Electrostatic and Conformational Effects on the Proton Translocation

Electrostatic and Conformational Effects on the Proton Translocation Steps in Bacteriorhodopsin: Analysis of Multiple M ... Biochemistry 2000 39 (2), ...
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J. Phys. Chem. 1995, 99, 7787-7800

7787

Electrostatic and Conformational Effects on the Proton Translocation Steps in Bacteriorhodopsin: Analysis of Multiple M Structures Christina Scharnagl,* Johannes Hettenkofer, and Sighart F. Fischer Institut fiir Theoretische Physik der Technischen Universitat Miinchen, James-Franck-Strasse, 0-85748 Garching, Germany Received: September 12, 1994; In Final Form: February 7, 1995@

Molecular dynamic, electrostatic, and quantum chemical calculations are applied in order to analyze in a model-independent approach the driving forces for the rise and decay of the M state in the bacteriorhodopsin photocycle. We find that a protein conformational change involving the reorientation of arginine R82 away from the chromophore binding site toward the extracellular region after the protonation of the primary acceptor aspartate D85 induces the development of several M subpopulations. They differ in the overall protein conformation and the total number and the distribution of protons and control the recovery of the ground state in different ways. This protein conformational change catalyzes extracellular proton release in the alkaline pH region and provides favorable electrostatic and structural features for speeding up the reprotonation of the retinal Schiff base, simultaneously slowing down its reisomerization. The de- and reprotonation steps are decomposed in single steps involving bound water molecules as intermediate proton binding sites. We show that, for each of the two overall translocations, the initial steps proceed near equilibrium, while further steps are unidirectional and fast.

Introduction Proton pumping by membrane proteins plays an essential role for the energy transduction in biological systems. Among the experimentally best characterized proteins is bacteriorhodopsin, a photoactive protein embedded in the purple membrane of Halobacterium salinarium. It transduces light energy into chemical potential by pumping protons from the cytoplasm to the exterior across the membrane. A protonated retinal Schiff base (RSB), covalently linked to the protein, is responsible for the light absorption in the visible spectral range (570 nm), which leads to its rapid isomerization from the all-trans to the 13-cis configuration. The subsequent relaxation of the photoproduct occurs via a complex series of thermal reactions including protein conformational relaxation, proton transfer, and reisomerization steps. The ground state intermediates are named according to the generic sequence BR-K-L-M-N-0-BR. The combination of optical and vibrational spectroscopy with mutagenesis experiments provides a framework for the determination of the temporal and spatial proton pathway (for recent reviews of the progress made toward an understanding of the structure and the transport steps and the variety of still open questions, see, e.g., refs 1-6). In the L to M step, the chromophore deprotonates. Aspartate D85 near the chromophore binding site acts as the primary proton acceptor. Its protonation is followed by release of a proton to the exterior from a so far experimentally unidentified group. In the M to N step, the retinal Schiff base is reprotonated from an internal donor, aspartate D96, which is located near the cytoplasmic region. The retinal all-trans configuration is restored with the formation of 0, while a proton is taken up from the cytoplasm in order to reprotonate D96. Transfer of the proton from D85 to the release group or the bulk recovers the initial state in the 0 to BR step. The pump works under a variety of environmental conditions (e.g., pH, salt concentrations), but the order and the rates for the individual steps differ. Several mechanisms for pathways have been proposed, leading to a variety of kinetic

* To whom correspondence should be addressed. @Abstractpublished in Advance ACS Abstracts, May 1, 1995.

scheme^.^-'^ All of them have to explain the existence of the biphasic rise and decay of the M intermediate.13 In a number of reports, the thermal reactions leading to the proton translocation are described as a linear sequence with transient equilibria between the intermediates (e.g., refs 2, 7, and 8). Alternative descriptions9-’*introduce parallel photocycles, each with its own M rise and decay time, originating from heterogeneities of the unphotolyzed protein. Despite the wealth of experimental information, it is necessary to analyze how the protein provides the driving forces for the individual steps. One of the still open questions is, how does the proton conduction pathway reorient during the lifetime of the M intermediate from the exterior to the cytoplasm in order to ensure vectoriality of the ion pump process? This reorientation is sometimes called the “reprotonation s w i t ~ h ’ . ~ As , ’ ~found, e.g., by changed Bragg scattering,l5,l6changes in the protein amid I/II vibrational bands,” and an altered projection map of the electron density in the M state,18 the protein undergoes significant changes in tertiary structure during the photocycle. It was a r g ~ e dthat ~ ~ these .~~ conformational changes play a vital role in the proton transport mechanism, but the molecular nature of the changes has not yet been established by experimental methods. Another open question addresses the involvement of internal bound water molecules in the transport process. On the basis of spectroscopic studies, it has been predicted that one or two water molecules interact with the protonated RSB20-24and favor its deprotonation in L. It has also been postulated that water is involved in the transfer of the proton from the internal donor D96 to the RSB across a hydrophobic cytoplasmic ~ h a n n e l ~in~ the % *M ~ to N step. The elucidation of the three-dimensional structure of bacteriorhodopsin at near-atomic resolution by electron cryomicroscopy (“Henderson model”:) establishes a basis for the theoretical modeling of the isomerization and pump process (e.g., refs 27-3 1). Molecular dynamic calculation^^^-^^-^^ allow one to discuss the position and flexibility of internal water molecules and to analyze what relaxation induced conformational changes are possible. The use of a macroscopic dielectric model with

0022-365419512099-7787$09.00/0 0 1995 American Chemical Society

7788 J. Phys. Chem., Vol. 99, No. 19, 1995 microscopic details including protein and water dipoles provides a reliable tool for the discussion of electrostatic interactions in the In this way it is possible to relate the relaxation induced structural changes to modified proton affinities of titratable groups that might participate and to identify the driving forces for the transfer steps. Together with the subsequent analysis of the structural relaxations induced by the protonation changes, it is possible to propose routes for proton transfer and define the reaction coordinate on a molecular basis. In our previous c ~ n t r i b u t i o nwe , ~ ~provided* analysis of the electrostatic and structural features in the chomophore binding site of the unphotolyzed protein and their changes upon isomerization. We discussed possible routes for the deprotonation step (L to M) and proposed a "working model" for a protein based reorientation switch that utilizes the positional flexibility of arginine R82. With this model we were able to account for the pH-dependent branching of the photocycle and to identify an extracellular proton release group. The reprotonation and thermal reisomerization process (M to N) after extracellular proton release are in the center of the present study. We analyze the relation between the proposed protein conformational change and the proton translocation mechanism. We show that it will really work as a reprotonation switch, as it provides favorable structural and electrostatic features for speeding up the reprotonation, simultaneously slowing down the reisomerization. For all structures discussed, we investigate how the extracellular and the cytoplasmic region of the protein respond to the conformational and electrostatic changes in the active site. The most prominent response is due to the cytoplasmic end of helix F, whose tilting during the cycle changes in a way that is favorable for adjusting the proton affinity of D96 to the actual requirement. Responsible for the speeding up of its deprotonation after extracellularproton release is a long range communication between the two opposing sides of the protein. Our analysis strongly argues for the development of heterogeneous populations differing in the o v e r 9 conformation and distribution of protons in the protein at the stage of the M intermediate. We analyze in which way these substates control the individual steps and show that the overall transfer mechanism for de- and reprotonation can further be resolved in discrete steps proceeding with a near-equilibrium initial step and subsequent fast and unidirectional steps.

