. In: Ebrey TG. Frauenfelder H, Honig B. Nakanishi K (Eds): Biophysicul Studies ofRetinal Proreins, University of Illinois Press, Urbana- Champaign. pp 188- 198. 1987. 34. Michaile S, Hong FT: Signal modulation via interfacial processes in molecular optoelectronic devices. Proc Annu I n t Conf I E E E EMBS, 11:1333-1335, 1989. 35. Michaile S, Hong FT: Component analysis of the fast photoelectric signal from model bacteriorhodopsin membranes: 1. effect of multi-layer stacking and prolonged drying. Bi{Je/KfITJc'hem Bioenerg, in press. 36. Hong FH, Hong FT: Analysis of the fast photoelectric signal generated by wild type bacteriorhodopsin: evidence for the polarity reversal of the B2 component at low pH. 19Y3 Meeting Int Soc. Molec Electron Bio-conzp (ISMEBCJ, Gaith- ersburg, MD. Abstr p 30, 1993. 37. Dancshazy Z, Karvaly B: Incorporation of bacteriorhodopsin into a bilayer lipid membrane: a photoelectric-spectroscopic study. FEBS Le// 72136- 138, 1976. 38. Drachev LA, Kaulen AD, Skulachev VP: Time resolution of the intermediate steps in the bacteriorhodopsin-linked electrogenesis. FEBS Lett 87:161-167, 1978. 39. Hong FT: Effect of local conditions on het- erogeneous reactions in the bacteriorhodop%in membrane: an electrochemical view. .I Elec,f iw chem Soc 134:3044-3052. 1987. 40. Ikonen M, Sharonov AY, Tkachenko NV, Lemmetyinen H: The kinetics of charges in dry bacteriorhodopsin Langmuir-Blodgett films--an analysis and comparison of electrical and optical signals. Adv Mat Optics Elec,tron 2:2 I 1-220, 1993. 4 1 . Hong FT: The bacteriorhodopsin model membrane system as a prototype molecular computing element. BioSy.sfen7.S 19:223-236, 1986. 42. Cone RA, Pak WL: The early receptor potential. In: Loewenstein WR (Ed), Handbook ofSen- sorj Ph?;sio/o,Ty. I'ol 1 , P rincip/es of Receptof' Physiology, Springer, Berlin, pp 345-356. 1971. 43. Matthews RG, Hubbard R, Brown PK, Wald G: Tautomeric forms of metarhodopsin. J Gen Physiol47:215-240. 1963. 44. Cone RA: Early receptor potential: photore- versible charge displacement in rhodospin. S(.i- enr'e (Wash.) 155:1128-1 131. 1967. 45. Ostroy SE: Rhodopsin and the visual process. Biochinz BiophysAr,ta 4639-125, 1977. 46. Hong FT: Intelligent materials and intelligent

t<JhiCJl 29:579-588, 1979.

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microstructures in photobiology. Nanohiology I :39-60, 1992. 47. Takagi T (Ed): The Concept of Infelligent Materials and the Guidelines on R&D Promotion, Science and Technology Agency, Government of Japan, Tokyo, 1989. 48. Kuhn H: Electron transfer mechanism in the reaction center of photosynthetic bacteria. P l y Rei, A 34:3409-3425, 1986. 49. Emeis D, Kiihn H, Reichert J, Hofmann KP: Complex formation between metarhodopsin I1 and GTP-binding protein in bovine photoreceptor membranes leads to a shift ofthe photoproduct equilibrium. FEBS Lett 143:29-34, 1982. SO. Stryer L: Cyclic GMP cascade of vision. Annu Rev Neuro.sc.i 9:87-I 19, 1986. 5 I , Yau K-W, Baylor DA: Cyclic GMP-activated conductance of retinal photoreceptor cells. A I I ~ U

52. Cafiso DS, Hubbell WL: Light-induced interfacial potentials in photoreceptor membrane. Bioph?s .I 30243.264, 1980. 53. Shevchenko TF, Kalamkarov GR, Os- trovsky MA: The lack of Hf transfer across the photoreceptor membrane during rhodopsin pho- tolysis. Sensory Systems. USSR Acad Sci, in Rus- sian, I : l 17-126. 1987. 54. Ostrovsky MA: Animal rhodopsin as a photoelectric generator. In: Hong FT (Ed): Molei.ular

