Cation Binding to Xanthorhodopsin: Electron Paramagnetic

In addition to the retinal chromophore, xR contains a carotenoid, which acts as a light-harvesting antenna as it transfers 40% of the quanta it absorb...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JPCB

Cation Binding to Xanthorhodopsin: Electron Paramagnetic Resonance and Magnetic Studies Elena Smolensky Koganov,† Gregory Leitus,‡ Rinat Rozin,† Lev Weiner,‡ Noga Friedman,† and Mordechai Sheves*,†,§ †

Department of Organic Chemistry and ‡Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel S Supporting Information *

ABSTRACT: Xanthorhodopsin (xR) is a member of the retinal protein family and acts as a proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber. In addition to the retinal chromophore, xR contains a carotenoid, which acts as a lightharvesting antenna as it transfers 40% of the quanta it absorbs to the retinal. Our previous studies have shown that the CD and absorption spectra of xR are dramatically affected due to the protonation of two different residues. It is still unclear whether xR can bind cations. Electron paramagnetic resonance (EPR) spectroscopy used in the present study revealed that xR can bind divalent cations, such as Mn2+ and Ca2+, to deionized xR (DI-xR). We also demonstrate that xR can bind 1 equiv of Mn2+ to a high-affinity binding site followed by binding of ∼40 equiv in cooperative manner and ∼100 equiv of Mn2+ that are weakly bound. SQUID magnetic studies suggest that the high cooperative binding of Mn2+ cations to xR is due to the formation of Mn2+ clusters. Our data demonstrate that Ca2+ cations bind to DI-xR with a lower affinity than Mn2+, supporting the assumption that binding of Mn2+ occurs through cluster formation, because Ca2+ cations cannot form clusters in contrast to Mn2+.

■

INTRODUCTION Xanthorhodopsin (xR)1 is a retinal-based proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber.2 XR exhibits high homology to the bR protein; however, in addition to the covalently bound all-trans retinal chromophore, through a protonated Schiff base (PSB) to Lys-240, it contains a carotenoid (salinixanthin), which acts as a light-harvesting antenna by providing additional excitation energy for the retinal isomerization and proton transport.1,3−5 Salinixanthin is a C40 polyene characterized by a glycoside residue and an acyl tail6 as well as a conjugated chain that consists of 11 double bonds and is attached to a ring bearing one double bond and a keto group in the C4 position.6 One of the distinctive features of eubacterial retinal-based proton pumps, such as xR, proteorhodopsin (pR), gloeobacter rhodopsin (gR), and a recently discovered protein ESR (isolated from Exiguobacterium sibiricum),7−9 is the presence of a complex counterion to the protonated Schiff base, which consists of an aspartate residue hydrogen-bonded to its neighbor histidine.8,10 The pKa value of this complex counterion, which serves also as the primary proton acceptor following light absorption, is not as low as 2.5 in bR but rather close to 7.8,10,11 The origin of the high pKa, which makes these proteins functional as pumps only at alkaline pH, is an intriguing question.7−9 In xR, His-62 (which is part of the counterion complex) is hydrogen-bonded to Asp-96 similarly to the active sites of the eubacterial pumps, pR,11 ESR,8 and gR.7 As this is an effective hydrogen bond, proton may be shared by the imidazole ring and the carboxylate in a single-well strong © 2017 American Chemical Society

hydrogen bond, leading to a higher pKa value than aspartate alone.11 Titration of the primary counterion to the PSB can be followed from its protonation, which is characterized by a high pKa of 612 and induces absorption change of the retinal chromophore. Lowering the pH induces a red shift in the absorption maximum from 568 to 585 nm12 (in bR, a shift of 40 nm13 is detected) and produces a species defined as “blue membrane”. Presently, limited knowledge is available related to the nature and properties of the various cation-binding sites in xR, its surface potential, as well as the involvement of cations in the proton-pumping activity and its effect on the local pKa of the protein residues. It was previously shown that regulating the pKa of residues in xR affects the CD and absorption spectra dramatically.14 Previous bR studies indicated one high-affinity and three weaker binding sites, which are characterized by similar binding constants and the cation-binding site.15 Several studies led to the suggestion that the binding site of the cation, which determines the state of protonation of Asp-85, is located in the vicinity of the retinal protonated Schiff base associated with Asp-85 and Asp-212 residues.16−18 A different approach revealed that the high-affinity binding site is located in the extracellular region on the exterior of the trimers and is associated with negatively charged lipids, whereas at least part of the three to four low-affinity cation-binding sites are located Received: December 16, 2016 Revised: April 3, 2017 Published: April 5, 2017 4333

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

Article

The Journal of Physical Chemistry B

The differences in the absorption spectra, at λmax (600 nm), of the retinal pigment were plotted against the pH, and the data were fitted to a modified Henderson−Hasselbalch equation (eq 1)

in the cytoplasmic surface17 and control the purple to blue equilibrium.17,19−21 A common approach, regarding bR, suggests a model in which cations bind to the negatively charged membrane surface and abolish the effect of high proton concentration on the membrane surface, thereby controlling the apparent pKa of the counterion Asp-8522−24 and influencing the purple to blue transition through the surface potential effects. According to the Gouy−Chapman theory, free or bound metal cations on the membrane surface compete with protons and thus determine the local proton concentration around the membrane. In the present study, we have focused on the characterization of the divalent cation Mn2+ binding to the deionized xR (DIxR) using electron paramagnetic resonance (EPR) spectroscopy. This is a first-row transition metal, which serves as an essential cofactor in metalloenzymes, which catalyze hydration or hydrolysis reactions. The role of the metal ion in these enzymes is to stabilize a highly reactive hydroxide ion, thereby ensuring that an activated nucleophile is available for catalysis at physiological pH.25 We found that xR can bind 1 equiv of Mn2+ in a high-affinity binding site followed by binding of 40 equiv in a positive cooperative manner and additional 100 equiv with lower affinity. Magnetic measurements of the Mn2+ cations within xR suggest that the Mn2+ cations can form clusters following binding to the DI-xR.