Schamagl et al. vector XI(^), ..., X N ( ~ ) ) ,whose elements xi are 1 (0) if the corresponding site i is protonated (deprotonated). The charge of the unprotonated residue is qp (- 1, acid; 0, base). Changes in self-energy and the interaction with other sites shift the acidbase equilibrium for the residue i in a given protonation state m of the protein:39

c N

pK,("'(i) = pK,,,(i) -

(4:

+ xi(")Wij /2.3kT

j )=i '1(

The free energy for a certain protonation state m of the protein in equilibrium with a solution of a given pH is31,39

I

-

c

(4;

2 j=l(+i)

+ xi'"')(qjo + Xj(m))Wij

The average protonation of site i is given by the Boltzmann weighted sum over all 2N possible protonation states of the protein:33

Proton transfer between two residues i and j connects the two protonation states = 0, ...) (m) = (..., Xy)= 1, ...) xJm) I

-

( n ) = (..., Xi(n) = 0,..., x y = 1, ...) For a donor-acceptor distance c3.5 A, the activation barrier for the transfer step is determined by the difference between the free energy of reactant and product state^.^^,^' Therefore, the local pK, values of the inferred groups define the energetics of each state in the pumping process. The rate for activated proton transfer from group i to group j within the hydrogenbonding distance can be estimated from the relation

Methods The factors that determine proton affinities in proteins are primarily e l e c t r o ~ t a t i c . ~If~ -there ~ ~ is only one titratable group i in the protein, its pK, is referred to as the intrinsic pKa (pKint(i) 33,39). It is given relative to the proton affinity of the group in aqueous solution pKmodel(i)as a reference:

Compared to the reference state, the solvation is altered and additional electrostatic interaction to permanent protein and water dipoles and pH-independent background charges32,33,39 are provided. AAGsolvand AAGack.account for the differences in these solvation and background terms for the protonated and unprotonated form of the titratable group and are subsumed under the name self-energy. With more than one titratable group in the protein, the mutual electrostatic interactions become pHdependent. This site coupling enters as extra free energy Wij for the protonation of group i in the presence of a proton on group j.39 A protein with N titratable residues will fluctuate among the 2Npossible protonation states ( m = 1, ..., 2N). We31 describe a certain proton configuration by the protonation state

with ko in the order of lo1*.. 1013 s-1.36 A measure for the stability of a special protonation state m and its change as a consequence of a conformational change is the free energy difference relative to the protein in the (hypothetical) neutral form, the ionization free energy AGion.40 It corresponds to the free energy of a spontaneous ionization process which proceeds to equilibrium and will, therefore, always be less than or equal to zero. The ionization energy is not purely electrostatic, because it depends implicitly upon the chemical potential and the bonding energy of the protons to the ionizable groups. Calculations of electrostatic interactions and reaction field contributions were carried out by the finite-difference PoissonBoltzmann (FDPB) method, using the program package DelPhi.32 For all calculations, only a subset of titratable groups (see Figure 1) was included: the retinal Schiff base RSB, members of the counterion complex (D85, D212, R82, Y157, Y185, and water molecule Xl), groups in the cytoplasmic channel (D96, T89, T46, and the bound water molecules, e.g., X2), and glutamate E204 in the extracellular region. The intramembrane water molecules and threonine side chains were

Proton Translocation Steps in Bacteriorhodopsin treated as bases with the corresponding pKmd,.(R-OH2+) = - 1.7.4* The actual setup and the limitations are the same as in our previous contribution3' with one exception: In order to obtain more reliable background interactions for aspartate and glutamate residues near the membrane surfaces (D96 and E204), we allowed the surrounding water molecules to reorient according to the two protonation states of the residues. For the discussion of the relaxation induced structural changes after isomerization or after changes in the charge distribution of the protein, we used the energy minimization and molecular dynamics facilities of the program package CHARMm (2.1 parameter see2). The detailed conditions for the calculations are given in ref 31. The torsional barriers for the C13=C14 dihedral of the retinal chromophore were calculated with MOPAC 6.0,43 using the AM1 Hamiltonian and multiple configuration interaction. They were separately determined for each counterion complex. The coupling of the counterion residues with the dihedral twist was first determined with an adiabatic torsional barrier calculation with CHARMm (30-deg steps). This provided the location of the surrounding charges for the quantum chemical calculation which allowed all other chromophore degress of freedom to relax in the fixed environment. Quantum chemical calculations using the INDO-SDTCI method4 were used for the calculation of electronic absorption spectra of the RSB, including surrounding charged and polar residues. We start with the original electron diffraction structure from Henderson et al.4, but the side chain of arginine R82, originally pointing to the extracellular side, was repositioned to face the chromophore binding site, as it was suggested by a former electrostatic calculation.3o The positions of intraprotein water molecules were carefully modeled using the information from the solvent accessibility algorithm, molecular dynamics, randomization, and an effective geometry optimization techn i q ~ e . ~The ' application of graduated harmonic constraints during the molecular dynamic simulation ensures that the helical part of the protein keeps close to the experimentally determined (for the BR ground state) or the minimum potential energy position (for subsequent structures). From the ground state structure we started the combined investigation of protein conformational changes and shifts in acid-base equilibria of the key residues, induced by the all-trans to 13-cis photoisomerization of the protonated retinal Schiff base. The names of the generated structures are chosen in accordance to the generic sequence. L-structures contain the 13-cis protonated RSB after isomerization, the chromophore in M-structures is the deprotonated RSB, and N-structures are those with the reprotonated RSB before thermal reisomerization. For each step, several substructures are analyzed according to the results of the electrostatic calculations. Results Deprotonation of the Protonated Retinal Schiff Base. Figure 1 shows the location of functionally important residues and water molecules in our equilibrated structure of the BR ground state. The theoretically modeled structure reproduces the NMR-spectroscopical1y proposed45quadrupole-like arrangement of the protonated Schiff base (RSBH+), the two aspartate residues D85 and D212 and the arginine residue R82. Recent FTIR and I 8 0 - and 2H-exchange experiment^^^ clearly prove that at least one water molecule is trapped in the active site in the BR ground state, which is able to exchange protons with the outside. Our structures show that a hydrogen-bond network can develop in the binding site. The Schiff base proton is in the hydrogen-bonding distance to the oxygen of one water

J. Phys. Chem., Vol. 99, No. 19, 1995 7789

/&.

. '..

R82

E204

Figure 1. Ground state of bacteriorhodopsin: Arrangement of the protonated retinal Schiff base (RSBH+) and functionally important groups in the transmembrane region (amino acids are labeled by their one-letter code; water molecules are labeled by X; hydrogen bonds are drawn with dotted lines).

molecule (X1 in Figure 1). whose hydrogen atoms are bound to the carboxylate groups of D85 and D212. Due to an unfavorable geometry, the bond between Xlo and RSBH+ will only be weak, despite the close proximity (3 A) of these groups. D85 is in direct hydrogen-bond contact to arginine R82 without a bridging water molecule. Two strong hydrogen bonds to the hydroxyl groups of the neighboring tyrosine residues Y57 and Y 185 solvate the negative charge on aspartate D212. One hydrogen-bonded chain consisting of water molecules and the hydroxyl side chain of threonine T89 spans the hydrophobic interhelical region and connects the binding site with the cytoplasmic region. There, water X2, aspartate D96, and threonine T46 are involved in a three-center hydrogen-bond complex. A second string of ordered waters, spanning the region from tyrosine Y 185 to the cytoplasmic side is discussed in ref 31. Five waters in the otherwise mainly hydrophobic protein interior below the active side are less ordered and cluster around the polar residues tyrosine Y79 and Y83 (compare Figure 1 in ref 31).

