Electrochemical Processes in Membranes That ... - ACS Publications

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Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch025

Electrochemical Processes in Membranes That Contain Bacteriorhodopsin Felix T. Hong Department of Physiology, Wayne State University, School of Medicine, Detroit, MI 48201

Pulsed-light illumination of reconstituted bacteriorhodopsin mem-branes elicits a fast photoelectric signal as a result of light-induced rapid charge separation. This signal is similar to the early receptor potential in a visual membrane. Nonelectrochemical interpretation of this type of signal reported in the literature has led to discrepancies in data reported by various laboratories and to conclusions that contra-dict established facts. This problem is addressed by applying the Gouy-Chapman diffuse double-layer theory to the electrokinetic pro-cess of rapid charge separation and recombination. This electrochemi-cal analysis leads to a universal equivalent circuit that offers a coherent interpretation of data, simple explanations for a number of otherwise inexplicable observations, and a predictive power that was lacking in the nonelectrochemical analysis. The interpretation is fur-ther extended to include photosynthetic and visual membranes and the design principles of molecular electronic devices.

I B A C T E R I O R H O D O P S I N IS U N I Q U E A M O N G M E M B R A N E P R O T E I N S because o f its

central role i n many different scientific disciplines a n d its use as the p r o v i n g g r o u n d f o r many different scientific approaches such as laser spectroscopy, m e m b r a n e biophysics, a n d molecular biology ( 1 - 6 ) . I n addition, bacteri orhodopsin has attained a n e w p r o m i n e n c e , not only because it is used as a m o d e l o f m e m b r a n e i o n p u m p s , b u t also because it is increasingly important for such technological applications as advanced photonic a n d photoelectronic 0065-2393/94/0235-0531 $10.70/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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BIOMEMBRANE ELECTROCHEMISTRY

materials ( 7 - 9 ) . I n this chapter, w e w i l l illustrate the p o w e r o f electrochem istry to elucidate b o t h the molecular a n d the u n d e r l y i n g physicochemical processes so as to enhance o u r understanding o f the basic mechanism o f bacteriorhodopsin function. W e w i l l also discuss possible technological appli cations. Bacteriorhodopsin, the o n l y p r o t e i n c o m p o n e n t o f the p u r p l e m e m b r a n e o f Halobacterium halobium, belongs to a family o f m e m b r a n e proteins that f o r m seven α-helical loops across the m e m b r a n e a n d that contain v i t a m i n A as the c h r o m o p h o r e ( J O , I I ) . R h o d o p s i n , the first k n o w n m e m b e r o f this family, is the visual pigment i n the r o d receptor cells o f vertebrates a n d i n the rhabdomeres o f invertebrates. R h o d o p s i n has b e e n k n o w n for more than 100 years. A l t h o u g h it was k n o w n ( 1 2 ) that a single p h o t o n absorbed b y a single rhodopsin molecule is sufficient to elicit a neural excitation o f the visual m e m b r a n e , the b i o c h e m i c a l mechanism o f visual phototransduction has only recently b e e n elucidated (for reviews, see references 1 3 - 1 8 ) . I n v i e w o f this n e w understanding, the absorbed p h o t o n energy serves m e r e l y as a trigger to unleash stored energy i n the f o r m o f a s o d i u m i o n gradient, a n d the visual m e m b r a n e functions as a p h o t o n signal transducer like a phototransistor. I n contrast, bacteriorhodopsin functions as a p h o t o n energy converter like a silicon photodiode. T h e absorbed p h o t o n energy is converted to electrochem ical energy i n the f o r m o f a transmembrane p r o t o n gradient, similar to the role o f the photosynthetic thylakoid m e m b r a n e o f chloroplasts o f green plants. I n other words, Halobacterium halobium uses a visual p i g m e n t to p e r f o r m photosynthesis. H o w e v e r , u n l i k e the reaction center o f a p h o totrophic p u r p l e b a c t e r i u m o r the two reaction centers o f higher plants, the reaction center o f Halobacterium halobium consists o f a single molecular component: bacteriorhodopsin.