F(x) = 1/(1 + 10n(pK a − x))

(1)

Preparation of Apo-xR. Apo-protein was prepared by incubating the pigment with 0.2 M freshly prepared hydroxylamine at pH = 7.2 and irradiating for 1.5 h with a Schott 250 W cold light source (Carl Zeiss Microscopy, Jena, Germany) equipped with a heat-absorbing filter and an optic fiber (level 4B). The light was filtered through a long-pass cutoff filter at λ > 550 nm (Schott, Mainz, Germany). The samples were thoroughly washed from hydroxylamine by dialysis procedure versus DDW and stored at 4 °C to avoid reconstitution with retinal originating from retinaloxime. UV−Vis Absorbance Measurements. All of the UV−vis absorption measurements were carried out using an Agilent 4583 diode-array spectrophotometer (Agilent Technologies, Palo Alto, CA) equipped with an Agilent 89090A thermostated cuvette holder (Agilent Technologies, Palo Alto, CA). The absorption spectra were corrected for light scattering. EPR Measurements. EPR spectra were recorded on a CW EPR spectrometer ELEXSYS-500 (Bruker) at room temperature in a flat quartz cell. The DI-xR samples were prepared as described above. The concentration of the xR samples was 5.5− 6.0 OD at 486 nm, which equals ∼1.4 × 10−5 M for xR pigment (the ratio of the absorbance at 486−567 nm is ∼7, as was shown in the bleach experiments). Mn2+ Titration and the Measurements of Its Binding Constant to DI-xR. The measurements were carried out in a flat cell using 120 μL of DI-xR protein at a concentration range of (0.9−0.8) × 10−5 M for xR pigment. The DI-xR samples were gradually titrated with an increasing concentration of MnCl2, starting with ∼0.2 equiv of MnCl2. The EPR signal originated mostly from unbound Mn2+ because the amplitude of the bound Mn2+ is ∼5% of that of an identical concentration of the free ion. A reference value of free Mn2+ signal at a concentration of 2 × 10−5 M was measured at the beginning of each experiment. The free Mn2+ concentration and the number of bound Mn2+ equivalents were calculated from the EPR signal intensity during each measurement and addressed to the calibration curve of the free Mn2+. The binding constant of Mn2+ to DI-xR and DI-Apo-xR was calculated using the Scatchard plot technique according to eq 217,29,30

■

EXPERIMENTAL SECTION Sample Preparation. Growth of S. ruber. S. ruber was grown using published methods.1,6,26 Sucrose (0.1%/L) was added to the growing medium according to the previous procedure.27 Purification of xR. XR membrane samples were prepared using published methods.1,28 The membranes were washed with 0.1 M NaCl, followed by three times washing with doubledistilled water (DDW). This treatment partially removed unbound salinixanthin. The xR membranes were dissolved in DDW. The process yielded a sample in which the ratio of the absorption at 280−568 nm was ∼3. All of the experiments were carried out with samples dissolved in DDW. DI-xR Preparation. The DI-xR was prepared by passing wellwashed xR suspension through Dowex 50 WX8 (Fluka) cation exchange column (∼1/2 of 1 mL tip). After the deionization, the pH of the sample became ∼3. As the DI protein is not stable at this pH, the pH was adjusted to 7−10 within 20 s by adding NaOH. The sample was precipitated, and to remove the access of NaOH, it was washed twice with DDW and resuspended in DDW. Titrations of DI-xR. The pH of the DI-xR samples was increased to ∼9.5 by adding NaOH (maximum concentration, 0.1 mM) and then decreased by adding HCl. A concentrated solution of 5 mM MnCl2·2H2O was prepared. The desired concentration of Mn2+ (20 and 100 equiv) for the titrations was calculated according to the real concentration of the retinal pigment that was evaluated from the “bleach” experiment at λmax = 567 nm (see Preparation of Apo-xR). For 20 and 100 equiv of Mn2+, appropriate volumes of the concentrated solution were taken and diluted to 1 mL in the presence of xR. Titration Experiments of xR. Titrations were performed in daylight, by adding small amounts of HCl to wt xR, followed by measuring the UV−vis absorption after the pH was adjusted. The titration process was confirmed by the reversibility of the process following a rapid pH increase.

