Comparison between the Binding of Ca2+ and Mg2+ to the Two High

J. Phys. Chem. 1995, 99, 11600-11604

11600

Comparison between the Binding of Ca2+ and Mg2+ to the Two High-Affinity Sites of Bacteriorhodopsin Seoung-Kyo Yoo, Elias S . Awad, and Mostafa A. El-Sayed* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 Received: January 9, 1995; In Final Form: May 17, 1995@

Bacteriorhodopsin contains Ca2+and Mg2+ ions whose removal inhibits its proton pump function. The binding constants of Ca2+ to the high-affinity sites were determined by the use of a calcium ion specific electrode. The unavailability of magnesium ion specific electrode prevented a similar determination for Mg2+. The binding constant of Mg2+ to the binding site of highest affinity is determined by using a calcium ion selective electrode to measure the concentration of free Ca2+ in competition with Mg2+ for the binding. The binding constant of Mg2+ to the second high affinity site is determined spectrally. The two high-affinity binding constants for Mg2+are compared with those obtained for Ca2+ in the absence and the presence of Mg2+. The fact that the presence of low concentration of one metal ion does not affect the binding constant of the other metal ion to the other binding site supports the assumption of the independence of the two high-affinity sites of one another. The difference in the observed values of the binding constants of the two high-affinity sites for Ca2+ and Mg2+ is qualitatively discussed in terms of the enthalpy and entropy changes in the binding equilibrium.

I. Introduction Bacteriorhodopsin (bR) is a retinal protein found in the purple membrane of Halobacterium salinarium. Under conditions of oxygen deprivation,bR functions as a photosynthetic system. The absorption of a photon by the chromophore retinal induces an isomerization from all-trans in the ground state to 13-cis, initiating a cyclic sequence of intermediates K, L, M, N, 0.4,5 The net effect of the photocycle is the translocation of hydrogen ions from the cytoplasm outwards, with high quantum yield under certain condition^.^.^ The hydrogen ion gradient produced across the bacterial cell membrane then serves as a source of chemical energy.3.4s8-9The structure and reactivity of bacteriorhodopsin have been the subject of a number of recent A detailed description of the structure of bR, based on highresolution electron cryomicroscopyIs reveals the juxtaposition of key amino acid residues which participate in the proton translocation mechanism, the “proton channel”. The sequence of the 248 amino acid residues of bR is k n o ~ n . ’ ~The - ~ retinal ~ chromophore is attached to Lys216 by formation of a protonated Schiff base (PSB) linkage.22-24 Even though the chemical bonding to Lys216 is not required for proton pumping, the anchoring of the PSB enhances its effectiveness in the proton translocation mechanism.25 Of all the intermediates, only in M410 is the Schiff base deprotonated. In the L-M step of the photocycle, H+ is transferred from the PSB to the carboxylate group of Asp85.26-28 The Schiff base (SB) is reprotonated in the M-N step by transfer of a proton from the carboxyl of Asp96 probably via an intervening prototropic group (possibly bound H20).29-32The release of a hydrogen ion into the bulk aqueous phase at the periplasmic side of the membrane occurs after a delay following the L-M step.33 The reisomerization of the retinal takes place prior to the 0 intermediate.34 This is followed by reprotonation of Asp96 in the 0-N step and the final conformation relaxtion of the protein around the retinal in the N-bR step.35.36

* To whom correspondence @

should be addressed. Abstract published in Advance ACS Abstracts, June 15, 1995.

