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Spectroscopic and Thermodynamic Characterization of the MetalBinding Sites in the LH1−RC Complex from Thermophilic Photosynthetic Bacterium Thermochromatium tepidum Yukihiro Kimura,*,† Yuki Yura,† Yusuke Hayashi,† Yong Li,† Moe Onoda,† Long-Jiang Yu,‡ Zheng-Yu Wang-Otomo,§ and Takashi Ohno† †

Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan § Faculty of Science, Ibaraki University, Mito 310-8512, Japan ‡

S Supporting Information *

ABSTRACT: The light-harvesting 1 reaction center (LH1− RC) complex from thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits enhanced thermostability and an unusual LH1 Qy transition, both induced by Ca2+ binding. In this study, metal-binding sites and metal− protein interactions in the LH1−RC complexes from wild-type (B915) and biosynthetically Sr2+-substituted (B888) Tch. tepidum were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopies. The ITC measurements revealed stoichiometric ratios of approximately 1:1 for binding of Ca2+, Sr2+, or Ba2+ to the LH1 αβsubunit, indicating the presence of 16 binding sites in both B915 and B888. The AA analysis provided direct evidence for Ca2+ and Sr2+ binding to B915 and B888, respectively, in their purified states. Metal-binding experiments supported that Ca2+ and Sr2+ (or Ba2+) competitively associate with the binding sites in both species. The ATR-FTIR difference spectra upon Ca2+ depletion and Sr2+ substitution demonstrated that dissociation and binding of Ca2+ are predominantly responsible for metal-dependent conformational changes of B915 and B888. The present results are largely compatible with the recent structural evidence that another binding site for Sr2+ (or Ba2+) exists in the vicinity of the Ca2+-binding site, a part of which is shared in both metal-binding sites.

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INTRODUCTION Photosynthetic purple bacteria convert light energy into chemical energy through a light-driven redox cycle. This process is initiated by a charge separation in a reaction center (RC) upon absorption of light energy captured by two types of light-harvesting (LH) complexes, LH1 and LH2.1 The LH1 complex surrounds the RC to form a core complex (LH1−RC), and the LH2 complexes are located nonstoichiometrically in the periphery of LH1−RC complexes. Three-dimensional X-ray crystallographic structures have been available for a long time for the RC2−4 and LH25,6 complexes at high resolution. In contrast, structural information on LH1−RC complexes was limited to low resolution.7,8 In the X-ray crystallographic structure of Rhodopseudomonas palustris at 4.8 Å resolution, the LH1 ring is composed of 15 αβ-subunits with a gap, where protein W is located.7 Single-molecule spectroscopic studies also supported the presence of the gap.9,10 In the structure of Rhodobacter sphaeroides at 8 Å resolution, each LH1 ring is an array of 14 αβ-subunits with a PufX protein, which promotes dimerization of the LH1−RC complexes.8 Recently, a 3.0 Å Xray crystallographic structure of the LH1−RC complex has © 2016 American Chemical Society

been reported for the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum,11 which can grow at temperatures up to 58 °C and exhibits an unusually red-shifted LH1 Qy absorption at 915 nm (B915).12,13 The LH1 forms a closed ring composed of 16 αβ-subunits, each of which binds one carotenoid and two bacteriochlorophyll (BChl) molecules.11 The electron density map reveals that each subunit binds one Ca2+ at the C-terminal region of the LH1 αβpolypeptide. However, it is unclear as to how many Ca ions are actually present in the native form of the Tch. tepidum LH1− RC complex because LH1−RC is usually crystallized from Ca2+-containing solutions (>20 mM). Previous studies demonstrated that Ca2+ is responsible for the unique thermophilic nature and the unusual spectroscopic properties of Tch. tepidum LH1−RC complexes.14,15 Upon removal of Ca2+, the LH1 Qy peak at 915 nm was blue-shifted to 880 nm, with a marked decrease in the thermal stability. The Received: October 5, 2016 Revised: November 18, 2016 Published: November 18, 2016 12466