1

T h e structural simplicity a n d the u n i q u e relationship o f bacteri orhodopsin to the t w o major photobiological systems—vision a n d photosyn t h e s i s — a l l o w us to consider several questions o f scientific a n d technological importance. W h a t is the m i n i m u m requirement o f a light energy converter? D o e s N a t u r e utilize a c o m m o n design for vision a n d f o r photosynthesis? H o w can w e technologically exploit bacteriorhodopsin b y "reverse engineering"? A l t h o u g h definitive answers m a y not always b e available, w e w i l l illustrate that a deeper insight is feasible b y using the p o w e r o f electrochemistry to analyze the relaxation kinetics o f electric signals e l i c i t e d f r o m a bacteri orhodopsin m e m b r a n e w h e n it is i l l u m i n a t e d w i t h a b r i e f (microsecond o r shorter) flash o f visible fight. T h e electric signals so generated b e l o n g to a class o f bioelectric signals k n o w n as displacement currents; the gating current o f a s q u i d axon is the best k n o w n example. U n l i k e other types o f bioelectric signals, displacement currents are not generated b y i o n diffusion i n water, but rather are generated b y charge displacements i n the m e m b r a n e . T h e fight-induced displacement currents are also k n o w n as the fast photoelectric signals. Specifically, b y invoking electrochemistry, a phenomenological d e -


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scription o f electrophysiology can be transformed into a rigorous molecular description o f the u n d e r l y i n g physicochemical processes. T h u s , some seem ingly inexplicable p h e n o m e n a w i l l have simple explanations based o n electro chemistry.


Fast Photoelectric Effect T h e fast photoelectric effect is the manifestation o f l i g h t - i n d u c e d r a p i d charge separation i n the d i r e c t i o n p e r p e n d i c u l a r to the m e m b r a n e surface (19-23). Because the photopigments maintain a preferential orientation w i t h respect to the m e m b r a n e , the charge separation is vectorial i n nature and, therefore, can be detected macroscopically as a photovoltage u n d e r o p e n circuit conditions or as a photocurrent ( k n o w n as a displacement photoeurrent) u n d e r short-circuit conditions. T h e best k n o w n example o f fast photo electric signals is the early receptor potential ( E R P ) f o u n d i n the retina o f the cymolgus m o n k e y (24, 25). H o w e v e r , earlier reports o f the early receptor potential and the E R P - l i k e signal i n reconstituted bacteriorhodopsin m e m branes were plagued w i t h inconsistencies a n d paradoxes. F o r example, the R 2 c o m p o n e n t o f the E R P was correlated w i t h the a c i d - b a s e reaction o f the metarhodopsin I to metarhodopsin II transition (25), yet the R 2 component was reported to have no significant p H dependence (26), w h i c h thus defies the law o f mass action. M o r e recently, w e p o i n t e d out a striking discrepancy i n the relaxation t i m e data reported by various laboratories o n the E R P - l i k e signal f r o m reconstituted bacteriorhodopsin membranes (Table I). A s w e w i l l show, these difficulties can be resolved b y an electrochemical analysis. I n fact, an electrochemical analysis o f l i g h t - i n d u c e d charge separation a n d recombination predicts these heretofore inexplicable observations.

Electrochemical Basis of the Fast Photoelectric Effect O u r electrochemical analysis is based o n a major conclusion f r o m decades o f investigation o f the E R P : T h e E R P is the electrical manifestation o f light-in d u c e d r a p i d charge displacements a n d the transient photocurrent satisfies the condition o f a zero time integral (33). I n other words, the displacement photocurrent is not generated b y diffusion o f ions t h r o u g h an aqueous channel i n the m e m b r a n e , but rather b y r a p i d charge separation a n d r e c o m bination. T h u s , the l i g h t - i n d u c e d f o r w a r d charge movement is subsequendy compensated b y an equal a n d reverse charge movement i n the opposite direction that results i n no net charge transport across the m e m b r a n e and, therefore, satisfies the zero time-integral c o n d i t i o n . C h a r g e recombination is a w i d e l y recognized p h e n o m e n o n i n photosyn thesis a n d solar energy conversion research that, u n t i l recently, was less appreciated i n bacteriorhodopsin research. C h a r g e recombination dissipates


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BIOMEMBRANE

ELECTROCHEMISTRY

T a b l e I. Relaxation T i m e Constants o f B a c t e r i o r h o d o p s i n Photosignals

2

T

Reference 27 28 29

(με) 1.3 25 4.4

31

a a

32

< 0.2

30 b

(με)