0022-3654/95/2099-11600$09.00/0

It is known that purple bR (A,, = 568 nm), in its native state, contains a number of tightly bound Mg2+ and CaZf ions. Chang et al.38reported 3-4 mol of magnesium and about one mol of calcium per mol of bR in the well-washed purple membrane. Removal of all ions by an ion-exchange column37-39 or by chelation with EDTA40 produces the blue form of bR (A,, = 608 nm). This blue bR (bbR), described by Oesterhelt and Stoeckenius,’ is also formed upon acidification of purple bR, which is controlled by a prototropic goup with pK, 2.05.4’ Regeneration of the purple color from blue bR is achieved at pH above 3.5 by addition of various ions, including lanthanides, and in particular by Ca2+and Mg2+ ions.37-39.42The blue form of bacteriorhodopsin undergoes photophysical transformations but does not pump protons.38 The precise role of metal cations is not known. Could it be that the cation binding serves merely to stabilize or to induce a favorable conformation for the photocycle activity, or is the bound metal ion an integral participant along the H+ translocation pathway, or both? Recent work has shown that the binding of Ca2+ions occurs at two independent high-affinity sites, one ion of Ca2+ at each site, and that the binding of Ca2+ to the second site correlates with the blue to purple color ~ h a n g e . 4About ~ ~ ~ 4-6 ~ additional Ca2+ ions are bound to bR with much lower affinity.44 Most of these sites (with the notable exception of one) are eliminated upon removal of the C-terminal segment of bR by the action of pa~ain.~.~~ In a recent report from this lab~ratory:~the binding constants, K1 and K2, of Ca2+ to the high-affinity sites of blue bR were determined using a calcium ion selective electrode to measure the concentration of free Ca2+ in equilibrium with calcium bound to bR. Analysis by a Scatchard plot established the oneto-one stoichiometry of calcium binding at each site and the pH dependence of K1 and K2 showed that 2 H+ ions are displaced by each Ca2+ bound. This data fits well with the conjecture that two COOH groups of Asp (or possibly Glu) are an integral part of the Ca2+ binding site. The special interest in the binding of calcium and magnesium to bR derives from the fact that these ions occur in the native state of bR and satisfy the metal ion requirement for the proton 0 1995 American Chemical Society

Binding of Ca2+ and Mg2+ to Bacteriorhodopsin pumping activity of the photocycle. It is expected that Ca2+ and Mg2+ are bound to the same high-affinity sites, but this is not a foregone conclusion. The present work provides direct evidence for the competition of Ca2+ and Mg2+ions in binding to the same particular high-affinity site.

11. Materials and Methods Bacteriorhodopsin was isolated from Halobacterium salinarium according to the procedure of Oesterhelt and Stoeckenius and Becher and C a s ~ i m . ~ Slants ~ * ~ ' of ET1001 were provided by Professor R. Bogomolni (University of Califomia, Santa Cruz). Deionization of native bR samples was done using a column of cation exchange resin in the hydrogen form (AG 50W-X4, Bio-Rad, Richmond, CA). The deionized bR samples emerging from the ion-exchange column had a pH of about 3.9. Samples of bR with and without added MgC12 in the desired molar ratio to bR were titrated by successive addition of microliters of M CaC12. After each addition the concentration of free Ca2+ was obtained from the electrical potential in millivolts (Model a 3 2 pH meter; Beckman, Fullerton, CA) using a calcium ion selective electrode (Orion 93-20; Cambridge, MA) measured against a double junction reference electrode (Orion, Cambridge, MA). Calibration of the calcium electrode was done using standard CaC12 solutions in deionized distilled water. The pH was monitored at various intervals during the titration. Spectra were measured on a diode array spectrophotometer (HP 8451A; Hewlett Packard, Palo Alto, CA). All experiments were done with light-adapted bR samples at 22 "C. Analysis of the binding data was obtained by means of the calcium ion selective electrode according to S ~ a t c h a r d ~(plot ~.~' of VICversus v) and the analysis of spectral data according to Awad and B a d r (plot ~ ~ ~of pR versus pCa) has been described previ~usly.~~

J. Phys. Chem., Vol. 99, No. 29, 1995 11601 (b) The model assumes that Mg2+ competes with Ca2+ for the same sites. However, no assumption need to be made as to which of the two sites has the higher affinity for Mg2+. (c) The competitive equilibrium for the two binding sites may be treated separately. In the above chemical equations, bbR denotes blue bacteriorhodopsin; Le., native bR which has been completely deionized by passage through an ion exchange column. It has been shown experimentally that n = 2 in the calcium equilibrium for each of the two sites. We expect that m = 2 also for the magnesium equilibrium, but this is subject to experimental verification. If titrations are performed at essentially constant pH, then one can write straightforward thermodynamic expressions for the equilibrium constants, namely Kc, = [CabR]/[Ca2+l[bbR] KMg= [MgbRl/[Mg2+l[bbRl Here [bbR] represents the concentration of unoccupied sites in bacteriorhodopsin, while [CabR] and [MgbR] represent the concentrations of sites occupied by calcium and magnesium. The association constants are Kca and K M ~respectively. , Let OA be the fraction of sites occupied by calcium and OB be the fraction occupied by magnesium. The fraction of unoccupied sites will then be 1- 6 A - OB. If CA and CB are the concentrations of free Ca2+ and free Mg2+ ions, respectively, and K A and KB are the respective association constants, then

e A / ( i - e, - e,) = KAcA

Rearranging eq 2 to isolate

OB,

(1)

we have

111. Determination of Affinity Constant of Mg2+ A. The Use of Ca2+ Specific Electrode: A Model for Competitive Binding. For the purpose of estimating the association equilibrium constants of Mg2+ to the two highaffinity sites of bacteriorhodopsin, the following simple model was assumed:

+ bbR t CabR + nH+ Mg2+ + bbR t MgbR + mHS Ca2+

The titrations of bbR with Ca2+ were performed by addition of successive aliquots of Ca2+ in the presence of a fixed total amount of magnesium (i.e., free Mg2+ plus Mg bound to bR). Since the measurements were done under equilibrium conditions, the concentration of free Ca2+and free Mg2+ depended on the total amount of calcium added at that point in the titration. The following derivation develops equations based on the above simple model for the competitive association equilibrium. The use of such a model will be justified by the success of extracting the association constants for magnesium from the experimental measurements of the calcium titration. We shall focus on the two high-affinity sites for the binding of Ca2+: (a) The model assumes that the binding of calcium at the two high-affinity sites is separable and noncooperative; i.e., Ca2+ ions bind to each of the sites independently and the association constants differ by a factor of about 5 or more. This aspect of the model has been confirmed for Ca2+ binding to blue bR by the separability of the association constants K I and K2 on the Scatchard plot (43).

Combining eqs 1 and 2, and substituting for

OB,

we have

If there are n identical binding sites on each bR molecule, then the number of sites occupied by calcium on each bR molecule is VA = neA . If we assume that magnesium is competing for the same sites, then similarly VB = neB. Multiplying eq 4 by n gives vA/cA

= (n - vA)(KA)/( 1

+ KBcB)

(5)

or

where

(7) Equation 6 has the form of a Scatchard plot equation for the binding of Ca2+ion to blue bR, except that KA' is not a constant but a function of KB, the association constant for Mg2+, and furthermore KA' depends on CB. The concentration of free Mg2+ ions, CB, is a variable quantity which at equilibrium is fixed by the total amounts in the system of each of calcium, magnesium, and bR. Equation 5 predicts that if the titration of deionized bR is carried out in the presence of an initial amount of magnesium, then as successive amounts calcium are added, the concentration of free Mg2+will increase successively, since the

Yo0 et al.

11602 J. Phys. Chem., Vol. 99, No. 29, 1995 binding of some Ca2+ ions will decrease the availability of sites for Mg2+ binding. Thus the value of KA' will decrease. The presence of a fixed total amount of magnesium will therefore perturb the linearity in the normal (magnesium-free) Scatchard plot. Instead, a curve is obtained with a slope continuously decreasing in magnitude. The slope at a particular value of V A gives a corresponding value for KA'. ,This in turn yields VB, and hence KB can be calculated as shown by the following equations:

8, = (1 - e,)( 1 - KA'/KA) cB = (1 -

eBpO

KB = &/Bo( 1 - e,)( 1 - e A - 6,)

I"

8 -

6-

4'

(8)

2-

(9) "

2

1

0

(10)

V

Bo is the total concentration of magnesium in the system. If Kl and K2 differ by about a factor of 5, then the two sites are separable on a Scatchard plot and each site may be treated independently; thus when n = I , n~ and 6 A have the same value. B. The Use of Spectrophotometric Method. If we assume a certain metal cation dependent equilibrium between blue and purple membrane with a single binding site determining the color of purple bR (pbR), an association constant of a metal ion and the blue bR (bbR) can be calculated. For the equilibrium

bbR

Figure 1. Curve A: Scatchard plot of CaZ+binding to blue bR in the absence of Mg2'. Curve B: Scatchard plot of Ca2+binding to blue bR in the presence initially of OSMghbR, where c = [free Ca*'] and v = [bound Ca*+]/[bbR]. This experiment was done at 22 "C and initial pH 3.9. Association constants of Ca2+ to pure blue bR (Kj = 1.30 x lo5 M-I and K2 = 0.22 x lo5 M-]) were determined from slopes of curve A. Association constants of Mg2+ to blue bR were calculated from eq 10 using the slopes at particular n values of curve B.