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7790 J. Phys. Chem., Vol. 99, No. 19, 1995

TABLE 1: Lowest Free Energy Protonation States for pH = 9 and pK, Values for Key Residues in the BR to N Sequence of the Bacteriorhodopsin Photocyle (0,Residue Protonated; 0, Residue Deprotonated; f, Deprotonated Form of Residue FixedY pK, value structure AGi,JkT RSB D85 D212 x1 E204 D96 BR -43.1 15.2 0 -2.3 0 -3.8 0 -10.5 0 12.7 0 12.6 0 Photoisomerizationb K -30.2 12.7 0 12.6 0 10.4 0 -2.4 0 -3.5 0 -13.5 0 Relaxation of 13-cis Chromophore and Protein‘ L‘ -26.3 11.7 0 -0.5 0 -10.4 0 12.3 0 12.7 0 2.8 0 Relaxationd L -22.1 10.8 0 3.8 0 0.4 0 -4.5 0 12.3 0 12.7 0 Deprotonation of the Retinal Schiff Base‘ MI* -26.9 4.1 0 -2.5 0 -11.6 0 18.8 0 11.7 0 14.6 0 -26.4 5.8 0 18.6 0 -1.1 0 -2.3 0 11.5 0 14.9 0 Protonation of Primary Acceptor Aspartate D85 MI -26.6 6.2 0 17.7 0 -3.5 0 - 19.8 0 11.7 0 14.7 0 Protein Conformational Change f MZ(0) -25.0 6.4 0 21.1 0 2.6 0 -13.1 0 8.5 0 12.0 0 -24.7 8.4 0 17.5 0 -1.8 0 -23.2 0 6.1 0 11.00 MA-) -26.1 8.4 0 23.9 0 6.1 0 -10.9 0 8.5 0 13.6 0 Md+) -18.2 6.0 0 14.7 0 -5.3 0 -25.7 0 6.1 0 10.3 0 For pH > 8.59 W-1 -12.0 9.8 0 25.4 0 3.8 0 -10.9 0 f 8.9 0 12.40 21.4 0 -2.2 0 -20.1 -15.9 0 f 6.2 0 Proton Transport from Aspartate D96h Initial Step: D96- + Xz+ (pKa(Xz)= 11.8) - 13.5 0 MNS-1 -14.6 10.1 0 23.8 0 3.5 0 -9.8 0 f 13.7 0 20.6 0 -1.8 0 -18.7 -13.8 0 f 7.8 0 Final Step: D96- XI+ MN*(-) -7.4 5.5 0 13.2 0 -6.1 0 8.0 0 f 1.9 0 -15.7 4.0 0 20.5 0 11.6 0 -9.7 0 f 2.2 0 13.7 0 -20.7 23.1 0 1.8 0 f 1.9 0 -0.2 0 Protonation of Aspartate D212’ -18.5 6.1 0 21.2 0 15.5 0 -14.3 0 f 0.9 0 16.5 0 22.4 0 5.1 0 f -20.8 -19.9 0 0.6 0 Reprotonation of Retinal Schiff Base N-1 -25.7 11.1 0 15.2 0 -5.0 0 -15.3 0 f 3.2 0 “The protein structures are generated by the procedure given in the text. The first row relates to the protonation state for the residues as assumed for the generation of the structure. Additional rows represent conformations with equal or lower ionization free energy. Values in parentheses indicate the number of protons as compared to the BR ground state. The values for the structures BR, K, and L’ are taken from ref 31. kT (298 K) corresponds to 0.6 kcal/mol. Structuralrelaxation of chromophore in “frozen” protein to twisted conformation. Equilibration leads to strong interaction of the retinal Schiff base N-H+ group to main chain oxygen of aspartate D212. Refined geometry incorporating optimal hydrogen bond interaction to the counterion region. e Proton transfer to bound water X1. f Reorientation of arginine R82 toward the extracellular side of the membrane. 8 Deprotonation of glutamate E204, extracellularproton release to bulk-attraction of helices C and G via E204 and R82 in the extracellular region increases the outward tilt of helices F (and E) in the cytoplasmic region-the forming cleft changes the hydration of aspartate D96 and induces its pK, reduction; the proton affinity of the retinal Schiff base increases due to the negative charge of aspartate D212 in the binding site. To the binding site along a chain of hydrogen-bonded water molecules. Requires strong structural changes involving an OH reorientation of tyrosine Y185; then it might serve as an intermediate proton binding site.

+

The first entry in Table 1 summarizes the electrostatic properties of the ground state proton configuration in bacteriorhodopsin. Our electrostatic calculation^^^ revealed that the stable arrangement of the three residues D85, D212, and R82 in a Schiff base counterion complex is necessary to hold the retinal chromophore in its protonated form over a wide pH range. A second positive charge in the binding site (R82) is also necessary to stabilize the negative charge of the two aspartate residues D85 and D212. Due to the hydrophobic environment in the cytoplasmic region the calculated pK, for D96 remains ’12, even with the refined procedure for the calculation of background terms for the protonated and the deprotonated form of the carboxylate group as compared to our previous calculation (see Methods and ref 31). From the two glutamate residues E204 and E9 near the extracellular side, only E9 is stabilized in the deprotonated form by a polar protein and water surrounding. E204 is exposed to the hydrophobic region below the active site and interacts with only a few water

molecules. This environments provides not enough solvation to compensate the large desolvation penalty for E204 and implies, therefore, the high pK, of 13. The simulations of the photoisomerization and the further ground state chromophore and protein relaxations31 lead to a protein structure which corresponds to the first intermediate with 13-cis protonated retinal Schiff base. We will refer to it as early L or L’. The interaction of the chromophore with the protein surrounding is determined by a strong bond of the Schiff base hydrogen to the main chain oxygen of D212 located on helix G. The movement of the Schiff base N-H+ group away from the direction of the proton transfer to aspartate D85 in the subsequent step is consistent with measurements of the fastest electrogenic signal after photoexcitation!6 The hydrogen-bond attack on helix G leads to a rearrangement of this helix and induces a slight outward tilt of helix F in the cytoplasmic region (0.6 A average deviation of backbone atoms compared to the BR ground state), thus indicating that structural changes in the