17 150

81 57 115 200

3

T

(ms) 0.06 2.4 2.5 1.06 4.5 2

4

T

(ms) 0.9 5.8 8 13 640 1000

T j not reported. Derived from Figure Id in reference 31. SOURCE: Reproduced with permission from reference 39. Copyright 1986.


a

the converted p h o t o n energy before it c a n b e further stored as a more stable and readily u t i l i z e d f o r m o f energy such as a transmembrane electrochemical gradient o f protons. T h e conspicuous manifestation o f charge recombination i n the f o r m o f the E R P possesses n o conceptual difficulty i n a visual m e m b r a n e . H o w e v e r , displacement photocurrents that are capacitative d o not lead to a net transmembrane

charge transport [direct current (dc)

photocurrent] a n d n o light energy is converted to a stable f o r m d u r i n g the process. T h e kinetic analysis that follows w i l l show that the net p r o t o n transport does occur despite the ever-present charge recombination. A strik i n g observation is that displacement photocurrents are m u c h m o r e p r o m i n e n t i n magnitude than the d c photocurrent i f a b r i e f (microsecond) light pulse is used to stimulate the m e m b r a n e . T h u s , a n o v e r w h e l m i n g fraction o f sepa rated charges appear to r e c o m b i n e . O n l y a negligible fraction is converted to the f o r m o f a p r o t o n gradient. O n the other h a n d , i f a l o n g rectangular light pulse is used, a displacement current c a n b e observed only briefly u p o n the onset a n d u p o n the cessation o f the light pulse; only the net charge transfer (forward transfer minus reverse transfer) is observed d u r i n g the steady state. Why

the magnitude o f displacement photocurrents tends to b e out o f

p r o p o r t i o n w h e n it is elicited b y a b r i e f light pulse c a n b e explained b y a universal equivalent circuit m o d e l that w i l l b e described later. T h e E R P was often attributed to intramolecular charge displacement. Because l i g h t - i n d u c e d conformational changes o f r h o d o p s i n are accompanied by r a p i d charge separation a n d because r h o d o p s i n maintains a fixed orienta tion i n the m e m b r a n e , a transient array o f electric dipoles w i t h net moments i n the d i r e c t i o n p e r p e n d i c u l a r to the visual m e m b r a n e w i l l appear u p o n i l l u m i n a t i o n . B y virtue o f the zero time-integral condition, such an electric dipole array must vanish as separated charges r e c o m b i n e u p o n cessation o f i l l u m i n a t i o n . T h i s process is tantamount to charging a n d discharging o f a capacitor. T h i s mechanism w i l l b e referred to as the oriented dipole

mecha

nism ( O D m e c h a n i s m i n F i g u r e 1) (34). H o w e v e r , this is not the only k i n d o f