TABLE 1: Association Constants 4 for Mg2+by the Competitive Method Using Scatchard Coordinates

+ M2+ * pbR

Here [M2+]fis the concentration of free metal. Let, [pbR]/[bbR] = R,

PK, = PR - P[M2+l,

eeb

K11105M-'

0.30

0.22 0.24 0.23 0.22 0.18 0.13 0.07

0.21 0.31 0.41 0.54 0.62 0.79 0.98

0.40 0.50 0.60 0.70 0.80

K2 = [pbR]/[bbR][M2+],

K2 = R/[M2+],

eAa

0.90

In this experiment successive amounts of Ca2' were added to bbR (55 pM) in the presence of Mg (27.5 pM), initially at pH 3.9 and 22 "C. OA is the fraction of sites occupied by Ca2+ cation. O B is the fraction of sites occupied by Mg2+ cation, computed from eq 8. a

(11)

When pR = 0,

changes as more Ca2+ is added during the titration. From the slope of the perturbed Scatchard plot at different values of VA. the equilibrium constant of the binding of Mg2+,KB, can be computed as discussed in section 111. Table 1 gives the results of this procedure as the value of K I for Mg2+ obtained from the slopes at different OA. Due to the approximations made and the fact that the binding to the second binding site begins to be filled at 8 A higher than OSCa/bR, it is best to calculate an average of KI for Mg2+ at different fraction of site occupied by Ca2+ below 0.5. A value of Kl = (0.3 f 0.1) x lo5 M-' is determined. The value of this affinity constant for Mg2+ is 4 times smaller than that found for Ca2+, in spite of the fact that Mg2+ has a smaller radius, 72 pm, as compared to 100 pm for Ca2+(49), that is Mg2+ has a much larger charge density. The standard free energy change of the binding reaction CB

PK, = -PCM2+lf The value of R at a given point in the titration was determined from the absorbance at 620 nm using the expression, R = (Ab620- A620)/(A620 -

(12)

where A620 is the absorbance after addition of a given amount of the metal ion, Ab620 is the absorbance of 100%blue bR (Amax = 608 nm), and Ap620 is the absorabance of 100% purple bR (Ama = 568 nm) obtained after complete regeneration. The value of p[M2+]f is calculated from the equation P[M2+lf= P[M2+l, - P[M2+lb

(13)

with p[M2+]b= (Ab620 - A620)/(fib620 - AP620), where [M2+Itis the total concentration of metal ions and [M2+]b is the concentration of bound metal ions.

IV. Results and Discussion A. Binding to the First Binding Site. Figure 1 compares the Scatchard plot of Ca2+ in the absence of Mg2+ (A) and in the presence of OSMg/bbR (B). As discussed in section 111, an apparent KA' that is obtained from eq 7 which predicts, as shown in Figure 1, that the slope is not constant, but depends on both KB and CB, the free Mg2+ concentration. Furthermore,

bbR

+ Mg2+(aq)

-

bbR-Mg

+ 2Hf(aq)

is given by the equation AGO1 = -RT In K1. The equilibrium constant is therefore related to AZi"1 and hS01 by the equation

K = e x p ( - W , / R T ) exp(ASQ,lR)

(14)

It is hard to know whether the driving force in the binding reaction is the enthalpy change or the entropy change or both, since neither the structure nor the location of the binding site is

J. Phys. Chem., Vol. 99, No. 29, 1995 11603

Binding of Ca2+ and Mg2+ to Bacteriorhodopsin

TABLE 2: Association Constants of the Second Binding Site of Ca2+and Mg2+ to Blue bR

1

sample

K~~(~o~M Ks,E~ - I ()1 0 5 ~ - 9 K ~ , (~1 c0 5 ~ - 7

+ bbR + (O.1MghbR) + (0.3MghbR) + (OSMghbR) + bbR

Ca2+ Ca2+ Ca2+ Ca2+ Mg*+ Mg2+

400

500

600

700

800

+ (0.5CdbbR)

0.22 0.22 -

0.25 0.16 0.42 0.36 -

0.22 0.22 0.25 0.25 0.25, 0.29

0.30

K2 was obtained from Scatchard plot using the pCa(free) from electrode measurements. KS,Ewas obtained using spectral pR data and pCa(free) from electrode measurements. KS,S was obtained using spectral pR data and pCa(free) from spectral measurements.

Nnm

Figure 2. Spectra taken after addition of successive amounts of IO-*

e

M CaC12 to blue bR in the presence of O.SMg/bbR, at pH 3.9 and 22 "C. The concentration of blue bR was 55 p M and the molar ratio of cations were (a) bbR only: (b) 0.5MghbR; (c) 0.5Ca (0.5Mg h b R ) ; (d) 1.OCa (0.5MghbR); (e) 1.5Ca (O.5MghbR); (f) 2.OCa i(0.5MghbR); (g) 3.OCa (O.5MghbR); and (h) 5.OCa (0.5Mg/ bbR).