DOI: 10.1021/acs.jpcb.6b10068 J. Phys. Chem. B 2016, 120, 12466−12473

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The Journal of Physical Chemistry B

biosynthetically Sr2+-substituted LH1−RC with an absorption maximum at 888 nm (B888) was eluted with a linear gradient of CaCl2 or SrCl2 from 10 to 25 mM, and fractions with AQy/ A280 ratios higher than 2.0 or 1.7 were collected as purified B915 or B888. For the AA analysis, crude OG extracts of LH1−RC in metalfree buffer containing 20 mM Tris−HCl (pH 7.5) and 0.08% (w/v) DDPC were purified by a 10−40% (w/v) sucrose density gradient ultracentrifugation at 150 000g and 4 °C for 7 h. The purified LH1−RC complexes were dissociated for 12 h at 95 °C in a metal-free vessel DigiTUBES (SCP Sciences) in the presence of nitric acid (Suprapur, Merck) and diluted appropriately with ultrapure water for each AA measurement. Metal-depleted LH1−RC complexes for ITC experiments were prepared as described previously.14,15 The purified LH1− RC complexes were passed through a size-exclusion column (Sephadex G25M PD10; GE Healthcare) to remove excess salts, followed by incubation at 0 °C for 1 h in the presence of 1−2 mM ethylenediaminetetraacetic acid (EDTA) to remove the metal cations tightly bound to the LH1−RC complexes. Then, the sample solution was extensively washed with a buffer containing 20 mM Tris−HCl (pH 7.5) and 0.08% (w/v) DDPC to remove residual EDTA and concentrated with Amicon Ultra 100 K filters (Millipore) to a concentration of 33 μM LH1−RC complex (0.53 mM of LH1 αβ-subunit). ATR-FTIR sample solutions were prepared as described previously.16 The concentrated LH1−RC complexes were diluted with buffer A (20 mM Tris−HCl, 20 mM CaCl2, pH 7.5) and ultracentrifuged at 195 000g and 4 °C for 30 min. The resulting pellet was suspended in buffer A containing 0.008% DDPC to an approximate concentration of 100 μM LH1−RC complex for the ATR-FTIR measurements. AA Analysis. Quantification of Ca2+ or Sr2+ content in B915 or B888 was conducted using a polarized Zeeman AA spectrophotometer (model Z-2010; HITACHI). All the vessels were rinsed with 0.1 M HCl and/or ultrapure water. Brushes used for sample suspension were washed with sonication in ultrapure water containing Chelex 100 (BioRad). ITC Analysis. ITC profiles were recorded on a MicroCal iTC200 microcalorimeter. A solution of metal-depleted LH1− RC complexes was titrated 30 times with 25 mM metal chloride solution at 25 °C and intervals of 5 min for CaCl2 or 2 min for SrCl2. The accumulated molar heat of the injectant was plotted as a function of time and analyzed by the one-site model of the Origin software to yield the thermodynamic parameters of the metal binding to the LH1−RC complexes. Perfusion-Induced ATR-FTIR Analysis. Perfusion-induced ATR-FTIR difference spectra were recorded on a Prestige-21 spectrophotometer (Shimadzu) equipped with a mercury−cadmium−telluride detector (Shimadzu) and a DuraSamplIR II ATR accessory with a three-bounce Si/ZnSe microprism (Smiths Detection) as described previously.16 An aliquot of the LH1−RC sample solution was dried using a stream of nitrogen gas on the ATR prism. A flow attachment was laid over the sample film and connected to a peristaltic pump via silicon tubing. The background spectra were measured after a perfusion of Ca2+ buffer (20 mM Tris−HCl, 25 mM NaCl, 20 mM CaCl2, pH 7.5). Then, the buffer was switched to Sr2+ buffer (20 mM Tris−HCl, 25 mM NaCl, 20 mM SrCl2, pH 7.5), and the sample spectra were recorded to yield the Sr2+/Ca2+ difference spectra. To obtain the ATR-FTIR difference spectra upon Ca2+ depletion, the background spectra