J. Phys. Chem., Vol. 99, No. 19, 1995 7791

Proton Translocation Steps in Bacteriorhodopsin chromophore binding site are communicated to this membrane surface. As a consequence, the residues threonine T46 and aspartate D96 are pulled into the intrahelical cytoplasmic region. The simultaneous reorientation of water molecules and the OH bond of threonine T89 leads to a strengthening of the hydrogen bonds in the chain extending toward the active site.31 A 50-ps molecular dynamic simulation revealed that the active site trapped water X1 easily reorients toward the chromophore, thus offering a competing favorable hydrogen-bond possibility for the Schiff base N-H+ bond. Relaxation of this structure by energy minimization leads to an intermediate termed L in our simulation of the photocycle. The new interaction pattern connects the protonated Schiff base and aspartate D85 via the intervening bound water X1. As the strong hydrogen bonds between D212 and the neighboring tyrosines Y57 and Y185 dominate, water XI may approach D212 only with an unfavorable angle, thus forming only a weak bond. The initial and crucial proton transfer event in the bacteriorhodopsin photocycle is the loss of the Schiff base proton in the L to M conversion. Microscopic evidence for the function of aspartate D85 as primary acceptor in the L to M transition was provided by a series of FTIR and mutagenesis experim e n t ~ , although ~ . ~ ~ ~ donor ~ and acceptor are not in direct hydrogen-bonding contact. The appearance of a negative FTIR difference band at 3642 cm-I in the L intermediate had shown that in the wild-type photocycle a previously weakly interacting water in the chromophore binding site became strongly interacting.24 The disappearance of this FTIR spectral change in the M intermediate (and in the D85N) mutant indicates that the hydrogen bonding of this water depends on the charge state of the retinal Schiff base and D85 and implicates it in the proton transfer r e a c t i ~ n . ~ ~ - ~ ~ The changed electrostatic interactions in the L structures introduce drastic consequences for the energetics of proton configurations. The destabilization of the ground state charge distribution shows that 10-12.5 kcallmol of the free energy acquired by light absorption is transformed in changed proton affinities for the key residues RSB, D85, D212, and X1 (Table 1). The calculated value corresponds rather well to the calorimetric determined 10 kcallm01.~~The direction of the proton affinity changes31induce the reduction of the free energy gaps between the ground state proton distribution and product states characterizing proton transfer from the protonated Schiff base to D85, X1, and D212. The calculated free energy gap for proton transfer from the protonated Schiff base to aspartate D85 in the BR state is 14.8 kcallmol; for the L’ and L state it reduces to 3.4 and 0.8 kcallmol, respectively. For proton transfer to D212 the corresponding values are 14.8 (BR), 5.0 (L’ ), and 3.8 kcallmol (L), thus energetically favoring transfer to D85 over transfer to D212. The comparison of the values for the structures L and L’ shows that the isomerization induced disrupture of the typical ground state electrostatic interaction pattern reduces the pKa for the Schiff base, simultaneously increasing the pKa for the two active site aspartates, although for structure L’ the movement of the Schiff base proton is away from the counterion region. The hydrogen-bond orientation between the Schiff base N-H+ group and the active site water molecule X1 modulates drastically the proton acceptor facility of X1. Compared to a free energy gap for transfer of 20 kcall mol in BR and L’, the gap reduces to 10 kcallmol after favorable orientation of donor and acceptor in L. We analyzed the involvement of water X1 as proton binding site in the deprotonation step in the photocycle, starting from

structure L according to the following stepwise reaction: RSBH+* OHOH.* aD85RSB.

*

RSB.

- .HOH*

.

-H30+. sD85*D85H

structure (L) structure (M1*) structure (M,)

- -

The approximate activation barrier ELM^* for the L MI* step is 10 kcaymol, the barrier EMI*Lfor the back-transfer MI* L is 9 kcallmol. Due to the proton sharing relationship between X1 and D85 in structure M1*,31the average protonationfH of X1 in MI* is only 0.6 for pH 2 7. With the approximate rates kLMI*= k, exp(-E,,,,lkr)

for the L

-

M1* transfer

for the M I *

kM,*L=fH(Xl)ko exp(-EMl,,/kT)

-

L step

we calculate a law of mass action [L]/[Ml*] = 4.3 for the first transfer step, corresponding to the [L]/[M] concentration ratio reported from a kinetics study at room temperature.2 Due to a low activation barrier for the MI* M1 step (0.3 kcallmol, Table l), MI* catalyzes fast transfer to aspartate D85 (structure MI). The high activation barrier of 18 kcallmol for back-transfer in the M1 structure (vide inffa) favors the unidirectionally of the second step. The alternative intermediate transfer step (RSB. * *OH-. * *D85H) is energetically less favorable, as i requires the stabilization of OH- near the negative charge of D212 in the active site. Protein Conformational Changes and Altered Proton Distribution after Deprotonation of the Retinal Schiff Base. A stable proton configuration with an ionization free energy of - 16 kcallmol (Table 1) develops after localization of the proton on D85 (MI state). In Figure 2 we compare the arrangement of key residues and water molecules with the precursor structure L’. The changed charge state of the active site has several consequences for the structure of the protein. First, the water molecule X1 reorients and enters in a strong hydrogen bond with the water channel spanning the interhelical region toward the cytoplasmic membrane side. Therefore, the hydrogen bond between X1 and D85 becomes weak, leading to the large free energy gap for back-transfer of the proton from D85 to X1 (18 kcallmol). Second, an increased outward tilt of helix F (averag? deviation compared to BR increases from 0.6 A in L’ to 1.O A in MI) shows that changes in the charge state of the active site due to the redistribution of protons are effectively communicated to the membrane surface. The widening of the cytoplasmic channel increases the torsional flexibility of D96, whose reorientation time is near 200 ps, as judged from an accompanying molecular dynamic simulation. The existence of more than one M state is now supported by spectros~opic~-~ 1~13,49and kinetic observation^.^^^,^ In the kinetic scheme with two sequential M states, its interconversion is attributed to a protein based “switch ~ t e p ” . ~ .In’ ~the scheme with independent parallel photocycles, different BR parent protein populations give rise to subpopulations with different decay (and rise) Additionally there was argued” that during the lifetime of the fast decaying population a transition between two protein states takes place. Despite the variety of experimental evidence (reviewed in ref 14) for different protein conformations, none of these experiments revealed the molecular nature of the conformations and the interconversion between them. In our previous c ~ n t r i b u t i o n ,we ~ ~ presented a “working model” for a protein heterogeneity, utilizing the positional flexibility of arginine R82. The reorientation of R82 away from

-

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7792 J. Phys. Chem., Vol. 99, No. 19, 1995

Figure 2. Comparison of the position of key groups in the MI structure (dark grey) and the precursor structure L’ (light grey).

the chromophore binding site toward the extracellular region generates the new structure developing after the protonation of aspartate D85 in the precursor MI state, which is referred to as the M2 structure. This model is able to connect the active site protonation changes to proton release on the extracellular side, defines glutamate E204 as extracellular release group and accounts for the pH-dependent branching of the photocycle as it is suggested in the kinetic scheme of? In particular, it explains the vectoriality of the release step despite the lack of an extracellular hydrogen-bonded water channel. Now we reexamined the possible molecular origin for the turning down of arginine R82 in the M1 structure. Although the arguments given here are only qualitative, they nevertheless lead to important insight. After protonation of D85 in the MI* to M1 step, no hydrogen bond holds R82 in the active site (see Figure 2). The electrostatic stabilization reduces from -26 kcaY mol in L to -14 kcaymol in MI and can be attributed to the interaction with the negatively charged D2 12. This interaction

Figure 3. Comparison of the position of key groups in the early M2 structure (dark grey) with the precursor structure MI (light grey). The M2) involves the folding conversion of the two M structures (MI back of arginine R82 away from the chromophore binding site toward the extracellular side of the membrane.