ν / c = K (n − ν )

(2)

The Scatchard plot of ν/c versus ν (ν is the ratio of the concentration of bound ligand to the total number of available binding sites; n is the number of Mn2+ equivalents bound to xR; and c is the concentration of free Mn2+) should give a straight line with a slope of −K (binding constant) while the x intercept yields the value of n (the number of binding sites with the same affinity). SQUID Magnetic Measurements. Sample Preparation. The concentration of wt xR and DI-xR samples was 5.5−6 OD at 486 nm (the ratio of the absorbance at 486/567 nm is ∼7), and it included various concentrations of MnCl2: 3 × 10−3, 10−3, 8 × 10−4, 2 × 10−4, and 5 × 10−5 M and 30% glycerol. The final volume of all samples was 80 μL. The DI samples were prepared as described above for the EPR measurements. The solutions of MnCl2 were prepared with 30% glycerol at 4334

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

The Journal of Physical Chemistry B

■

concentrations of 1, 0.3, 2 × 10−2, 1 × 10−3, 8 × 10−4, 2 × 10−4, 5 × 10−5, and 2 × 10−5 M. Magnetic Measurements. The magnetic measurements were preceded with the SQUID magnetometer MPMS XL. The magnetic moments of frozen MnCl2 solutions were measured at different concentrations with or without DI-xR protein. The temperature-independent diamagnetic contribution of xR protein was measured repeatedly before and after deionization as control solutions. All samples were placed in special sealed plastic capsules, which were measured beforehand as a reference. The volume and mass of sample components were thoroughly controlled. Some samples were prepared and measured repeatedly. The magnetic-field dependences of magnetic moment, at definite temperatures, were measured for several samples. The temperature dependence of magnetic moment of each sample was measured in the temperature interval 2 < T < 200 K (solutions are frozen), with step ΔT = 1 K at an applied magnetic field (He) of 4000 Oe. Different modes were applied for the measurements: (1) The external field (He) was applied at 200 K, and then the sample magnetic moment was measured when the sample was cooled to 2 K (field-cooling measurements − FCM). (2) The sample was fast-cooled to 2 K under zero magnetic field, and then a magnetic field (He) was applied for measurements during sample warming from 2 to 200 K (zero-field-cooled mode − ZFC). (3) The sample was fastcooled to 2 K under the applied field (He), and then it was measured during warming from 2 to 200 K (field-cooled mode − FC). Data Processing. The magnetic susceptibility (χs) of the sample was obtained from the values of magnetic moment as χs(T) = Ms(T)/H, where Ms is the magnetization of the sample after reference signal has been subtracted. The χs(T) dependences were described as a sum of two superimposed contributions: χs(T) = χMn(T) + χadd(T), where χMn(T) is the paramagnetic contribution, which originated from the Mn2+ ions and χadd(T) is the sum of the contributions, which originated from other molecules and ions present in the solution. To refine the χMn(T) from other contributions, we applied 1. Direct separation of contributions suggesting that χadd is independent of temperature but, as it was observed, paramagnetic χMn is close to be proportional to 1/T. 2. Pascal coefficients,31 which represent diamagnetic contributions of different atoms, ions, and molecules (but not of the xR protein) in the studied samples. 3. We have performed control measurements of wt xR and DI-xR without MnCl2 as well as different concentrations of MnCl2 water solutions without xR. Each of the methods has its obvious drawbacks. They are mainly linked to the need to obtain accurate values of the magnetic contribution of the Mn2+ ions and to eliminate the contribution of various other molecules and complexes, whose amount is relatively defined. However, comparing, analyzing, and averaging the calculated data for χMn(T) obtained from different modes of the magnetic measurements, different repeatedly measured samples, and different methods of refinement allowed us to obtain χMn(T) values with high accuracy, as it is possible, even for samples with low Mn concentration. The dependences were normalized according to 1 mol of Mn2+ cations.

Article

RESULTS AND DISCUSSION

EPR Studies of Mn2+ Binding to DI-XR. It was previously shown that the pH decrease of xR was accompanied by a red shift of the pigment absorption maximum by 511 cm−1, from 568 to 585 nm (blue membrane), 12 due to Asp-96 protonation.12,14 The titration of xR, in DDW and in 1 M of NaCl (Figure S1), showed that the pKa of the counterion in DDW is 6.3, whereas in 1 M concentration of NaCl, the pKa value is 4.5. To explore possible specific binding sites for divalent cations and their affinity, we have carried out EPR measurements to monitor the binding of paramagnetic cation Mn2+ to DI-xR. The DI-xR was obtained using a cation exchange column; however, unlike bR,17 the DI-xR sample was unstable at low pH. Therefore, immediately following the deionization process, the pH of the DI-xR suspension (pH 3) was increased to ∼6 by adding NaOH. The binding of increasing concentrations of Mn2+ (starting with ∼0.2 equiv) to DI protein membranes was followed by an EPR spectroscopic measurement. The EPR spectrum mostly originates from unbound Mn2+, as the amplitude of the EPR signal of Mn2+ protein bound is only ∼5% of that of an identical concentration of the free ion in solution (Figure 1B).17 To exclude the possibility that the effect on the EPR spectrum during the binding of Mn2+ is originated from a protein conformational change due to the blue to purple transition,17 we carried out a control experiment (as Ca2+ does not affect the EPR spectrum), in which we followed the binding of Ca2+ instead of Mn2+ to the blue membrane. However, no effect on the EPR spectrum was observed (data not shown). The obtained Scatchard plot for the binding of Mn2+ to the DI-xR is unique. We have detected a free Mn2+ EPR signal only after the addition of 1 equiv of Mn2+, implying that the first equivalent binds to a very strong binding site. The plot indicates binding in a positive cooperative manner of ∼40 equiv of Mn2+ cations (positive slope, region I) and binding of strong ∼100 equiv of Mn2+ with a binding constant of K = 3.2 × 104 (region II). Moreover, not only that the Scatchard plot does not follow a characteristic straight line for the Scatchard plot,30 but also it has a positive slope followed by a negative slope (Figure 1B). These features cannot be explained in terms of multiple classes of independent binding sites, as in these cases, the obtained Scatchard plots are always curves with a negative slope.30 Therefore, the obtained region I in the Scatchard plot of xR (Figure 1B) can be interpreted as representing interactions between the binding sites, which leads to cooperative binding of Mn2+ and involves xR structural changes induced by the binding of cations. A similar pattern of cation binding has also been recently shown for the phR protein.32 It is plausible that the cooperative binding of a large cation equivalents number is due to the formation of clusters by the Mn2+ cations.33 Because protonation of the counterion red-shifts the absorption maximum of the retinal pigment, it is possible to determine the counterion pKa by titration. To check whether binding of Mn2+ ions to DI-xR affects the pKa of the complex counterion of the pigment, we have monitored the absorption maxima of the retinal pigment after the addition of different MnCl2 concentrations to DI-xR. Figure 2 indicates that 20 equiv of MnCl2 shift the pKa of the DI-xR counterion from 7.1 to 5.8, whereas 100 equiv shift it to 5.2. We note that the addition of 1 M NaCl to xR shifts the pKa value of the counterion to 4.5 (Figure S1). Therefore, 100 equiv of Mn2+ ions have a significant effect. 4335