modified properties were almost completely restored by reconstitution with Ca2+ or to a lesser extent by reconstitution with Sr2+ or Ba2+. We have conducted a Fourier transform infrared (FTIR) study on the Tch. tepidum LH1−RC complex and detected the reversible structural and/or conformational changes of the LH1 moiety induced by metal exchanges between Ca2+ and other divalent cations (Sr2+ or Ba2+).16 The FTIR difference bands generated by the Ca 2+ -to-Sr 2+ substitution were almost identical to those generated by the Ca2+-to-Ba2+ substitution, in spite of the fact that the Ca2+binding site is highly sensitive to the size and/or binding properties of the metal cations.14,15 Therefore, it is unclear whether Sr2+ (or Ba2+) binds to the same site that binds Ca2+ in the Tch. tepidum LH1−RC complex. Tch. tepidum cells can grow in a medium containing Sr2+ instead of Ca 2+.17 The resulting biosynthetically Sr2+substituted LH1−RC complexes exhibited a blue-shifted LH1 Qy maximum at 888 nm (B888) and lower denaturing temperature of 67 °C compared to the wild-type LH1−RC complex, which exhibits the Qy maximum at 915 nm and denaturing temperature of 75 °C. Intriguingly, B915 and B888 exhibited different spectroscopic and thermodynamic properties in their Ca2+-bound forms, although both peptide sequences are largely identical. The LH1 Qy peak position and the denaturing temperature of Ca2+-substituted B888 were 908 nm and 72 °C, respectively, which were close to but not the same as those of the Ca2+-reconstituted B915 (915 nm and 75 °C).17 However, the previous resonance Raman study revealed that the hydrogen-bonding interactions between the Trp residues of LH1 αβ-polypeptides and BChl-a molecules were almost identical between the Ca2+-reconstituted B915 and the Ca2+-substituted B888.18 These results indicate that some factors other than the hydrogen-bonding interactions might be crucial for the enhanced thermal stability and the unusual Qy redshift of the wild-type LH1−RC complexes from Tch. tepidum. In this study, the metal-binding sites and metal−protein interactions in B915 and B888 were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and perfusion-induced attenuated total reflection (ATR) FTIR spectroscopies. On the basis of the present results and the structural information, the metal-binding site and metal− protein interactions responsible for the enhanced thermal stability of the Tch. tepidum LH1−RC complex are discussed.

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EXPERIMENTAL SECTION Sample Preparation. The Tch. tepidum LH1−RC complexes were purified as described previously17 with minor modifications. Wild-type and biosynthetically Sr2+-substituted Tch. tepidum cells were cultured anaerobically at 50 and 45 °C, respectively, for 7−10 days. The harvested cells were disrupted in 20 mM Tris−HCl buffer (pH 8.5) at 0 °C by sonication (Sonopuls HD3200, Bandelin). The resulting chromatophores were treated with 0.325−0.350% (w/v) lauryldimethylamine Noxide (Anatrace) to remove a large portion of the LH2 complexes. After ultracentrifugation, the pellets were further treated with 0.9% (w/v) n-octyl-β-D-glucopyranoside (OG, Anatrace) to extract the LH1−RC components. The solubilized LH1−RC complexes were loaded onto a DEAE anion-exchange column (Toyopearl 650S, TOSOH) equilibrated at 4 °C with 20 mM Tris−HCl buffer (pH 7.5) containing 0.08% (w/v) dodecylphosphocholine (DDPC, Anatrace). The wild-type LH1−RC with an absorption maximum at 915 nm (B915) or 12467