-.

is, however, screened by the protein dipoles of the neighboring tyrosine residues Y57 and Y 185. The repulsive interaction to the protonated aspartate D85 (approximately 2 kcal/mol) forces the arginine side chain in a strained conformation which CpCy and Cy-Cfi torsional angles near their maximum potential energy values. For fluctuations, that overcome the remaining small barrier, new stabilization can be gained through a reorganization of the water molecules in the extracellular region (compare, e.g., Figure 4 in ref 31). This favorable solvation stabilizes the position of arginine R82 near the extracellular region. Figure 3 shows the arrangement of selected residues for the structure after repositioning of R82 and subsequent protein relaxation. Due to the distinct solvation of the negative charge on aspartate D212 by the protein dipoles of tyrosines Y57 and Y 185, the chromophore binding site remains stable, even after removal of the positive charge of R82, which ensured

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Proton Translocation Steps in Bacteriorhodopsin

4.0

5.0

6.0

7.0

8.0

0

RSB

&

E204

9.0 10.0 11.0

PH Figure 4. Average protonationfH of the retinal Schiff base (RSB)and glutamate E204 in the protein structure M2, calculated for a Boltzmann weighted distribution of ionization states.

electrostatic neutrality in MI. The concomitant change of the electrostatic interactions in the active site leads to further rearrangement in the cytoplasmic region in the vicinity of helices E and F. We find an increased average outward tilt of the cytoplasmic end of helix F to 1.5 A. This tilting is near the value proposed from a recent electron microscopic study,18 which reports a movement of up to 2 A. Similar changes in the tertiary structure are also reported by neutron and X-ray diffraction studies. l5,I6 The approximately equal ionization free energies (Table 1) for the two protein conformations MI and M:! (before proton release) indicate comparable electrostatic stability. The enlarged cleft in the cytoplasmic region leads to an easier reorientation of D96 and opens the possibility for a change in hydration, thus inducing a slight decrease in its pKa. The single negative charge in the binding site leads to an increase in the proton affinity of RSB. The approaching positive charge of R82 destabilizes the proton on E204 in the extracellular region. Close lying protonation states31 give rise to charge heterogeneities. These states differ not only in the distribution of protons among the key residues but also by the total amount of protons in the protein: M2(0), Mz(+), Mz(-) (the number of protons in the protein relative to the BR ground state is given in parentheses; see Table 1). The pH-dependent population of these states account for a branching of the proton translocation steps for the M2 protein conformation. The calculated midpoint between net release and net uptake (pH 7.2 31)corresponds to the apparent pKa = 6.3 for proton release reported in ref 2. Figure 4 shows the fractional protonation for the retinal Schiff base RSB and glutamate E204 for a Boltzmann weighted distribution of ionization states. For pH > 8.5 the proton configuration Mz(-) with deprotonated RSB and deprotonated glutamate E204 determines the titration behavior of the protein. Between pH 6 and 8.5 the average protonation for both E204 and the RSB is near 0.5 (populations Mz(0)). For pH < 6 no release occurs; the Schiff base tends to reprotonate (M:!(+)). Reprotonation Steps after Extracellular Proton Release. As a next step we have to analyze whether and in which way our protein conformation Mz can really act as switch for the reprotonation of the RSB. It was that aspartate D96 plays an important role in the reconstitution steps of the photocycle. In mutants, where D96 was substituted with nontitratable g r o ~ p s , ~ lthe -~~ rates for the proton pumping drastically decreased. This has stimulated the idea that D96 acts as an internal donor for the reprotonation of the retinal Schiff base. Since the distance between the donor D96 and acceptor RSB is approximately 10 A, the proton has to be transported across the hydrophobic protein interior along the

chain of hydrogen-bonded titratable residues and water molecules. The inhibition of the transport due to dehydration of the p r ~ t e i nsupports ~~,~~ the involvement of water molecules in this proton translocation step. Independent of the kinetic scheme used for the interpretation of spectroscopic data,2,7,8s11 it was found that an intermediate with protonated 13-cis retinal Schiff base (N intermediate) can only be accumulated in the alkaline pH region. FTIR changes occurring before the appearance of the following intermediate argue for the existence of additional transient states after proton release in the alkaline. It is proposed55 that, in these states, the proton on D96 becomes ~ , ~ ~ that the destabilized. Time-resolved FTIR s p e ~ t r a lindicate deprotonation of D96 is coincident (on the 2 0 9 s time scale of the experiment) with the reprotonation of the Schiff base. In other pH regions, reisomerization may occur before or competing with (e.g., ref 11) reprotonation. An evaluation of the order of the two crucial steps for the recovery of the BR ground state is possible when the determining activation parameters can be calculated. The unambigiousness of the experimental results for the high-pH region facilitates the comparison to our theoretical results. Favored deprotonation of E204 occurs according to our calculations in the alkaline pH region for pH > 8.5. The relaxation of the protein structure after extracellular release leads to structure M2(-), which is compared in Figure 5 with its precursor before release. The docking of the helices G and C due to the attractive interaction between the positive R82 (on C) and the (now) negative E204 (on G) enhances the outward tilt of helix F in the cytoplasmic region to an average deviation of 1.8 A, thus arguing for a communication between the two sides of the membrane along the helices. Figure 6 compares the orientation of the a-helices in the relaxed M2(-) structure with the BR ground state. This movement of helix F opens the region around D96 and allows additional water molecules to enter. The widening of the channel favors the twist of the COOH group in order to adopt an energetically more favorable orientation of its proton toward the interhelical water chain. This reorientation is coupled to a rearrangement of water X2, which is held in a three-center hydrogen-bond complex with D96 and threonine T46. These local changes in its dipolar environment destabilize the proton of D96, thus reducing its pKa from values '12 to 8.9 (see Table 1). The experimental pKa values are between 7 and 9.50,57The closer approach of water molecules in the region above D96 provides a pKa reduction by 2 units. The same downshift can be attributed to the reorientation of water X2. The orientation of the hydroxyl group of T46, which helps to fix water X2 near D96 is responsible for an upshift of the pKa of D96 by 1 unit. This finding is consistent with experimental indicating that the substitution of this residue with a neutral one speeds up the reprotonation step. The structural changes in the cytoplasmic end of the water chain are communicated to the active site, where they lead to a reorientation of water molecules. The induced relaxations give rise to an increase in the proton affinity of RSB to pKa = 9.8. The calculated difference in proton affinity for D96 and RSB of 1 pK, unit is also reported for the high-pH cycle of wild-type bacteriorh~dopsin.~~ The relative order of proton affinities of D96 and RSB is in accordance with the appearance of a N-like proton configuration as the most stable one (Table 1). Our calculations give no clue for a pKa reduction of D85, that could favor an altemative reprotonation of the retinal Schiff base, as it was proposed by a vibrational analysis of the photocycle."

Scharnagl et al.

7794 J. Phys. Chem., Vol. 99, No. 19, 1995

CYTOPLASM

s ‘ ! /-.:

‘

Figure 5. Comparison of the position of key groups in the relaxed M2( -) structure after extracellular proton release from glutamate E204 (dark grey) with the early M2 structure (light grey).

Using the step-by-step model in Chart 1, we analyzed the energetics of the transport of the proton from D96 to the active site: CHART 1 (X2)

(Xl)

(active site)

D96-H ... HOH ... (HOH), ... HOH (RSB, D212-) structure M2(-) D96- ... H3O+... (HOH), ... HOH (RSB, D2127 “injection”:MN,(-) ...transport along (HOH),. .. D96-H ... HOH... (HOH), ... HsO+ (RSB, D2127 “ejection”:MN,(-)

The injection region is the three-center hydrogen-bonded complex of D96, T46, and water molecule X2. From there the transport takes place along a single file of water molecules, including the hydroxyl side chain of threonine T89. The last element in the sequence is water molecule X1 in the active site, which is in hydrogen-bonding distance (2.4 A) to the Schiff base nitrogen. This favorable arrangement of X1 suggests its bivalent function as an intermediate proton binding site also

EXTERIOR Figure 6. Comparison of the orientation of the transmembrane a-helices in the BR ground-state structure (light grey) and the relaxed M2(-) structure (dark grey). Shown are the retinal Schiff base, arginine R82, and aspartate D96. Changes in the active site charge distribution and near the extracellular side of the membrane after proton release are effectively communicated to the cytoplasmic region.