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

Article

The Journal of Physical Chemistry B

Figure 2. pH-induced absorption changes of DI-xR in the presence of 0, 20, and 100 equiv of Mn2+. The pKa value was determined by using the equation F(x) = 1/(1 + 10n(pKa−x)). The solid lines represent the best fits, with pKa values of 7.1, 5.8, and 5.2, respectively. Curves are normalized to the maximum. The graph represents the normalized difference absorption changes during the titration of the counterion cluster at 600 nm in the pH range of 4.1−9.4. The inset represents the different absorption spectra of the DI-xR sample during the titration without Mn2+, in the pH range of 5.4−9.4. All spectra were subtracted from the spectrum that was measured at the highest pH (pH 9.4 for the sample without Mn2+). The arrows represent the direction of the titration from high to low pH. The maximum concentration of Na+ in the samples is 0.1 mM.

Figure 1. EPR Measurements of DI-xR titration with Mn2+. The concentration of free Mn2+ in the solution was determined by the EPR spectra. (A) EPR spectra of the DI-xR titration with MnCl2. Curve 1: control solution of Mn2+ in a concentration of 1.4 × 10−5 M; curve 2: DI-xR after addition of 0.2 equiv of Mn2+; curve 3: DI-xR after addition of 8 × 10−4 M Mn2+. (B) The Scatchard plot of Mn2+ binding to DI-xR. The measurements were carried out for DI-xR protein at a concentration of ∼1.4 × 10−5 M for xR pigment. The DI-xR samples were gradually titrated with an increasing concentration of Mn2+, starting with 0.2 equiv of Mn2+.

Binding of Mn2+ to DI-xR-Apo. To pinpoint the role of the retinal chromophore in the binding of Mn2+ cation to DIxR, we studied the binding of the Mn2+ cation to DI-apo-xR (xR in which the retinal−protein covalent bond was cleaved by hydroxylamine). According to the obtained Scatchard plot (Figure 3), the DI-apo-xR lacks the strong binding site for the first equivalent of Mn2+, in contrast to DI-xR, which has a very strong binding site for the first equivalent. The first equivalent binds to the DI-apo-xR via a positive cooperative way (region I) of the Mn2+ cations. The EPR signal was observed even when less than 1 equiv was added, implying that the DI-Apo-xR lacks the first strong binding site, as was detected for DI-xR. The positive cooperative binding of Mn2+ to the DI-apo-xR is only for 8 equiv of Mn2+, in contrast to DI-xR, which binds ∼40 equiv in a cooperative manner. It is also evident that the binding strength of Mn2+ is considerably reduced in the apo-xR relative to that of wt xR (binding constant (K), 8.8 × 103 vs 3.2 × 104). These results can be attributed to the change in protein conformation due to the cleavage of the PSB linkage. It was

Figure 3. Scatchard plot of Mn2+ binding to DI-Apo- xR. The measurements were carried with apo-DI-xR protein at a concentration of ∼1.4 × 10−5 M, as determined before the bleach process. The apoDI-xR samples were gradually titrated with an increasing concentration of MnCl2, starting with 0.2 equiv of MnCl2.

previously shown that the formation of apo-bR induces protein conformation alteration and therefore eliminates strong cationbinding site.17 The question arises as to the location of the large number of Mn2+ ions binding. Extensive studies were carried out on bR, and several models were put forward, as described in the Introduction. A common approach, regarding bR, suggested that cations bind to the negatively charged membrane surface and abolish the effect of high proton concentration on the membrane surface, thereby controlling the apparent pKa of the 4336