DOI: 10.1021/acs.jpcb.6b10068 J. Phys. Chem. B 2016, 120, 12466−12473

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around 915 nm at temperatures up to 60 °C but releases a portion of bound Ca2+ on further elevation of the temperature during the differential scanning calorimetry (DSC) measurement. This is supported by the present AA result, which indicated that the B915 complexes purified in Ca2+-free buffer contain 18 ± 1 Ca2+. Typically, the Tch. tepidum cells are cultivated at 48−50 °C in the presence of 340 μM Ca2+, which is over 350-fold than the concentration of LH1 αβ-subunits (∼0.93 μM) in the mature cells. Therefore, it is strongly indicated that the LH1−RC complex retains all the bound Ca2+ in the native state. In contrast, the content of Sr2+ in B888 was half the number of Ca2+ in B915. This suggests that only half of the sites are occupied by Sr2+ in the native state of B888, or a portion of Sr ions was released during the sample preparation, because of the lower binding affinity compared to that of Ca2+. To confirm this possibility, the number of metal-binding sites and their affinities to metal cations in B915 and B888 were examined by ITC analysis. Metal-Binding Sites and Metal−Protein Interactions. The upper panels of Figure 1 show ITC profiles obtained by titration of Ca2+-depleted B915 (A) and Sr2+-depleted B888 (B) with 25 mM Ca2+ solution. Both profiles exhibited negative exothermic peaks upon Ca2+ binding to the metal-depleted LH1−RC complexes although the intensity was relatively lower in Sr2+-depleted B888. Consistent with the previous result,14 the exothermic signals upon Ca2+ binding to Ca2+-depleted B915 exhibited a two-phase change: a rapid return due to the fast Ca2+-binding reaction and a slow recovery due to the conformational rearrangement within the core complex. In contrast, binding of Ca2+ to the Sr2+-depleted B888 exhibited a relatively smaller contribution of the slow recovery process (Figure 1A inset). These results indicate that the conformational rearrangement after the binding of Ca2+ is distinctively different between B915 and B888. The lower panels of Figure 1 are plots of the integrated heat per mole of injectant upon binding of Ca2+ to Ca2+-depleted B915 (A) or Sr2+-depleted B888 (B) as a function of the Ca2+/ αβ-subunit molar ratio. Both plots exhibited sigmoid curves with inflection points at approximately 1.0, suggesting that the Ca2+-binding reactions in B915 and B888 can be described by a one-site model. Similar ITC profiles were obtained by titration of Ca2+-depleted B915 and Sr2+-depleted B888 with Sr2+ or Ba2+ (data not shown). The thermodynamic parameters for binding of Ca2+, Sr2+, or Ba2+ to the metal-depleted LH1−RC complexes are summarized in Table 2. Both species exhibited a metal/αβ-subunit molar ratio (N) of 1.0 for Ca2+, demonstrating that the number of metal-binding sites in LH1 is 16 in both B915 and B888. The result for B915 is compatible with the number of Ca2+ detected by AA analysis (Table 1). The molar ratio for Sr2+ or Ba2+ was 0.9 or 0.9−0.8, indicating that B915 and B888 essentially possess the same number of binding sites for Sr2+ or Ba2+ as that for Ca2+. One would expect that residual Ca2+ ions bound to the metaldepleted LH1−RC complexes are responsible for the reduced number of the ratio for Sr2+ or Ba2+. However, this is not the case because AA analysis of the Ca2+ -depleted B915 demonstrated that the number of residual Ca2+ was 1.1 ± 0.5 per LH1−RC and Ca2+ ions bound to the LH1 are almost completely removed. Therefore, the slightly reduced molar ratios are presumably attributable to lower occupancies for Sr2+ and Ba2+. Such a lower occupancy for Sr2+ (∼0.7) in the Ca2+binding site was also reported for the biosynthetically Sr2+substituted photosystem II complexes from Thermosynechococ-

were measured for the LH1−RC samples equilibrated in the Ca2+ buffer, and the samples were treated with EDTA buffer (20 mM Tris−HCl, 25 mM NaCl, 5 mM EDTA, pH 7.5) for 10 min. Then, the buffer was switched to Ca2+-free buffer (20 mM Tris−HCl, 25 mM NaCl, pH 7.5), and the sample spectra were collected after washing out EDTA. Therefore, contribution of EDTA to the spectra was negligible. All of the buffers were perfused at a rate of 1 mL/min. Each spectrum was accumulated at 25 °C for 1.5 min (150 scans) at 25 °C, and 30−40 spectra (4500−6000 scans) from several fresh samples were averaged to improve the signal-to-noise ratio.

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RESULTS AND DISCUSSION Quantification of the Metal Content. The recent X-ray crystallographic structure of the Tch. tepidum LH1−RC complex revealed that one LH1 complex is composed of 16 αβ-subunits, each of which binds one Ca2+ at the C-terminal binding site.11 However, it is unclear as to how many Ca2+ ions are originally present in the Tch. tepidum LH1−RC complex because the crystals were prepared from a solution of purified LH1−RC in the presence of Ca2+ (>20 mM).11 In addition, biosynthetically Sr2+-substituted LH1−RC complexes are also supposed to contain the same number of Sr2+ if Sr2+ fully substituted Ca2+ in vivo. Therefore, the metal content within the LH1−RC complexes was evaluated by AA spectrophotometry. Table 1 shows the concentration of Ca2+ or Sr2+ in the Table 1. Numbers of Metal-Binding Sites in B915 and B888 As Determined by AA Analysis Ca2+-binding site 2+

Sr2+-binding site 2+

sample

[Ca ] (μM)

number of site

B915 B888

6.7 ± 0.5

18 ± 1

a

a

a

a

3.2 ± 0.6

9±2

a

[Sr ] (μM)

number of site

Under the limits of detection.