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charge separation a n d recombination that c a n b e f o u n d i n the visual m e m brane a n d i n the p u r p l e m e m b r a n e o f H. halobium. T h e metarhodopsin I - m e t a r h o d o p s i n I I transition, w h i c h occurs concur rently w i t h the R 2 signal, is a key step i n the photobleaching o f rhodopsin. I n this reaction, one p r o t o n is b o u n d to the pigment ( 3 5 ) . S u c h a p r o t o n b i n d i n g reaction is r e q u i r e d for p r o t o n p u m p i n g i n the p u r p l e (bacteriorhodopsin) m e m b r a n e . I f w e assume that a reverse reaction exists i n each a n d every b i o c h e m i c a l reaction, then a p r o t o n b i n d i n g reaction must b e accompanied b y a reverse reaction i n w h i c h the b o u n d p r o t o n is released into the adjacent aqueous phase; that is, the p r o t o n is released into the same aqueous phase w h e n c e it was b o u n d previously. U p o n l i g h t - i n d u c e d p r o t o n b i n d i n g b y the pigment, the counterions must b e left b e h i n d i n the adjacent aqueous phase. T h i s abandonment constitutes another k i n d o f charge separation. T h e reverse interfacial p r o t o n transfer c a n thus b e regarded as charge recombination. S u c h interfacial p r o t o n transfer reactions a n d the accompanying reverse reactions are also tantamount to charging a n d discharging o f a capacitor. W e refer to this m e c h a n i s m as the interfacial proton transfer mechanism ( I P T mechanism i n F i g u r e 1). Because the objective o f p r o p o s i n g these models is to understand the macroscopically measured displacement photocurrents i n terms o f the u n d e r lying mechanistic a n d molecular processes, the connection between macro scopic electrical parameters a n d microscopic parameters must b e d e m o n strated. T h i s linkage c a n b e p r o v i d e d b y an analysis based o n the G o u y - C h a p m a n diffuse double layer theory a n d the derivation o f a n equiva lent circuit. A s d o c u m e n t e d elsewhere (34, 36), analysis o f the t w o models shown i n F i g u r e 1 gives rise to a space charge density profile a n d a n electrical potential profile across the m e m b r a n e . T h e equations that describe these profiles can b e reinterpreted as c h a r g e - p o t e n t i a l relationships i n terms o f three fundamental capacitances: a geometric capacitance ( C ) a n d t w o d o u ble layer capacitances ( C ) . T h i s interpretation allows the spatial relationship o f these capacitances and the (microscopic) photovoltage source ( E ) a n d its internal resistance ( R ) to b e d e d u c e d . T w o slightly different equivalent circuits that were obtained for the two mechanisms c o u l d b e further r e d u c e d to a c o m m o n irreducible equivalent circuit, i n w h i c h a composite capacitance, C , is connected i n series w i t h the macroscopically measured photoelectromotive force (photoemf), £ . C o n t r a r y to conventional w i s d o m , this capaci tance, w h i c h w e n a m e d chemical capacitance, is physically distinct f r o m the ordinary m e m b r a n e capacitance. A detailed argument, p u b l i s h e d elsewhere (20), proves this point o f view. H e r e it suffices to point out that whereas an ordinary m e m b r a n e capacitance is i n parallel w i t h the photoemf, the capaci tance C is i n series w i t h the photoemf. I n other words, the photosignal is ac-coupied (alternating-current coupled) through a b u i l t - i n series capaci tance, w h i c h , together w i t h the internal resistance ft , forms a high-pass filter for the photosignal. g

d

p

p

p

p

p


536


OD Mechanism

IPT Mechanism Ρ

Η 0 ΝΊ/-Ρ* +

3

PhT

Η 0
>a»o

e

R >0

Tm=0

e

R =0

p

E (t) = E -Ht)

BREIF PULSE STIMULATION



540


(32) u n d e r o p e n circuit condition» is i n d e e d the m e m b r a n e R C relaxation (Table I). T h i s effect was recognized a n d reported b y D r a c h e v et al. (32), b u t the distortion o f the remaining t i m e constants was not appreciated. Because almost all reported E R P data i n the literature are measured u n d e r o p e n circuit conditions, this effect explains, at least partially, w h y the R 2 c o m p o nent o f the E R P was f o u n d to be insensitive to p H changes i n the aqueous phases. A similar p r o b l e m can also b e identified i n the literature o f bacteri orhodopsin: the E R P - l i k e signal f r o m a reconstituted bacteriorhodopsin m e m b r a n e is insensitive to p H i n the physiological range (32). H o n g a n d M o n t a i (38) have demonstrated that such a lack o f p H sensitivity i n bacteri orhodopsin membranes is, i n part, due to the c o m m o n l y used o p e n circuit measurement m e t h o d . W e f o u n d that at least two chemically distinct c o m p o nents exist i n a reconstituted bacteriorhodopsin membrane: a B l component that is insensitive to b o t h temperature a n d p H changes a n d is thus analogous to the R l component o f the E R P , a n d a second temperature sensitive component, B 2 , that is analogous to the R 2 component, w h i c h was f o u n d to be highly sensitive to p H u n d e r a near short-circuit c o n d i t i o n but that is insensitive to p H u n d e r an o p e n circuit c o n d i t i o n ( F i g u r e 4).