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

Article

The Journal of Physical Chemistry B

Figure 4. Competition studies of Mn2+and Ca2+ binding to DI-xR. (A) Scatchard plot of the competition between Mn2+and Ca2+. Mn2+cations were added to DI-xR that was preincubated with 40 equiv of Ca2+. (B) Competition study when Ca2+ cations were added to DI-xR that was preincubated with 40 equiv of Mn2+.

counterion.22−24 It is feasible that a similar effect occurs in DIxR and the Mn2+ ions bind to the surface of the protein− membrane complex and affect the local proton concentration in the vicinity of the counterion. The membrane−xR complex may contain some components that are not related to xR; therefore, it might be possible that some of the Mn2+ cations bind to these components. However, the fact that the binding of the cations is significantly modified in the apo-membrane and the strong effect of 20 and 100 equiv on the counterion pKa, strongly support that the ion binding is associated with xR. However, the possibility that some cations bind to other components cannot be completely ruled out, and future studies are required to clarify this issue as well as the location of binding sites. Binding of Ca2+ Cations. To check if the Ca2+ cation binds to the DI-xR as well, we have carried out competition studies by adding MnCl2 to protein that was preincubated with 40 equiv of Ca2+ (Figure 4A). Mn2+ cations could bind to the Ca−xR complex; however, the presence of Ca2+ weakened the binding of Mn2+, and only 10 equiv of Mn2+ bind cooperatively compared with 40 equiv that bind to xR, as can be deduced from the Scatchard plots (Figures 1B and 4A). These results indicate that the Ca2+ cations bind to the DI-xR but not as strong as the Mn2+ cations. As the binding pattern of this competition resembles the regular binding of Mn2+ (although much weaker; K = 8.5 × 103 vs 3.2 × 104) and the positive cooperative is retained, it may suggest that binding of Ca2+ cations induces a different conformation of the protein, which binds Mn2+ weaker than DI-xR. We have also carried out a reverse experiment, in which various concentrations of Ca2+ were added to the DI-xR sample incubated with 40 equiv of Mn2+ (the number of Mn2+ cations that bind cooperatively, see Figure 1). The results presented in Figure 3B indicate 5% of free Mn2+ before the addition of Ca2+. The addition of 650 equiv of Ca2+ increased the fraction of free Mn2+ by only 5%, and additional ∼1200 equiv increased it to 40%. These results suggest a very weak competition between Ca2+ and Mn2+. Once the Mn2+ cations form the clusters by cooperative binding, as the Ca2+ cations cannot form clusters, its ability to compete with the formed Mn2+ clusters is very low. Magnetic Measurements of the Bound Mn2+ Cations. As free Mn2+ cations in solution and those organized in clusters within the DI-xR show different paramagnetic behaviors, we

performed magnetic measurements to shed further light on the possible formation of Mn2+ clusters within the DI-xR. As expected, the susceptibility of MnCl2 water solutions (Figure 5A) shows satisfactory agreement with Curie law for weakly coupled magnetic moments of Mn2+ ions with S = 5/2. The observed magnetic exchange at low temperatures is mainly of antiferromagnetic type. Curie (C) and Weiss (θ) constants are derived by fit of the Curie−Weiss equation: χMn = C/(T − θ). These constants are shown in Table S1 together with the calculated values of effective magnetic moments ( μeff = 3CkB/NAμB2 ). The derived values of C and μeff are close to the theoretical value of C0 = 4.38 cm3 K mol−1 and μeff ≈ 5.92 BM for Curie-like paramagnetism of Mn2+ cations.34 Unlike aqueous MnCl2 solutions, the temperature dependence of the magnetic susceptibility of the MnCl2−DI-xR complex solutions (Figure 5B) cannot be satisfactorily described by Curie-like law, which corresponds to weakly coupled magnetic moments of Mn2+ ions with S = 5/2. Thus, it is plausible that the magnetic moments in the MnCl2−DI-xR complex are localized on not only weakly coupled Mn2+ free cations but also small clusters comprising several Mn2+cations, which are strongly coupled by means of exchange interaction inside the clusters. Although the exact structures of the protein−Mn2+ complexes are still not known, the complicated dependence could be approximately described by the Curie− Weiss law. The difference in the C values resulted for solutions with and without the DI-xR−protein complex is more pronounced in diluted MnCl2 solutions (Table S1). The same difference is directly detected by comparing Figure 5A,B. The dissimilarity of the Curie constants, C, obtained for xR solutions from C0 = 4.38 cm3 K mol−1 increases with the reduction of MnCl2 with the concentration (Table S1). The DI-xR solution with a low Mn2+ concentration, 5 × 10−5 M (4 equiv of Mn2+), shows the highest C value (χMnT values in Figure 5B). Thus, it is conceivable to assume the following: (i) There are almost no “free” Mn2+ cations with noninteracting magnetic moments in the xR solution concentration of 5 × 10−5 M; (ii) The paramagnetic contribution originated from the cluster formation, in which several magnetic moments of Mn2+ cations are sufficiently strongly coupled via exchange interactions; (iii) The interactions are predominantly of ferromagnetic type such that each Mn2+ cation cluster member 4337