purified state of B915 or B888 directly determined by the AA measurements. The LH1−RC concentration was calculated to be 0.37 μM based on the absorbance and the molar extinction coefficient of the carotenoid band at 514 nm (ε514 = 2736 mM−1 cm−1).14 The number of metal-binding sites was assessed to be 18 ± 1 and 9 ± 2 for B915 and B888, respectively. The former coincides with the number identified in the crystal structure of the Tch. tepidum LH1−RC complex, which showed 16 Ca2+ in the LH1 and one Ca2+ in the RC.11 It is noteworthy that the samples for AA analysis were carefully prepared without Ca2+ or Sr2+ contamination during all the procedures, as described in the Experimental Section. Therefore, the present results support the hypothesis that B915 originally contains 18 ± 1 Ca2+, and the binding sites are fully occupied in the native state. These Ca ions are tightly bound to the LH1 complex because the Qy peak was little affected upon removal of free Ca2+ by gel filtration or extensive washing with Ca2+-free buffer but was largely blue-shifted to 880 nm upon complete removal of bound Ca2+ by metal chelators (Figure S1A). A similar trend was confirmed for Sr2+ depletion from B888 complexes (data not shown). The thermal stability of B915 was markedly decreased upon Ca2+ depletion by EDTA treatment and also by gel filtration alone (Figure S1B), although the latter little affected the spectral property of the LH1−RC complex (Figure S1A). This indicates that the desalted B915 tightly binds Ca2+ to exhibit the Qy maximum 12468

DOI: 10.1021/acs.jpcb.6b10068 J. Phys. Chem. B 2016, 120, 12466−12473

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Figure 1. ITC profiles for the binding of Ca2+ to Ca2+-depleted B915 (A) and Sr2+-depleted B888 (B). Upper panel: ITC titrations of 25 mM CaCl2 into 0.53 mM LH1 αβ-subunit of the metal-depleted LH1−RC complexes at 25 °C. The inset figure shows an expanded view of the titration profiles for each complex (solid line: Ca2+-depleted B915, dotted line: Sr2+-depleted B888). Lower panel: binding isotherms of the titrations as a function of the Ca2+/αβ-subunit ratio. The solid line represents the best fit to a one-site model.

Table 2. Thermodynamic Parameters of the Binding of Metal Cations to the Metal-Depleted LH1−RC Complexes from Tch. tepidum, Evaluated by ITC Analysisa sample 2+

Ca -depleted B915

Sr2+-depleted B888

2+

Ca Sr2+ Ba2+ Ca2+ Sr2+ Ba2+

K (104 M−1)

N

metal cation 1.0 0.9 0.9 1.0 0.9 0.8

± ± ± ± ± ±

0.1 0.2 0.2 0.1 0.2 0.1

3.7 0.6 1.1 2.3 0.6 1.0

± ± ± ± ± ±

0.7 0.2 0.2 0.9 0.2 0.1

ΔH (kcal/mol) −3.4 −3.0 −4.4 −2.3 −2.8 −4.0

± ± ± ± ± ±

0.1 0.8 0.6 0.2 0.8 0.3

TΔS (kcal/mol) 2.8 2.1 1.1 3.5 2.3 1.5

± ± ± ± ± ±

0.1 0.9 0.7 0.3 0.9 0.3

ΔG (kcal/mol) −6.2 −5.1 −5.5 −5.8 −5.1 −5.5

± ± ± ± ± ±

0.2 1.7 1.3 0.5 1.7 0.6

N, molar ratio of metal cations to αβ-subunit; K, binding constants of metal cations to αβ-subunit; ΔH, change in enthalpy; ΔS, change in entropy; T, reaction temperature (298 K); ΔG, change in Gibbs energy.

a

cus vulcanus.19 However, the number of Sr2+ in B888 detected by AA analysis was much lower than that obtained by ITC analysis. This can be interpreted by the lower binding constant K, 0.6(±0.2) × 104 M−1 for Sr2+ binding to Sr2+-depleted B888 compared to 3.7(±0.7) × 104 M−1 for Ca2+ binding to Ca2+depleted B915. Considering the relatively weak binding affinity, release of Sr2+ from B888 during the sample preparation is probably responsible for the smaller number of Sr2+ detected by AA analysis. However, we cannot completely exclude the possibility that the Sr2+-binding sites of B888 are not fully occupied in the native state despite the maximum binding potential of 16 Sr2+. The thermodynamic parameters (ΔH, TΔS, ΔG, and K) for Sr2+ or Ba2+ binding were largely similar between Ca2+-depleted

B915 and Sr2+-depleted B888. In contrast, the Ca2+-binding constants for B915 and B888 were 3.7(±0.7) × 104 and 2.3(±0.9) × 104 M−1, respectively, and the resulting ΔG values also demonstrated that the Ca2+-binding reaction is more favorable in B915 than in B888. These results support the previous findings that the small but significant differences between B915 and B888 originate from some modifications caused by the biosynthetic replacement of Ca2+ with Sr2+.17 Furthermore, ΔH and TΔS values for Ca2+ binding to the metal-depleted LH1−RC complexes were significantly different between B915 and B888. In B915, the ΔH and TΔS values for Ca2+ binding were −3.4 ± 0.1 and 2.8 ± 0.1 kcal/mol, respectively, indicating that the structural changes result from favorable enthalpic contributions, possibly due to the changes 12469

