861 — Charge motion inside the bacteriorhodopsin molecule

437

Bioelectrochemiktry and Bioenergetics, 15 (1986) 437-445 A section of J. Electroanal. Chem., and constituting Vol. 211 (1986)

Blsevier Sequoia S.A., Lausanne - Printed in The Netherlands

861-

CHARGE MOTION INSIDE THE BACTERIORHODOP!!3IN MOLECULE *

L. KESZTHELYI Institute of Biophysics, Biological Research Center, 6701 Szeged (Hungary)

(Manuscript received June 29th 1985)

SUMMARY The bacteriorhodopsin molecules in Halobacteria membranes pump protons after light absorption. During this action light energy is transduced into electrochemical energy. The importance of proton pumping in bioenergeticsprompted us to work out three different macroscopically oriented systems of bacteriorhodopsin molecules to study the molecular events of charge motion inside the molecules. The flash-excited absorption changes and electric signals were measured simultaneously. It has been established that the time constants of the absorption and electric signals (evoked as displacement currents of moving charges) are the same in the pH range of 4-8; the analysis of amplitudes of electric signals gives the distances of charge motion inside the protein; the first step of charge motion is faster than 30 ps; proton translocation needs water molecules to be present; the stoichiometry does not change with pH until pH 10.5; tyrosin modification blocks the pumping activity but modest protein digestion does not influence it in the pH range of 4-10.

INTRODUCTION

Cells, even cell organelles, are capable of sustaining a potential across their membrane. The membrane potential is the result of an unequal build-up in the concentration of different ions internally, in the closed cells or organelles and externally in the environment. The membrane potential, though, would fade if it were not sustained constantly by special means existing ‘in living cells. This is accomplished by protein units contained within the membranes. In exchange for energy consumption, these protein units translocate ions through the membranes, thus constantly reestablishing the membrane potential. Bioelectrochemistry studies the membrane potential, its variation under different

Invited lecture delivered at the VIIIth International Symposium on Bioelectrochemistry and Bioenergetics, Bologna, June 24th-29th 1985. l

0302-4598/86/$03.50

Q 1986 Blsevier Sequoia S.A.

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conditions, correlates its value with the ion concentration differences, etc. The variables studied are those related to the products of the ion translocation processes performed by the special proteins. The process itself is a molecular event: to understand the mechanism of ion translocation in proteins is a very important but difficult project. In this paper we report the study of a proton translocator molecule, the bacteriorhodopsin (BR) protein, which seems to be the simplest known ion pump [l]. The aim of these experiments is to collect data on the proton’s motion inside the molecule in order to learn more about the actual mechanism by which the membrane potential is developed. EXPERIMENTAL

It is well known that charges moving in vacuum, or dielectrics produce a displacement current which may be picked up even by far-lying electrodes [2]. Accordingly, displacement currents should be generated during the proton translocation in the BR protein (which is considered as a dielectric medium). In order to measure the displacement current within a particular system, the charge motion must be unidirectional throughout, i.e. all the proteins must be oriented in one direction. The BRs contained within a single cell are oriented so as to pump protons across the cell membrane, creating a potential difference between the inside and outside of the cell. The current, though, is not measurable because it is impossible to put electrodes inside and outside of a cell. Furthermore, because of the spherical symmetry of the cells, and thus the net orientation of the BRs within the cells, the current is not measurable by electrodes immersed within a cell suspension. The BR molecules are located in concentrated form in the plasma membrane of the cells. The purple membrane (pm) contains the molecules ordered into a two-dimensional crystal in the lipid matrix. During the first observation of charge motion inside BR, the purple membranes were attached in a more or less oriented manner to model membranes [3-61. The data produced by flash illumination showed a negative and a positive component of charge motion (relative to the direction of proton translocation). Because the pm layers were very thin, light absorption measurements could not be done. It was therefore not possible to correlate the electric data with the photocycle of the BR, as characterized by light absorption changes [l]. We have found that pms have a large permanent electric dipole moment perpendicular to the membrane plane [7]. A rather small (15-20 V/cm) electric field turned out to be sufficient to saturate the orientation of pms in suspension. A laser flash excited - lOI oriented BR molecules to pump protons in one direction [8]. In later developments, after orientation the membranes were immobilized in gel [9] or dried on a transparent electrode [lo]. Again, upon laser flash - 1015 BR molecules are excited to proton translocation, producing a macroscopic displacement current. Furthermore, the quantity of BR molecules is sufficient for light absorption measurement.