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

Article

The Journal of Physical Chemistry B

contributes to the C value (and to μeff) significantly more than the noninteracting Mn2+ ion. This observation may suggest that the binding of the Mn2+ cations to the DI-xR triggers cluster formation. As the multinuclear clusters have their own magnetic moment and behave as small magnetic particles, they weakly interact with either each other or the free Mn2+ cations in the surrounding solution.33 It is plausible that at 5 × 10−5 M concentration, only a minor fraction of Mn2+ cations is not part of the clusters. These results and suggestions are consistent with the above-described EPR results. The Scatchard plot (Figure 1B) indicates that when 4 equiv of Mn2+ are added to DI-xR (concentration, 5 × 10−5 M), the Mn2+ cations bind in a cooperative manner and almost no signal derived from free Mn2+ ions was detected. The χMnT (T) values detected following the addition of ∼16 equiv of Mn2+ (concentration, 2 × 10−4 M) to DI-xR are close to the values obtained at 5 × 10−5 M although somewhat smaller. Namely, in this concentration, a fraction of Mn2+ is free and does not bind to the protein; however, most of the Mn2+ cations are arranged in clusters. The EPR results indicate that following the addition of 16 equiv of Mn2+, DI-xR still binds the Mn2+ cations by a highly cooperative manner and only a weak signal of free Mn2+ is observed in the solution. Thus, we can conclude that the high cooperative binding of Mn2+ cations to DI-xR is due to the formation of clusters. Nevertheless, we cannot determine the exact size and structure of the resulting clusters. To shed further light on the cluster structure, we have tried to separate the magnetic susceptibility contribution of the Mn clusters from that of Mn2+ ions, which do not participate in DIxR-bound Mn2+ clusters. We assume χMnT = (1 − α)χclus Mn T + 2+ T, where α is the fraction of Mn ions in the solution, αχfree MN which do not participate in the cluster formation. Accordingly, clus 2+ χfree MN and χMn are contributions in the susceptibility of free Mn cations and the cations included in the clusters. The values χfree MN clus T = C0 = 4.38 cm3 K mol−1 and χclus = 4C0 = 17.52 cm3 Mn T = C K mol−1 were taken (avoiding overparameterization) in keeping with our previous consideration. This assumption does not imply that all of the clusters in the DI-xR solutions with different Mn2+ concentrations consist of exactly four Mn2+

Figure 5. Paramagnetic contribution (χMn) in magnetic susceptibility of MnCl2 solutions vs temperature, presented by χMnT (T). (A) Paramagnetic contribution of χMn in magnetic susceptibility of MnCl2 water solutions in different concentrations (1, 0.3, 2 × 10−2, 1 × 10−3, 8 × 10−4, 2 × 10−4, 5 × 10−5, 2 × 10−5 M), vs temperature. (B) Paramagnetic contribution of χMn for the magnetic susceptibility of DIxR with MnCl2 in different concentrations (3 × 10−3 M (gray), 1 × 10−3 M (red), 8 × 10−4 M (blue), 2 × 10−4 M (green), 5 × 10−5 M (pink)), vs temperature, presented by χMnT (T) dependences. The concentration of xR pigment is 1.4 × 10−5 M. Both graphs have the same scale. The statistical errors of all of the measured values are shown by error bars. The level χMnT = 4.38 cm3 K mol−1 is shown by the black line.

Figure 6. Estimated molar separation of the Mn2+ cations remained free (blue symbols, right scale) and of Mn2+ cations included in the clusters (black symbols, left scale) vs concentration of MnCl2 in the suspensions of DI-xR. 4338

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

Article

The Journal of Physical Chemistry B

the cluster formation. This phenomenon is supported by the magnetic measurements described above. The exact location of the clusters in the membrane−xR complex and the factors that control their formation cannot be identified by the present results and should be the subject of future studies.

cations; however, it only suggests that the resulting average magnetic moment of the clusters is close to the sum of the magnetic moments of the four Mn2+ cations. The α values derived from such estimation were used to separate the concentrations of two types of the Mn2+ cations in the studied DI-xR suspensions. The result of such separation is shown in Figure 6. In spite of the inaccuracy of the above-described calculations, we can still make the following assumptions: (i) Formation of Mn2+ cation clusters is initiated at very low Mn2+ concentrations; (ii) As Mn2+ concentration increases, clusters and/or their number related to one xR molecule grow as long as all molecules of the DI-xR protein will be saturated by the Mn2+ cations; (iii) The number of Mn2+ cation equivalents, which is not included in the clusters, is much lower at low concentration and increases linearly with Mn2+ concentrations. We can also suggest that a saturation of the DI-xR protein clusters by Mn2+ cations takes place when about 16 equiv of Mn2+ cations are bound to each DI-xR molecule, that is, the concentration of Mn2+ cations involved in the cluster formation is 2 × 10−4 M. It is evident that the MnCl2 concentration reaches about 5 × 10−4 M only once. The Mn clusters can be characterized by different sizes and may interact with protein carboxylate groups and water molecules.33 The exact size and structure of these clusters require further studies.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b12670. Detailed information about the fitting of the χMn results for MnCl2 solutions with or without xR as well as the pH-induced absorption changes of membrane suspension of xR during its titration (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +972-8-9344320. Fax: +972-8-9343026. ORCID

Mordechai Sheves: 0000-0002-5048-8169 Funding

This work was supported by grants from the Kimmelman Center for Biomolecular Structure and Assembly, the Benoziyo Endowment Fund for the Advancement of Science, and J & R Center for Scientific Research.