+

+

+

+

+

known. Qualitatively, one may conclude that the smaller value of K1 for Mg2+ suggests that the reaction is probably enthalpy, and not entropy, driven. This conclusion is based on the following argument: Mg2+has a much larger hydration enthalpy than Ca2+: at 25 "C AHohyd = -1920 kJ/mol for Mg2+ and -1620 kJ/mol for Ca2+(42). Of course, the binding of Mg2+ to blue bR is also expected to be stronger than that of Ca2+. However, the difference in the hydration energy is expected to be larger than the difference in the binding energy. This would reduce AWl for the overall binding process for Mg2+ as compared to Ca2+. The Mg2+ ions are more capable of organizing the aqueous medium than Ca2+. This leads to a larger increase in the entropy of the binding reaction with Mg2+ than with Ca2+,and thus a larger equlibrium constant, contrary to observation. B. Binding to the Second Binding Site. It was determined previously that binding to the second binding site changes the color of blue bR to p ~ r p l e . ~ *Furthermore, -~~ the binding constant K2 is computed from eq 11. If there is no coupling between metal ions in the different sites, spectroscopy can be used to determine the [bbR] and [pbR] from the observed spectral changes as metal ions are added. The free metal cation can be either determined directly by potentiometric technique for Ca2+ or calculated indirectly from the initial [M2+]and the stoichiometry of binding as deduced from [bbR] and [pbR] determined spectrally. Figure 2 shows the changes in the absorption spectrum of blue bR as metal cations are added. The presence of a well-defined isosbestic point provides strong evidence that there is only one equilibrium involved between two absorbing species in this optical region. Table 2 lists the values of K2 determined for the binding of Ca2+ (the top row) as determined by three methods. In the second column, the results of the Scatchard method using the Ca2+ selective electrode are listed. In the third column, the results are shown using a combination of the spectral method to determine [bbR] and [pbR], while the potentiometric method is used to determine [Ca2+]f. In the last column, the results are given of the combination of the spectrophotometric method with stoichiometry to determine the concentration of [bbR], [pbR], and [Ca2+]f. The three methods give similar results. In the second, third, and fourth rows, the results of the experiments that test the effect of added Mg2+ ions on the binding affinity of Ca2+to the second high-affinity site are given. The fact that similar results are obtained by use of the

-1

4.5

4.0

5.0

5.5

pCa(free) or pMg(free) Figure 3. Spectral ratio plot, pR v s pCa(free) or pMg(free): (0)Ca (OSMghbR); ( 0 )Mg (OSCdbbR). The concentration ratio, R,

+

+

of blue to purple bR and pCa(free) or pMg(free) were determined from spectral data using eqs 12 and 13. A slope of n =1 in this plot indicates that the binding of 1 mole of Ca2+ per mole blue bR is responsible for the observed blue to purple color change.

spectroscopic method to those obtained in the absence of Mg2+ suggests that in the presence of Mg2+ in one site does not alter the binding of Ca2+ in the other site and that the two sites are independent (i.e., uncoupled). The experiment that uses the Ca2+ specific electrode gives different results. The reason for this is not quite understood since we have found that in aqueous medium, Mg2+ does not interfere with the determination of the Ca2+ concentrations. In the last two rows, the results of the spectrophotometric method in the determination of K2 for Mg2+ in $e absence and in the presence of Ca2+ are given. The value of K2 for Mg2+ is found to be not too different from K2 for Ca2+ and the presence 0.5Ca2+/bR had no effect on K2 for Mg2+. These results are also shown in Figure 3. While K , and K2 for Ca2+ are found to be different, K I and K2 for Mg2+ seem to be of comparable magnitude. This might reflect differences in the structure of the two high-affinity sites and their different responses to the binding of the Ca2+ and the Mg2+.

Acknowledgment. This work was supported by the Department of Energy (Office of Basic Energy Sciences) under grant DE-FG03-88ER13828. References and Notes (1) Oesterhelt, D.; Stoeckenius, W. Proc. Nuti. Acud. Sci. U.S.A. 1973, 70, 2853-2851. ( 2 ) Oesterhelt, D. Angew. Chem., Inr. Ed. Engl. 1976, 15, 16-24. (3) Henderson, R. Annu. Rev. Biochem. Bioeng. 1977, 6, 81-109. (4) Lozier, R.; Bogomolni, R. A.; Stoeckenius. W. Biophys. J. 1975, 15, 955-962.

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