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A=408 nm Fig. 1. Comparison of the electric and absorption signals. (a) The large, faster negative component corresponds to BR-K transition, the slower negative component is due to K-L transition (T = 5°C); (b) L-M transition (T= 22°C); (c) M-O and O-BR transitions (T = 22°C). The electric signals measured in these systems contained five time-resolved components with different amplitudes and rates. They are ordered according to the intermediates of the photocycle of BR named K, L, M, O [1]. In Fig. 1 a set of data is reproduced. The signal starts with a fast component: its rise time of 1 Fs duration is due to the width of the exciting laser flash while its decay of 0.3-0.4 #s is the time constant of the electronics. (The main components of the R C time constants are: the resistance of the solution, Rs, the measuring resistance, R,,, coupled in parallel, R = (RsRm)/(R ~ + R,,), and the measuring capacitance C = C e + C i , , where Ce is

440

the capacitance of the electrodes and C,,, the input capacitance of the amplifier. The bandwidth of the amplifier is large enough to transmit the signal without distortion. The signal is negative, showing a charge motion opposite to the final direction of proton motion. In a special experiment the appearance of the first component was found to be faster than 30 ps [ll], and was assigned to the BR-K transition. The second component, which is still fast and has a negative amplitude, is followed by three components with positive amplitudes. Light absorption measurements performed in parallel to the electrical measurements showed that the lifetimes of the components of the electric signal coincide with the lifetimes of the intermediates of the photocycle [8] (within the pH range of absorbance of 4-8 [12]). Thus, we may state the following: The absorbance changes of the intermediates of the photocycle are time-correlated with charge motion inside the protein. A simple analysis of the displacement current [13] made it possible to correlate the amplitudes of the different components of the electric signal U,(t) with the displacement of the charge, which is assumed to be a single proton. Here we write the expression for a single exponential with rate constant k: U,(t)

= NQF;

1 _kfRC

where N is the number of excited photocycles, Q is the elementary charge, D is the distance of the electrodes, the factor F contains the dielectric constant of the protein (for the region in which the proton moves), the degree of orientation and a calibration factor for geometry. Using equation (l), many important results related to the BR proton pump could be obtained. RESULTS AND DISCUSSION

The results obtained by evaluating the data may be classified into two categories: (a) Results which are obtained when one proton is assumed to move during the photocycle; (b) Results which are valid independent of the above assumption. (a) To analyse the electrical data, equation (1) is used. R, C and D are known, N may be determined from the absorption change at X = 410 nm, Q is the elementary charge if the motion of a single proton is assumed and ki is determined from the time dependence of the signal. Inserting the data into equation (l), the unknown quantities of ed, can be calculated for all five transitions. With the further assumption of 4. = constant = F, the d, values can be determined by normalizing the quantity FZdi to the membrane thickness (5 nm). The data, with their assignments to the photocycle, are given in Table 1. A detailed argument is given in Ref. 14, showing the validity of the above assumptions. Here we mention only that equation (1) reflects the essence of the problem, being capable of reproducing the protein thickness uia FZdi, using a value of l/4 for F. . The scenario of the proton translocation using the d, values in Table 1 may be the following:

441 TABLE 1 Distances the proton traverses during translocation in BR Transition

Assignment

Distance (MI)

4

BR-K K-L

-0.13 - 0.02 +o.s + 3.1 +1.5

4 d, 4

L-M M-O

d,

0-bR

(1) The Schiff-base protons move 0.13 nm inwardly due to rruns-cis isomerization during BR-K transition [15]; (2) d, - 0.02 nm is a small rearrangement of charges during K-L transition; (3) The proton releases from the Schiff-base and jumps 0.5 nm forward, possibly attaching itself to a COO- group; (4) During the M-O decay the proton gets into a long conducting path of d, = 3.1 nm and reaches the surface; (5) A proton is picked up at the other side of the membrane and reprotonates the Schiff-base (0-BR, d, = 1.5 nm). (b) The most important assumption-independent result is that A = EJm UN,(t)dt 0