■

CONCLUSIONS In the present study, we aimed at shedding light on cation binding to xR. EPR measurements indicated the ability of DIxR to bind a large amount of Mn2+ cations. The first equivalent of the Mn2+ cation binds very strong such that it was not possible to detect the EPR signal of free Mn2+ cation. A similar phenomenon was found also for the hR protein.32 In addition, we have revealed that ∼40 equiv of Mn2+ cations bind in a positive cooperative manner along with 100 strong binding sites (with a binding constant (K) of 3.2 × 104) and ∼100 weak binding sites. The number of bound Mn2+ equivalents to DI-xR is much higher than that for the DI-bR protein, which has one high-affinity and three to four low-affinity binding sites.17 SQUID measurements for DI-xR with different concentrations of Mn2+ cations imply that the cations added to the protein form clusters. However, the exact size of these clusters should be further studied. The fact that Ca2+ cations bind in a much lower affinity than Mn2+ also supports our assumption that the binding of Mn2+ occurs through cluster formation, as Ca2+ cations cannot form clusters. A similar pattern of cation binding was also found for the hR light-driven Cl− pump protein, which contains two chromophores retinal and bacterioruberin as xR. It was found that hR has two high-affinity binding sites, 5 equiv that bind in a cooperative way, and 20 equiv that bind in a low affinity.32 As for DI-xR, it was also suggested that DI-hR binds the Mn2+ cations via clusters. Although DI-hR has the same pattern of Mn2+ binding, DI-xR is able to bind significantly more cations. It is very plausible that the Mn2+ cations bind the negative charges of the membrane−xR complex surface and affect the apparent pKa of the complex retinal protonated Schiff base counterion. According to the Gouy−Chapman theory, free or bound metal cations on the membrane surface compete with protons and thus determine the local proton concentration around the membrane. This will affect the proton concentration in the complex counterion vicinity to the retinal protonated Schiff base, thereby affecting the apparent counterion pKa. The cooperative manner of the binding of Mn2+ is consistent with

Notes

The authors declare no competing financial interest. § M.S. holds the Katzir-Makineni professorial chair in chemistry.

■

ABBREVIATIONS: xR, xanthorhodopsin; bR, bacteriorhodopsin; PSB, protonated Schiff base; EPR, electron paramagnetic resonance; DI-xR, deionized xR; DDW, double-distilled water

■

REFERENCES

(1) Balashov, S. P.; Imasheva, E. S.; Boichenko, V. A.; Anton, J.; Wang, J. M.; Lanyi, J. K. Xanthorhodopsin: A Proton Pump with a Light-Harvesting Carotenoid Antenna. Science 2005, 309, 2061−2064. (2) Antón, J.; Oren, A.; Benlloch, S.; Rodriguez-Valera, F.; Amann, R.; Rossello-Mora, R. Salinibacter ruber Gen. Nov., Sp Nov., a Novel, Extremely Halophilic Member of the Bacteria from Saltern Crystallizer Ponds. Int. J. Syst. Evol. Microbiol. 2002, 52, 485−491. (3) Slouf, V.; Balashov, S. P.; Lanyi, J. K.; Pullerits, T.; Polivka, T. Carotenoid Response to Retinal Excitation and Photoisomerization Dynamics in Xanthorhodopsin. Chem. Phys. Lett. 2011, 516, 96−101. (4) Gdor, I.; Zhu, J. Y.; Loevsky, B.; Smolensky, E.; Friedman, N.; Sheves, M.; Ruhman, S. Investigating Excited State Dynamics of Salinixanthin and Xanthorhodopsin in the near-Infrared. Phys. Chem. Chem. Phys. 2011, 13, 3782−3787. (5) Zhu, J.; Gdor, I.; Smolensky, E.; Friedman, N.; Sheves, M.; Ruhman, S. Photoselective Ultrafast Investigation of Xanthorhodopsin and Its Carotenoid Antenna Salinixanthin. J. Phys. Chem. B 2010, 114, 3038−3045. (6) Lutnaes, B. F.; Oren, A.; Liaaen-Jensen, S. New C-40-Carotenoid Acyl Glycoside as Principal Carotenoid in Salinibacter ruber, an Extremely Halophilic Eubacterium. J. Nat. Prod. 2002, 65, 1340−1343. (7) Tsukamoto, T.; Kikukawa, T.; Kurata, T.; Jung, K. H.; Kamo, N.; Demura, M. Salt Bridge in the Conserved His-Asp Cluster in Gloeobacter Rhodopsin Contributes to Trimer Formation. FEBS Lett. 2013, 587, 322−327. (8) Balashov, S. P.; Petrovskaya, L. E.; Lukashev, E. P.; Imasheva, E. S.; Dioumaev, A. K.; Wang, J. M.; Sychev, S. V.; Dolgikh, D. A.; Rubin, A. B.; Kirpichnikov, M. P.; et al. Aspartate-Histidine Interaction in the