for the reprotonation step. The active site contains the two possible acceptors D212- and the Schiff base. To calculate the energetics along of the transport on a molecular level, we treat water molecules and threonine side chains as protonatable sites in the low dielectric region in the protein interior.58 We follow the individual relaxation steps by short molecular dynamic runs (10 ps, a time which allows for the rotational reorientation time of water molecules59)and energy minimization. The most energy demanding step is the separation of the proton from D96 and its stabilization on water X2, thus creating two charged groups in a region of low dielectric constant. The desolvation penalty for these groups adds up to 42 kcal/mol. As the calculated actual free energy gap for the transfer is 16 kcal/mol, 26 kcal/mol of electrostatic stabilization is provided by solvent and protein dipoles, mainly due to the reorientation of the OH bond from nearby T46 and the hydration provided by the surrounding water dipoles. Both structural rearrange-

J. Phys. Chem., Vol. 99, No. 19, 1995 7795

Proton Translocation Steps in Bacteriorhodopsin ments are facilitated by the widening of the cytoplasmic region. The transfer of the proton back to D96- in MN,(-) requires 3 kcdmol. The energy of injection is given by the difference of the free energy gaps for forward and backward transfer. The calculated energy of 13 kcallmol is in the order of the experimentally determined barrier for the proton transfer from D96 to RSB (10 kcal/mol).60 The step by step transport of the proton along the chain of R-OH groups involves members of equal desolvation penalty. Therefore, the energetics of the transport is determined only by the electrostatic interaction of the positive charge with protein and water dipoles and the two negatively charged aspartates, D96 in the cytoplasmic region and D212 in the chromophore binding site. Like in the previous steps, the deprotonated form of D212 is stabilized by the hydrogen bonds with the two neighboring tyrosines Y57 and Y185. As no polar side chains line the transport channel, the positive charge can be stabilized without large energy costs by the fast reorientation of neighboring water dipoles. For the steps toward T89, the sum of electrostatic attraction excerted on the proton from the two negative charged ends of the channel is constant (approximately 15 kcallmol). The activation barriers are small (1.2-2 kcall mol); the steps proceed fast and near equilibrium. The fast transfer corresponds to proton conduction via a string of water molecules, as found, e.g., in g r a m i ~ i d i n .Near ~ ~ the active site (next to threonine T89) the transport goes downhill due to a negative free energy gap (approximately -2.5 kcaymol). The large attraction from the negative D212 speeds up the transfer. This shows that the presence of a negative charge near the chromophore, which is a result of the proceeding protein conformational change, is a prerequisite for the vectoriality of the reprotonation step. Without this structural element, the attraction by D96 in the cytoplasmic region enhances backtransport in the first steps, as no stabilization by protein dipoles can be provided. Additionally, the removal of the positive charge of arginine R82 from the binding site allows the proton to reenter in a surrounding with protonated D85. For all protein structures along the transport chain, the retinal Schiff base is the residue with the highest proton affinity, leading to a N-like lowest free energy proton configuration (Table 1). The final transient proton binding site in our calculation is the water molecule X1 (structure MN*(-)). From there, the proton will be ejected into the chromophore binding site, where RSB and D212 with graduated affinities (pKa(X1) < pKa(D212) < pKa(RSB) (Table 1)) are possible acceptors. The involvement of D212 as intermediate proton binding site has been discussed,6l but its protonation during the photocycle is now excluded by investigations of NMR chemical shifts62 and FTIR.63 The protonation of D212 at that stage of the cycle would require large structural changes, involving the breaking and reorientation of the strong hydrogen bonds to the tyrosine neighbors. We enforced this structural change using molecular modeling and energy minimization. The results of the electrostatic calculation (included in Table 1) reveal, that even in this case, the proton will not be stabilized on D212. These calculations show that the barrier, which determines the rate of proton transfer from D96 to RSB, consists largely of the enthalpy cost of separating the proton from D96 and amounts to 13 kcallmol. In order to prove consistently that reprotonation proceeds the reisomerization and that, therefore, our structure is really capable of accumulating an N intermediate, it is necessary to compare the activation parameters for reprotonation and thermal reisomerization of the unprotonated retinal Schiff base in the M2( -) structure. Generally, this barrier depends strongly on the charge environment of the chro-

mophore.@ By use of an extensive quantum chemical calculation, we determined this barrier for the chromophore in the charge environment given for structure Mz(-). The calculated value (27 kcdmol) is twice the value of the reprotonation barrier and shows unambiguously that reprotonation preceeds reisomerization. The orientation of key residues in the protein structure after reprotonation of the RSB (structure N(-)) is compared to precursor M2(-) in Figure 7. The (-) sign indicates that no uptake of a proton has taken place up to now. As in the L‘ structure, the chromophore enters in a strong hydrogen-bond interaction with the protein backbone of helix G. As a result, helix G is pulled down, whereas the outward tilt of the cytoplasmic end of helix F reduces to the value before the deprotonation of E204 (1.6 A). This induces a slight reorientation of the water molecules near D96 and shifts its pKa up (Table 1). Due to the absence of R82 from the binding site, the nature of the counterion for N(-) and L differs. Despite this difference the calculated proton affinity of RSB is comparable to L’ (Table 1). Additionally, we probed the influence of the different nature of the counterion in our L and N(-) protein conformations on the electronic structure of the chromophore by quantum chemical calculations. The calculated absorption wavelengths (L’, 525 nm; N(-), 529 nm) are nearly identical. As it is generally expected that changes in the vibrational spectra in the C=C stretch region are correlated with wavelength shifts of the visible absorption,” this argues for a largely unaltered C=C stretch pattern of the protonated retinal Schiff base in N compared to L as it is indeed reported from a resonance Raman study.” The theoretical value for the C13=C14 torsional barrier of the protonated retinal Schiff base in this N(-) environment is 18 kcallmol, indicating that the N intermediate will live long enough to allow for further protein conformational changes that will, e.g., induce the reprotonation of D96.

Discussion The principal question for the photocycle of bacteriorhodpsin is in which way the “reaction center” at the chromophore binding site is coupled to the protein environment in order to induce vectorial proton pumping. The use of combined molecular dynamic, electrostatic, and quantum chemical calculations allows the analysis of the connection between isomerization induced geometry changes in the retinal photoreceptor chromophore, the accompanying protein relaxations, and changes in proton affinities of protonatable residues. The generation of the structures of the intermediates has to rely on the experimental information about the unphotolyzed p r ~ t e i n .A ~ careful modeling of the interhelical water molecules by means of molecular dynamics29-31~35 leads to some insight in their possible locations and interactions with protein residues. Starting from one ground state protein conformation, which is near the experimentally determined one, a molecular dynamic analysis shows what kind of conformational changes might occur upon i s o m e r i z a t i ~ n .A~reliable ~~~~~~~ electrostatic calculation like the FDPB method connects the population of protonation substates with the overall protein conformation, the interaction to permanent dipoles and other titratable group^.^^,^^ The explicit treatment of water molecules as members of the low dielectric protein interior is necessary for a reliable predition of pKa values,33thus allowing for direct hydrogen-bond interaction to charged groups. Their inclusion as titratable sites facilitates the discussion of their role as intermediate proton carriers in the de- and reprotonation step.31,58 The geometrical relationship of the donor-acceptor pair and

7796 J. Phys. Chem., Vol. 99, No. 19, 1995

Scharnagl et al.

.

."