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Figure 2. Double-reciprocal plots of absorbance changes of B915 (A) and B888 (B) monitored at 937 nm as a function of Ca2+ concentration in the presence of 0.1 mM (square), 1.0 mM (circle), or 5.0 mM (triangle) Sr2+.

progress to understand the contribution of the LH1 C-terminal extrinsic region to the unusual properties of the LH1−RC complex from Tch. tepidum. Competitive Metal Binding. The ITC analysis revealed marked differences in the binding affinities of Ca2+ and Sr2+ to the LH1−RC complexes. However, it is possible that the two metal cations associate with different binding sites with different affinities. To confirm this, competitive metal-binding experiments were conducted in both B915 and B888. Figure 2 shows double-reciprocal plots of the rate of the absorption change at 937 nm for B915 (A) and B888 (B) as a function of the Ca2+ concentration in the presence of different concentrations of Sr2+. The least-squares linear fitting of the experimental data exhibited a crossing point on the y-axis, strongly indicating that Sr2+ competitively binds to the Ca2+binding site in both B915 and B888. The apparent inhibition constants for Sr2+ were estimated to be 0.56 and 0.36 mM for B915 and B888, respectively. The difference in the inhibition constant can be explained by the binding constant K obtained from ITC analysis (Table 2). The binding constant of Ca2+ to Ca2+-depleted B915 was significantly greater than that to Sr2+depleted B888 despite the identical binding constants of Sr2+ in both species, resulting in the effective inhibition by Sr2+ of Ca2+ binding in B888. Structural Changes Induced by Metal Binding and Metal Depletion. To further investigate the differences between B915 and B888, structural and/or conformational changes upon Sr2+/Ca2+ substitution were analyzed by ATRFTIR spectroscopy. Figure 3 shows the Sr2+/Ca2+ ATR-FTIR difference spectra of B915 (spectrum a) and B888 (spectrum b) generated by the metal exchanges from Ca2+ to Sr2+. The characteristic difference bands of B915 reflect the structural modifications of the metal-binding sites and their surroundings between the Ca2+-bound state (negative peaks) and the Sr2+bound state (positive peaks).16 These signals appeared at the same positions with comparable intensities but with inverse signs in the subsequent difference spectra generated by the Sr2+-to-Ca2+ replacement, indicating fully reversible conformational changes upon the exchange (Figure S2). Unexpectedly, Sr2+/Ca2+ difference spectra for B888 (spectrum b) were almost identical to those for B915, as can be clearly seen in the doubledifference spectrum (spectrum c) obtained by subtracting

in the electrostatic interactions within the LH1 C-terminal region. On the other hand, Ca2+ binding to Sr2+-depleted B888 was rather entropically favorable (ΔH = −2.3 ± 0.2 and TΔS = 3.5 ± 0.3 kcal/mol), suggesting increased hydrophobic interactions in the LH1 C-terminal region and/or release of water molecules associated with the charged residues involved in the metal-binding sites. The former is consistent with the previous reports that the largest part of the driving energy leading to the LH1 αβ-heterodimer association arises from hydrophobic interactions occurring in the terminal domains.20−25 In our preliminary study, N-terminal domain of the LH1 αβ-polypeptides from B888 was not altered; however, C-terminal modifications were detected by matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry (unpublished results). This suggests that the LH1 C-terminal domain is responsible for the difference in the thermodynamic properties between B915 and B888. A C-terminal domain swap study of Rba. sphaeroides strongly indicated that the C-terminal extrinsic region of the LH α-polypeptide contributes significantly to the structural and functional properties of LH complexes.26 The crystallographic structure of the Tch. tepidum LH1−RC complex demonstrated that the LH1 C-terminal extrinsic region of an α-polypeptide includes a coil extending into the next αβ-polypeptide through the loop, a part of which comprises the Ca2+-binding site.11 However, the present ATRFTIR study demonstrated that the conformation of the Ca2+binding site in the loop remains largely the same in B915 and B888, as described later. Furthermore, our previous resonance Raman study strongly indicated that hydrogen-bonding interactions between BChls-a and αβ-polypeptides are almost identical in B915 and B888 in their Ca2+-bound forms.18 Notably, the structural information indicated that C-terminal coils are circularly arranged on the membrane surface and serve the side chain of α-Tyr55 to interact with the main chain of the next β-Pro44 through hydrogen bonding. Therefore, the Cterminal coil of the LH1 α-polypeptide is a potential structural origin responsible for the unusual properties of the Tch. tepidum LH1−RC complex, and the differences in the thermodynamic parameters for the Ca2+ binding between B915 and B888 result from some modifications in the Cterminal extrinsic region of the LH1 α-polypeptides induced by the biosynthetic Sr2+-substitution. Further investigations are in 12470