num Press, New York, pp 187-201, 1989. 55. Wilden U, Kiihn H: Light dependent phosphorylation of rhodopsin: number of phosphorylation sites. Riochenzistry 21:3014-3022, 1982. 56. Drain CM, Christensen B, Mauzerall D: Photogating of ionic currents across the lipid bilayer. PTO(, Annu Int ConflEEE EMBS 11:1336- 1336, 1989. 57. Drain CM, Mauzerall D: An example of a working molecular charge sensitive ion conduc- tor. Bioelectrockenz Bioenerg 24:263-268. 1990. 58. Simmeth R, Rayfield GW: Evidence that the photoelectric response of bacteriorhodopsin occurs in less than 5 picoseconds. Bic~phy .~ .I 57:1099-1101, 1990. 59. Mathies RA, Lin SW, Ames JB, Pollard WT: From femtoseconds to biology: mechanism of bacteriorhodopsin's light-driven proton pump. Am7rr Rev Biophys B i o p l q Chem 20:491-5 18. 1991. 60. Miyasaka T, Koyama K, ltoh I: Quantum conversion and image detection by a bacteriorhodopsin-based artificial photoreceptor. Science (Wash.) 255342-344. 1992. 61. Haronian D, Lewis A: Elements of a unique bacteriorhodopain neural network architecture. Appl Optics 30597-608, 1991. 62. Birge RR: Protein-based optical computing and memories. Computer (IEEE) 25( I 1):56-67. 1992. 63. Oesterhelt D, Brauchle C, Hampp N: Bac- teriorhodopsin: a biological material for information processing. Q Rev B i o p h ~ 4:425-478. 199 l . 64. Ni B, Chang M, Duschl A, Lanyi J, Needle-

Rev Neirrosci 12:289-327, 1989.

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IEEE ENGINEERING IN MEDICINE AND BIOLOGY

man R: An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halohium. Gene 90:169-172, 1990. 65. Hong FT, Hong FH, Needleman RB, Ni B, Chang M: Modifying the photoelectric behavior of bacteriorhodopsin by site-directed mutagene- sis: electrochemical and genetic engineering ap- proaches to molecular devices. In: Aviram A (Ed): Molecular Electronic.sScience and Technology, American Institute of Physics. New York, pp 204- 217, 1992. 66. Hong FH, Chang M, Ni B, Needleman RB, Hong FT: Component analysis of the fast photoelectric signal from model bacteriorhodopsin membranes: 3. Effect of the point mutation ASP212 + ASN212. Bioelei,trochern Bioenerg, in press. 67. Gergely C, Varo G: Charge motions in the D85N and D212N mutants of bacteriorhodopsin. In: Rigaud J-L (Ed). Structures and Funr,tions Retinal Proteins, Colleque INSERM, Vol 221, John Libbey Eurotext, Ltd, Montrouge, pp 193- 196, 1992. 68. Moltke S, Heyn MP, Krebs MP, Mol- laaghababa R, Khorana HG: Low pH photovoltage kinetics of bacteriorhodopsin with replacements of Asp-96. -85, -212 and Arg-82. In: Rigaud J-L (Ed), Structures andFunctions cfReti- nul Proteins, Colleque INSERM, Vol 221, John Libbey Eurotext, Ltd, Montrouge, pp 201 -204, 1992. 69. Greenbaum E: Platinized chloroplasts: a novel photocatalytic material. Sc,ieirce (Wush.) 230:1373-137.5, 1985. 70. Hong FT: Relevance of light-induced charge displacements in molecular electronics: design principles at the supramolecular level. .I Molec Electron 5:163-185, 1989. 7 1 . Keszthelyi L: Intramolecular charge shifts during the photoreaction cycle of bacteriorhodopsin. In: Bolis CL. Helmreich EJM. Passow H (Eds): Information uiid €ner,qy Transduction in Biological Mrnzhrunes, Alan R. Lisa, Inc. New York, pp51-71, 1984. 72. Birge RR, Cooper TM: Energy storage in the primary step of the photocycle of bacteriorhodopsin. Biophys J 4 2 6 - 6 9 . 1983. 73. Michaile S, Duschl A, Lanyi JK, Hong FT: Chloride ion modulation of the fast photoelectric signal in halorhodopsin thin films. Proc 12rhAnnu

74. Shen Y, Safinya CR, Liang KS, Ruppert AF, Rothschild KJ: Stabilization o of the membrane protein bacteriorhodopsin to 140 C in two- dimensional films. Nuturr (L<JII~oI?) 366:48-50, 1993. 75. Hong FH, Chang M, Ni 8, Needleman RB, Hong FT: Genetically modified bacteriorhodopsin as a bioelectronic material. In: Alper M, Bayley H, Kaplan D, Navia M (Eds): Biomolecrl- lor Materials bji D e s i ~ n , Mor Res Synip Prnc Vol 230, Materials Research Society. Pittsburgh, Pa., in prew.

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