4339

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340

Article

The Journal of Physical Chemistry B Retinal Schiff Base Counterion of the Light-Driven Proton Pump of Exiguobacterium sibiricum. Biochemistry 2012, 51, 5748−5762. (9) Luecke, H.; Schobert, B.; Stagno, J.; Imasheva, E. S.; Wang, J. M.; Balashov, S. P.; Lanyi, J. K. Crystallographic Structure of Xanthorhodopsin, the Light-Driven Proton Pump with a Dual Chromophore. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16561−16565. (10) Tsukamoto, T.; Kikukawaa, T.; Kurataa, T.; Jungb, K. H.; Kamoa, N.; Demura, M. Salt Bridge in the Conserved His−Asp Cluster in Gloeobacter Rhodopsin Contributes to Trimer Formation. FEBS Lett. 2013, 587, 322−327. (11) Luecke, H.; Schobert, B.; Stagno, J.; Imasheva, E. S.; Wang, J. M.; Balashov, S. P.; Lanyi, J. K. Crystallographic Structure of Xanthorhodopsin, the Light-Driven Proton Pump with a Dual Chromophore. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16561−16565. (12) Imasheva, E. S.; Balashov, S. P.; Wang, J. M.; Lanyi, J. K. PhDependent Transitions in Xanthorhodopsin. Photochem. Photobiol. 2006, 82, 1406−1413. (13) Kimura, Y.; Ikegami, A.; Stoeckenius, W. Salt and Ph-Dependent Changes of the Purple Membrane Absorption-Spectrum. Photochem. Photobiol. 1984, 40, 641−646. (14) Smolensky Koganov, E.; Brumfeld, V.; Friedman, N.; Sheves, M. Origin of Circular Dichroism of Xanthorhodopsin. A Study with Artificial Pigments. J. Phys. Chem. B 2015, 119, 456. (15) Duñach, M.; Seigneuret, M.; Rigaud, J. L.; Padros, E. The Relationship between the Chromophore Moiety and the Cation Binding-Sites in Bacteriorhodopsin. Biosci. Rep. 1986, 6, 961−966. (16) Jonas, R.; Ebrey, T. G. Binding of a Single Divalent-Cation Directly Correlates with the Blue-to-Purple Transition in Bacteriorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 149−153. (17) Eliash, T.; Weiner, L.; Ottolenghi, M.; Sheves, M. Specific Binding Sites for Cations in Bacteriorhodopsin. Biophys. J. 2001, 81, 1155−1162. (18) ElSayed, M. A.; Yang, D. F.; Yoo, S. K.; Zhang, N. The Effect of Different Metal Cation Binding on the Proton Pumping in Bacteriorhodopsin. Isr. J. Chem. 1995, 35, 465−474. (19) Eliash, T.; Ottolenghi, M.; Sheves, M. The Titrations of Asp-85 and of the Cation Binding Residues in Bacteriorhodopsin Are Not Coupled. FEBS Lett. 1999, 447, 307−310. (20) Szundi, I.; Stoeckenius, W. Purple-Blue Transition in LipidDepleted Purple Membrane. Biophys. J. 1987, 51, A414. (21) Szundi, I.; Steckenius, W. Effect of Lipid Surface-Charges on the Purple-to-Blue Transition of Bacteriorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 3681−3684. (22) McLaughlin, S. The Electrostatic Properties of Membranes. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113−136. (23) Ehrenberg, B.; Ebrey, T. G.; Friedman, N.; Sheves, M. The Surface-Potential on the Purple Membrane Measured Using a Modified Bacteriorhodopsin Chromophore as the Spectroscopic Probe. FEBS Lett. 1989, 250, 179−182. (24) Szundi, I.; Stoeckenius, W. Purple-to-Blue Transition of Bacteriorhodopsin in a Neutral Lipid Environment. Biophys. J. 1988, 54, 227−232. (25) Christianson, D. W.; Cox, J. D. Catalysis by Metal-Activated Hydroxide in Zinc and Manganese Metalloenzymes. Annu. Rev. Biochem. 1999, 68, 33−57. (26) Smolensky, E.; Sheves, M. Retinal-Salinixanthin Interactions in Xanthorodopsin: A Circular Dichroism (Cd) Spectroscopy Study with Artificial Pigments. Biochemistry 2009, 48, 8179−8188. (27) Oren, A.; Mana, L. Sugar Metabolism in the Extremely Halophilic Bacterium Salinibacter ruber. FEMS Microbiol. Lett. 2003, 223, 83−87. (28) Smolensky Koganov, E.; Hirshfeld, A.; Sheves, M. Retinal BIonone Ring-Salinixanthin Interactions in Xanthorhodopsin: A Study Using Artificial Pigments. Biochemistry 2013, 52, 1290−1301. (29) Danchin, A. Transfer-RNA Structure and Binding-Sites for Cations. Biopolymers 1972, 11, 1317−1333. (30) Danchin, A.; Gueron, M. Cooperative Binding of Manganese (II) to Transfer RNA. Eur. J. Biochem. 1970, 16, 532−536.

(31) Bain, G. A.; Berry, J. F. Diamagnetic Corrections and Pascal’s Constants. J. Chem. Educ. 2008, 85, 532−536. (32) Dutta, S.; Weiner, L.; Sheves, M. Cation Binding to Halorhodopsin. Biochemistry 2015, 54, 3164−3174. (33) Gatteschi, D.; Sessoli, R.; Villain, J. Molecular Nanomagnets; Oxford University Press Inc.: New York, 2006; p 408. (34) Ashcroft, N. W.; Mermin, N. D. Solid State Physics; Holt, Rinehart & Winston: New York, 1976.

4340

DOI: 10.1021/acs.jpcb.6b12670 J. Phys. Chem. B 2017, 121, 4333−4340