DOI: 10.1021/acs.jpcb.6b10068 J. Phys. Chem. B 2016, 120, 12466−12473

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The Journal of Physical Chemistry B

Figure 3. Sr2+/Ca2+ ATR-FTIR difference spectra of B915 (a) and B888 (b) resulting from the metal exchange between Ca2+ and Sr2+. Spectrum c shows the double-difference spectrum obtained by subtracting spectrum a from spectrum b.

spectrum a from spectrum b. This result indicates that the structure and/or conformation of the metal-binding sites are mostly similar in B915 and B888. The Sr2+/Ca2+ difference spectra reflect the structural modifications of the LH1−RC complex induced by the removal of Ca2+ and binding of Sr2+. To separate the contribution of these two components, the effects of metal depletion from LH1−RC complexes were examined. Figure 4 shows the ATRFTIR difference spectra of B915 upon Ca2+ depletion by EDTA (spectrum a) and upon Ca2+-to-Sr2+ substitution (spectrum b). Interestingly, most of the difference bands resulting from Ca2+ depletion largely coincided with the characteristic Sr2+/Ca2+ difference bands, although some differences were confirmed in the amide I and II regions (1700−1500 cm−1). Similar results were observed when B915 was treated with another chelator, EGTA (data not shown). These results support that the Sr2+/ Ca2+ difference bands predominantly originate from the structural and/or conformational changes around the Ca2+binding site following the release of the bound Ca2+. Furthermore, the difference bands upon Ca2+ depletion were fully reversed by the subsequent addition of Ca2+ (Figure S2), indicating that the structural and/or conformational changes of LH1 complexes are interconvertible between the Ca2+-bound and Ca2+-depleted forms. In the previous far-UV CD analysis, no significant change was observed in the secondary structures of the LH1 polypeptides upon Ca2+ depletion.14 However, the present ATR-FTIR approach enabled the detection of the significant conformational changes in the polypeptide main chains and amino acid side chains comprising the Ca2+-binding site upon Ca2+ depletion. In contrast, the ATR-FTIR difference spectrum of B888 upon Sr2+ depletion (spectrum c) was largely different from the Ca2+-depleted spectrum of B915 (spectrum a). Most of the characteristic bands found in the Ca2+-depleted spectrum of B915 were lost and several mid-to-low intensity bands were detected upon Sr2+ depletion of B888. These bands were

Figure 4. ATR-FTIR difference spectra of B915 upon Ca2+ depletion by EDTA (spectrum a) and upon Ca2+-to-Sr2+ substitution (spectrum b). The latter which is the same as spectrum a in Figure 3 is presented for comparison. Spectrum c was obtained for B888 following Sr2+ depletion by EDTA. Spectrum d was calculated by subtracting spectrum b from spectrum a.

almost completely reversed when reconstituted with Sr2+ (Figure S2), reflecting the structural and/or conformational changes originating from the Sr2+−protein interactions. Furthermore, a similar pattern was observed for B915 in the double-difference spectrum d obtained by subtracting spectrum b from spectrum a, corresponding to the Sr2+-depleted spectrum of B915. These results indicate that the structural and/or conformational changes induced by depletion and binding of Sr2+ are insignificant in both species. Metal-Binding Sites for Ca2+ and Sr2+. The present ATRFTIR data revealed that the structural and conformational changes of the Tch. tepidum LH1−RC complex upon Ca2+-toSr2+ substitution are predominantly induced by Ca2+ depletion and that the contribution of Sr2+ binding is insignificant. However, if both Ca2+ and Sr2+ bind to the same site as indicated by the present competitive metal-binding experiments (Figure 2), it is incomprehensible why the metal-depleted difference spectra were markedly different between B915 and B888 despite their almost identical Sr2+/Ca2+ and Ca2+/Sr2+ difference spectra (Figures 3 and S2). On the basis of the structural information of the Tch. tepidum LH1−RC complex, the Ca2+-binding site includes carboxylate residues of α-Asp49 and β-Leu46 ligating Ca2+ in monodentate and bidentate manners, respectively.11 Generally, the symmetric νCOO− modes of these residues are sensitive to the coordination of metal cations and alter the peak positions and band intensities upon binding and depletion of metal cations, as reported previously.27−29 This is also confirmed with aqueous solutions 12471