a true charge translocation, because in the case of internal charge or dipole motion, a zero value for A should be obtained, as has been shown in the case of gating currents of the nervous membranes [16]. Measurements performed on the pH dependence of the electric signals showed that A is constant between pH 4.5 and 10.5. Above pH 10.5 the value of A begins to fall suddenly [12]. Furthermore, different enzymatic treatments, which digest only the outer portions (trypsin and papain digestion) of the BR protein, do not influence the value of A within the pH range of 4.5-10.5 [17]. The data are shown in Fig. 2. In these experiments the area A is always related to the amplitude of the first negative signal, which is considered to be proportional to the number of excited photocycles. Thus the efficiency of a BR protein in converting proton energy into a proton translocation is affected by neither a wide variation in pH, 4.5-10.5, nor by the digestion of residues on the exterior of the protein. These data seem to be in contradiction with previous results. Many authors have reported that the number of protons per photocycle is dependent on the pH (a review may be found in Ref. 18) and also upon alteration of the protein by enzymatic treatments (Refs. 19, 20). The differences between our results and others, though, are probably due to the different methods of observation. While electric signals measured in oriented pm systems represent the moving charges, in the other systems the change of proton concentration in the solution was registered by pH dyes or electrodes, which do not distinguish between pumped or simply released protons (so-called Bohr protons). >

0. This shows clearly that the electric signals demonstrate

442

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Fig. 2. pH dependence of the quantity A (see text) for (a) normal, (b) trypsin-modified and (c) papain-modified purple membranes. In (c) the papain/BR concentration is @) 1:200; (A.) 1:20; (A) 1:2.

While the electrical signals obtained for the pH range of 4.5-10.5 and for enzymatic digestions of the outer portions show that the BR proteins are capable of pumping protons with the same efficiency, several methods have been discovered for blocking the proton translocation.

443

I 20

0

Fig. 3. Humidity

Relative humidity % I I I 40 60 80 dependence

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1 100 A (see text).

b)

0 A

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Fig. 4. Humidity dependence of the activation enthalpy of the transitions in BR: (a) K-L; M-BR. The different symbols in (b) and (c) relate to repeated measurements.

(b) L-M;

(c)

444

Iodination of one or two tyrosine residues in BR results in A = 0, meaning that in this case charges are not translocated [21]. From this and also from many other experiments it follows that tyrosines participate in proton translocation. A = 0 was also found in dried oriented samples [22]. More precisely, the electric signals showed a nom1 behaviour before the photocycle reached the M intermediate. After this state negative signals appeared instead of positive ones. The dependence of A on humidity is shown in Fig. 3. The data show that in the absence of water molecules the protons do not enter into the proton-conductive pathway from the M state but fall back to their original position: the Schiff-base. If the humidity is increased above 608, A takes on increasingly positive values. It is interesting to note that characteristic changes appear in the activation enthalpy values of the different transitions at the same humidity value (Fig. 4, [23]). All these observation point out the immense role of water in the mechanism of proton translocation. The problems have been recently discussed by us and a simple model for proton translocation was worked out [14]. According to our model, the protons in the M state can be released from the carboxyl group to water molecules approaching it. In a water-filled subcompartment they are conducted outwards in the existing electric field (due to charge separation in BR and probably to surface charges) similarly to the proton current in water. Further experiments are needed to corroborate the hypothesis. CONCLUSIONS

The displacement current induced by moving charges inside the bacteriorhodopsin offers a possibility for studying the molecular mechanism of proton translocation. The data show that: Charge motions and optical changes are coupled (pH 4-8); Assuming the motion of a single proton, five discrete steps can be resolved; The stoichiometry does not depend on pH (4.5-10.5) or on trypsin and papain digestion of the residues on the exterior of the protein; Tyrosins are involved in proton translocation; Water is needed for an efficient proton pump. ACKNOWLEDGEMENTS

My colleagues K. Barabas, J. C&g&, Zs. Dancshazy, A. DQ, G. Groma, S. Hristova, P. Ormos, J. Posfai, G. V&r6 and L. Zim&nyi took part in the above investigations. Their contributions are gratefully acknowledged. The work was partially supported by cooperative grants between the Hungarian Academy of Sciences and the National Science Foundation under grants INT 78-27606 and INT 82-17761. REFERENCES 1 W. Stoeckenius, R.H. Lazier and R.A. Bogomolni, Biochim. Biophys. Acta, 505 (1979) 215. 2 K. Siionyi, PhysikaJische Elektronik, Teubner, Stuttgart, 1972, pp. 649.

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