Component Analysis T h e p r e c e d i n g analysis clearly indicates that the practice o f decomposing a photosignal i n terms o f its i n d i v i d u a l components as shown i n T a b l e I is not r e c o m m e n d e d . I n fact, o u r analysis indicates that a photosignal that repre sents a single exponential relaxation w i l l b e manifest as a biexponential decay except u n d e r true short-circuit conditions. T h u s , decomposition of the photosignal into components that correspond to the u n d e r l y i n g c h e m i c a l processes must be based o n the equivalent circuit analysis a n d taken into account the presence o f a nonzero access impedance. T h e equivalent circuit analysis indicates that all o f the circuit parameters can b e either d e t e r m i n e d experi mentally or d e r i v e d b y computation using measurable parameters a n d are, therefore, not freely adjustable (see A p p e n d i x III i n reference 36). T h u s , unlike some kinetic models w i t h many parameters that must be d e t e r m i n e d i m p l i c i t l y b y curve fitting, the present equivalent circuit m o d e l offers a stringent test o f the validity o f o u r electrochemical analysis. T h e previously p u b l i s h e d (34, 36) derivation o f the equivalent circuit implies that the equivalent circuit is i n t e n d e d for a photocurrent that arises f r o m a single relaxation process; that is, either first-order or pseudo-first-order processes. T h u s , a composite photosignal that consists o f b o t h B l a n d B 2 is not expected to agree w i t h the equivalent circuit. Therefore, a prerequisite to test the validity o f the equivalent circuit analysis is to devise a successful m e t h o d to separate the two components, for example, b y elimination o f the B 2 c o m p o nent completely but leaving the B l component intact.


HONG


25.

541


ο χ I

0

ni

5

1

I

10

15

I n ,

20 TIME

I

I

I

25 38 35 (MICROSEC)

I

40

In,

45

I

50

C

0

50

100

150

200 250 300 350 TIME (MICROSEC)

400

450 500

Figure 4. Temperature and pH dependence of the Bl and the B2 components. The photosignals generated in Trissl-Montal films are shown in A and in C for their pH and temperature dependence, respectively. The corresponding photosignals obtained from a multilayered thin film are shown in Β and in D. The plot in Β shows superposition of photosignals obtained from the same film at pH 7, 8, 9, and 10. Continued on next page.


542


D ο χ


0

10

20

30

40 50 60 70 TIME (MICROSEC)

80

90

100

Figure 4.—Continued. The small temperature dependence shown in D corresponds to an activation energy of 2.5 kcal/mol. (Reproduced with permission from references 39 and 4L Copyrights 1986 and 1987.)

O n e such m e t h o d was reported b y O k a j i m a a n d H o n g (39). M u l t i p l e layers o f p u r p l e m e m b r a n e sheets were first deposited onto a t h i n (6-μιη) T e f l o n film that h a d been previously coated w i t h a mixture o f azolectin a n d n-octadecylamine. T h i s t h i n film assembly was allowed to d r y f o r at least two days, t h e n m o u n t e d i n a t w o compartment chamber, a n d t h e n rehydrated. Pulse light stimulation consistently gave rise to a photosignal that was p H insensitive ( F i g u r e 4B) a n d that was consistently i n agreement w i t h the equivalent circuit ( F i g u r e 5). I n fact, w h e n the access impedance was varied, the change o f the photosignal t i m e course c o u l d be fully p r e d i c t e d b y the equivalent circuit analysis ( F i g u r e 5 i n reference 39). H o w e v e r , i f the p u r p l e m e m b r a n e sheets are deposited onto the T e f l o n film by means o f a m e t h o d originally developed b y T r i s s l a n d M o n t a i (40), a photosignal o f considerably smaller magnitude is observed; this photocurrent consists o f b o t h a temperature- a n d p H - i n s e n s i t i v e component and a temper ature- a n d pH-sensitive component ( F i g u r e 4A a n d C ) . H e r e , a composite signal w i t h at least two components ( B l a n d B2) is b e i n g dealt w i t h , a n d the decomposition can b e tricky. T h e foregoing analysis indicates that each o f the two components decays i n two exponentials, as shown i n F i g u r e 6. Because o f the partial overlap o f the decay o f B l a n d the rise o f B2 a n d because o f their opposite polarities, i n h i b i t i o n o f B2 alone also c a n b e interpreted as a concurrent i n h i b i t i o n o f B2 a n d enhancement o f B l (cf., F i g u r e 4A a n d C ) . I n fact, the concurrent i n h i b i t i o n a n d enhancement was the interpretation presented b y D r a c h e v et a l . (32). T h i s interpretation further contributes to the apparent lack o f p H dependence o f the B2 component, because part o f the p H dependence o f B2 was attributed to B l instead. O u r use o f t w o separate methods o f reconstitution a n d o u r electrochemical analysis p e r m i t us to avoid this pitfall. T h u s , i f w e define the p H - i n s e n s i t i v e photosignal f o u n d i n the multilayered t h i n film as the B l component a n d the pH-sensitive p o r t i o n o f the


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Electrochemical Processes in Membranes That ... - ACS Publications

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