Figure 7. Comparison of the position of key groups in the N(-) structure (dark grey) with its precursor structure M2(-) (light grey). The insert shows only a selected part of the retinal around the Schiff base linkage and aspartate D212 from a slightly different perspective.

the ionization free energy gap between reactant and product states provide a guideline for proton transfer possibilities, the energetics of the different steps, and the driving forces for a change of the ionization states. Following this guide, the relaxation of the protein conformation according to the new distribution of protons followed by the analysis of the new distribution of ionization states allows the generation of steps leading from one intermediate to the other without additional input, e.g., from proposed kinetic schemes (model-independent approach). At each step, the rates of forward transfer and backtransfer can be examined and the free energy gaps can be compared to those from competing steps. In our starting structure for the unphotolyzed protein, arginine R82 is positioned near the chromophore binding site, thus providing a complex hydrogen-bonded counterion complex for the protonated RSB.31 The calculated proton affinities (Table 1) are in reasonable agreement with experiment; e.g., we calculate a pKa value of 15.2 for the retinal Schiff base, while the experimental value is 13.7.@ The overall arrangement of

the charged members R82,RSB,D85,and D212 in the binding site resembles a quadrupole, as it is corroborated by N M R spectro~copy.~~ The titration behavior of these four groups is determined mainly by strong local interaction^.^^+^^-^^ The same interaction pattern is found for another photochemically active protein, which might serve as a sensory pigment for the chromatic adaptation of the photosynthetic antenna of cyanobacteria.65 Unlike bacteriorhodopsin, this prdtein is globular, thus indicating that the strong local interactions might be typical for sensory and energy transducing pigments. A recent study dealing also with the calculation of proton affinities in the BR ground state35shows that the electrostatic potential generated by a cluster of ordered water molecules in the extracellular region near the active site might also be able to stabilize the protonation of the retinal Schiff base and the two negative charges of the active site aspartate residues D212 and D85.As already suggested by Henderson et a1.: arginine R82 might fluctuate between the positions near the extracellular side and the active site. The different populations of these two confor-

Proton Translocation Steps in Bacteriorhodopsin mations could be the reason for a heterogeneity of the parent state. However, the close proximity of the two negative charges of D85 and D212 without the balancing positive charge of R82 nearby argues for a blue-shifted visible absorptionM of the protonated retinal Schiff base. An appreciable population of the second state can then be ruled out. Besides the structural heterogeneity due the possible flexibility of arginine R82, fluctuations among different protonation substates on different time scales can also be d i s c ~ s s e d . ~ ~ The position of the structurally active water in the binding site as predicted by our modeling procedure31 is corraborated by a recent FTIR Water molecule X1 is trapped in the center of the cavity built by the members of the quadrupole. The presence of several strongly bound water dipoles near the Schiff base is supported by a variety of experiment^.^^,^^,^^,^^ Numerous studies have also predicted, that water and hydroxylbearing side chains ( e g , threonine T89) will form a hydrogenbonded network which conducts the proton along the hydrophobic cytoplasmic channel to the retinal Schiff base in the reprotonation step.25,26,54The present analysis of the proton transfer shows that the active site caged water can serve as an intermediate proton binding site in both steps involving transfer of the proton from or to the chromophore. Its action as orientational flexible proton shuttle in the reprotonation step is functionally important for the completion of the photocycle under those conditions where the reisomerization preceeds reprotonation. The dense packing of residues and water molecules in the active site ensures the coupling of the retinal isomerization to movements of protein residues and the protein backbone. The changed geometrical relationship between the protonated RSB and members of the hydrogen-bonded counterion complex is a critical determinant for the stabilization of ionization substates with changed proton distributions after retinal isomerization. Our investigation shows how the formation of a strong and favorable oriented hydrogen bond between the protonated RSB and the water molecule in the counterion region, X1, favors deprotonation. The rather unfavorable hydrogen-bond geometry between X1 and the second aspartate in the binding site, D212, is the reason for the unidirectional transfer to D85, whose role as primary stable proton binding site is experimentally establ i ~ h e d . ~ , With ~ , ~ ’ X1 as the intermediate proton binding site, the deprotonation is resolved in two steps, the transfer from RSBH+ to X1 (L to MI*) and the subsequent transfer to D85 (MI* to MI). The calculated equilibrium constant for the first step is near the experimental value for the [L]/[M] concentration ratio of 4 calculated from a kinetics analysis at room temperature.2 The second step (MI* MI) is almost barrierless and unidirectional. At the stage of the M intermediate, the vectoriality of the pumping process will be decided. The existence of several M substates which differ in protein conformation and/or ionization state of titratable residues, thus controlling the transfer steps in different ways, is established by a variety of experimental e ~ i d e n c e . * . ~ -Recently, ’~ we presented a working modeP1 for a protein conformational change developing after the primary transfer step that proposes a reorientation of arginine R82 away from the chromophore binding side toward the extracellular region. The actual molecular dynamic and electrostatic analysis gives qualitative arguments for the destabilization of R82 in the active site after protonation of D85. Although these arguments are only preliminary, the consequences of this conformational change, which are reported in this and the previous paper,31account for a variety of experimental observations. Additional support is provided by our recent quantum chemical calculation (to be published), which shows that the

-

J. Phys. Chem., Vol. 99, No. 19, 1995 1191 removal of arginine R82 from the binding site results in a blueshifted visible absorption by 10 nm. This is consistent with the experimental wavelength shift for the two M intermediates reported in ref 68. We argue for its functional role as a control switch for the further proton translocation steps. P r e v i o ~ s l y ~ ~ we analyzed how the switch to this protein conformation favors extracellular proton release. This contribution shows that it really acts as a reprotonation switch by providing the requirements for speeding up the transport of the proton to the chromophore and delaying its reisomerization. A main feature of the protein structure with reoriented arginine R82, which we term Mz, is a change in the chromophore’s binding site charge state, leaving this region with a single negative charge on aspartate D212, which is stabilized due to the solvation by the neighboring protein dipoles of tyrosine residues Y57 and Y185. Arginine R82 now interacts with glutamate E204 in the extracellular region of the protein. An ensemble of close lying ionization states are switching the protonation of RSB, E204, and D96. Their pH-dependent population induces the branching of the phot~cycle.~’: For pH > 8.5 glutamate E204 releases its proton to the extracellular aqueous phase (structure Mz(-)). Therefore, we identified E204 as a release group for the bacteriorhodopsin photocycle. For pH < 6 a proton is taken up in order to reprotonate the retinal Schiff base (structure M2(+)). In the intermediate pH range the proton is located either on RSB or E204 without a change in the total number of protons compared to the precursor structures (structures Mz(0)). The midpoint for the switch between net uptake and net release is 7.2,31near the experimental value of 6.3 reported by a thermodynamic study of the wildtype photocycle.2 The existence of this variety of ionization substates during the lifetime of the M intermediate could kinetically or spectroscopically merge the IR signal due to the deprotonation of E204 with signals from other residues, thus explaining the fact that this residue has not been identified by IR experiments as release group; eg., an unresolved bond pattern around 1732 cm-l in M has been analyzed as the composition of D115 and D96 difference bands.” Although our calculations rationalize the role of E204 as the release group, there are a few other residues with hydroxyl groups nearby (e.g., threonine T205). Their IR pattern due to deprotonation will be blurred with water structural changes. The molecular mechanism for this release step has to be investigated further. To study the completion of the cycle after release, we traced the protein conformational relaxation and the redistribution of ionization states associated with the deprotonation of E204 (relaxed structure Mz(-)). This leads to relevant predictions for the completion of the photocycle in the pH region above 8.5, termed as the “high-pH’ region for our theoretical study. Two structural features are favorable for speeding up the RSB reprotonation. First, the additional widening of the cytoplasmic channel favors hydration of aspartate D96 and leads to an increased destabilization of its proton as compared to the structure before release. Second, along its way during the cytoplasmic channel, the proton is pulled forward by the single negative charge of D212 in the active site. Additionally, this negative charge increases the proton affinity of the retinal Schiff base to a value near the pK, of D96. The overall rate for the transfer of the proton from D96 to the Schiff base is determined by the energy cost for the stabilization of the pair of oppositely charged groups in the injection step (13 kcdmol). As the torsional barrier for reisomerization of the Schiff base is twice as large, reprotonation preceeds reisomerization for the relaxed M2(-) subpopulation, thus favoring the formation of an intermediate with reprotonated Schiff base but one proton deficit

7798 J. Phys. Chem., Vol. 99, No. 19, 1995

Scharnagl et al.

A-D RSB

D212

R82

E204

L(0)

(0)

positively charged residue

1

*.........................

N (=)

€3- neutral residue

O--

T

negatively charged residue

Figure 8. Molecular model: Schematic representation of chromophore and protein conformational changes and proton translocation steps during the bacteriorhodopsin photocycle in the alkaline pH region. The minus sign (in brackets) indicates the temporary loss of a proton after extracellular release compared to the number of protons in the BR ground state (indicated by (0)). The steps for the recovering of the BR ground state from the N(-) structure are only speculative.

as compared to the precursors: the N(-) intermediate. These results can be applied in order to discuss qualitatively the decay behavior for other M subpopulation. The most important activation parameter for the reprotonation, the injection energy, is proportional to the proton affinity of the internal donor D96 and, therefore, correlated to the hydration of its protonated carboxylate group. If the cytoplasmic channel is wide enough, additional water molecules can enter and easily reorient. A favorable hydration of D96 will destabilize the proton. The large PKa reduction speeds up the reprotonation. This is the case for the relaxed M2(-) structure, e.g., after extracellular release induced a large cytoplasmic cleft. If the channel is narrower due to the reduced outward tilt of helix F (e.g., in structure M2(0) and all precursors), a higher PKa of D96 increases the energy cost for injection and, therefore, the lifetime of the M intermediate. In this connection, we can correlate the relaxed M2(-) structure with a fast decaying population of M, the M2(0,+) structures with the slower decaying ones. As the altered charge distribution in the M1 active site favors backtransfer of the proton to D96 and delays, therefore, the transport to RSB, M1 structures would belong to the slow decaying M populations. The existence of M subpopulations with different decay times has long been p~b1ished.l~Our analysis shows that they can be attributed to a heterogeneity developing after the first proton transfer step. They need not to be an inherent property of the unphotolyzed BR protein, as it is for example argued in refs 9 and 11. The prediction of our calculation, that for the fast decaying M compound release proceeds uptake, should be verified by suitable electrogenicity measurements. The relative order of reprotonation and reisomerization is determined by the activation energies for the two processes. Our quantum chemical calculations and related papersa revealed that the barrier for thermal reisomerization strongly depends on the protonation state of the chromophore and its charge environment. Due to the high torsional barrier for the retinal

Schiff base in the M2( -) structure, reisomerization succeeded the reprotonation, thus leading to an N intermediate (protonated, 13-cis RSB). The result that a fast decaying M is followed by an accumulation of N is corroborated by all experiments (e.g., refs 2 and 11). For other M subpopulations, e.g., without deprotonation of the release group and higher injection energy due to less favorable solvation of D96, the situation will not be so obvious. With comparable reisomerization and reprotonation parameters, these steps will compete. This will explain why in the lower pH photocycle the chromophore in the next stable intermediate (0)is already the protonated, all-trans RSB (e.g., ref 2). Under these conditions, it will also be possible that two M intermediates with different configuration of the C 13=C14 retinal bond are present, as it was proposed in ref 11. It would also indicate that the proton in the M BR sequence has to be transported either to the 13-cis or to the all-trans Schiff base, supporting therefore our transport model where an active site bound water with different flexibility acts as a transient binding site for reprotonation. According to our results, the long lifetime of the N intermediate is a direct consequence of the charge environment of the protonated retinal Schiff base, which increases the reisomerization barrier. For the completion of the photocycle, the negative charge of D212 has to be screened partially in order to reduce the barrier for thermal reisomerization of the retinal Schiff base around the C13=C14 bond. The recovery of the ground state requires further that the proton affinity of D96 has to increase to favor uptake of a proton from the cytoplasm. This can be accomplished by partial closing of the cytoplasmic channel, as is indicated by the upshift of its PKa in our N(-) structure, occurring together with a partial inward tilt of helix F in that region. It is conceivable that the proton uptake by D96 primarily induces further conformational changes in the protein backbone, e.g., the repositioning of helix F. Due to the

-

Proton Translocation Steps in Bacteriorhodopsin long range communication between cytoplasmic and extracellular parts of the protein, this could start the reorientation of arginine R82 toward the active site, where overall neutrality of the counterion complex catalyzes the reisomerization. Further consequences would be the increase of the proton affinity of E204 to its original value and the destabilization of the proton of D85 in the active site. Its proton could then be transferred to the release group. Our model for the photocycle requires, therefore, a second interconversion between the two global protein conformations after the reprotonation step. This proposal is supported by experimental evidence (e.g., crystallographic and spectroscopic method^,'^,^^ thermodynamic arguments7s8) for large scale protein conformational changes which return the protein to the original state as N decays. The extension of our calculations has to establish the order and energetics of these steps. The existence of two protein conformations that change access of the retinal Schiff base from the exterior to the cytoplasm has been proposed by several g r o ~ p s . ' ~The J ~ link of the charge state of an active site to global protein conformations has recently been postulated as a paradigm that applies generally for proteins with sensory as well as energy-transducing funct i o n ~ . Our ~ ~ contribution presents a molecular model for mechanisms of an effective communication between the active site and the protein. The first communication after isomerization is a strong hydrogen-bond contact (short range) between the Schiff base proton and the backbone of helix G. The response of the protein is a slight outward tilt of helix F in the cytoplasmic half. The redistribution of protons, e.g., the protonation of D85 in direct contact to arginine R82 in the active site, destabilizes this residue. Its switch to the extracellular side changes the overall charge in the chromophore binding site. The protein responds again with an outward tilt of helix F in the cytoplasmic region. The same response occurs after the deprotonation of the extracellular release group (E204), thus indicating an effective communication also between the two opposite sides of the membrane. After reprotonation, the short range mechanism reverts the tilt partially. Our model for the protein conformation changes and proton translocation steps in the bacteriorhodopsin photocycle is summarized in Figure 8. According to this model, the population of the two principal protein conformations with arginine R82 in the active site or near the exterior, respectively, is shifted at several stages of the cycle. A consequent extension of this concept leads to the existence of these states also in the unphotolyzed BR protein. The results of a recent electrostatics calculation indicate that the charge state of D85, D212, and the RSB in the BR ground state can also be stabilized with R82 near the extracellular side.35 This would indicate that both states could be populated already in the ground state, thereby giving rise to the existence of several parallel pathways?-'* where the transfer steps might be controlled in a different way. The X-ray diffraction of D85N mutants with deprotonated retinal Schiff base shows14the same changes as they occur in wild type after deprotonation in the M state, thus arguing for the population of the same global protein conformation. An effective test calculation should probe the relative stability of the two arginine orientations in both proteins by molecular dynamic and electrostatic calculations in order to develop a detailed model for the driving forces leading to the interconversion of the two conformations. Just like our preliminary analysis, this X-ray study argues for the dominant role of the charge state of aspartate D85 for the switch between the population.

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