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The Journal of Physical Chemistry B

crystallographic structure of the Sr2+-substituted LH1−RC complex from Tch. tepidum.30

of metal acetate complexes (Figure S3). The symmetric νCOO− band of calcium acetate at 1441 cm−1 was downshifted to 1412 cm−1 in sodium acetate, namely, upon Ca2+ depletion. Similar downshifts were observed for strontium acetate from 1431 to 1412 cm−1 upon Sr2+ depletion, although the band intensities were reduced compared to those for Ca2+ depletion. These carboxylate band shifts upon depletion of divalent cations are incompatible with the present result of symmetric νCOO− bands in the 1450−1300 cm−1 region appearing only in the Ca2+-depleted spectra of the LH1−RC complexes. Thus, the present experimental evidence strongly indicates that the binding sites for Ca2+ and Sr2+ are not the same. Considering the numbers of binding sites determined by the ITC analysis (Table 2) and the competitive binding of both metal cations (Figure 2), a possible interpretation for the present spectroscopic and thermodynamic results is that Sr2+ associates with a different binding site in the vicinity of the Ca2+-binding site, but both sites are not occupied simultaneously by the metal cations. Recently, an X-ray crystallographic structure of the Sr2+substituted LH1−RC complex from Tch. tepidum has been resolved at 3.3 Å resolution and a different binding site for Sr2+ (or Br2+) was identified.30 Intriguingly, the site is located at the C-terminal side of each LH1 subunit and in proximity to the Ca2+-binding site at an average distance of 5 Å and comprises amino acid residues from only α-polypeptide; αn-Asp49, αnAsn45, αn+1-Gln56, and αn+1-Tyr55. Among the ligands for Sr2+, αn-Asp49 is involved in the Ca2+-binding site of the wild-type LH1−RC complex11 and therefore, shared in both metalbinding sites. This is very consistent with the present conclusion that Sr2+ binds to the different site in the vicinity of the Ca2+-binding site, but both sites are not occupied simultaneously by the metal cations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10068. Absorption spectra and DSC profiles of B915 in the presence or absence of Ca2+ (Figure S1); ATR-FTIR difference spectra of B915 and B888 upon metal exchanges between Ca2+ and Sr2+ and upon metal depletions/reconstitutions (Figure S2); ATR-FTIR absorption spectra of metal acetate complexes (Figure S3) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +81-78-8035819. ORCID

Yukihiro Kimura: 0000-0003-3747-0367 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants-in-aid for Scientific Research (C) (24570158 and 16K07295) (Y.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We appreciate the Research Facility Center for Science and Technology of Kobe University and the Instrument Center of the Institute for Molecular Science for assistance with ITC measurements.

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CONCLUSIONS In this study, metal-binding sites and metal−protein interactions in B915 and B888 were investigated by AA, ITC, and ATR-FTIR analyses. The AA analysis provided experimental evidence that B915 and B888 originally contain Ca2+ and Sr2+, respectively. The number of metal-binding sites was estimated by ITC measurements, to be approximately 16 per LH1 complex in both B915 and B888. The binding affinity and the metal−protein interaction modes for Ca2+ binding were significantly different between B915 and B888, despite identical conformational changes around the Ca2+-binding site, as revealed by the ATR-FTIR analysis. These results and the structural information indicate that the C-terminal extrinsic coils of the LH1 α-polypeptide are a potential structural origin for the differences in the thermal stability and LH1 Qy redshift of both LH1−RC complexes in their Ca2+-bound forms. The present ATR-FTIR data further demonstrated that release and binding of Ca2+ are predominantly responsible for the metaldependent structural/conformational changes of Tch. tepidum LH1−RC complexes, and the contribution of depletion/ binding of Sr2+ is insignificant. On the basis of the structural information of the Ca2+-binding site and IR data for model compounds, the presence of another metal-binding site for Sr2+ (or Ba2+) is proposed. Considering the number of binding sites derived from the ITC analyses and the competitive metalbinding properties, a plausible interpretation is that the Sr2+binding site exists in the vicinity of the Ca2+-binding site, and both sites are not occupied simultaneously by the metal cations. The present conclusion is supported by the recent X-ray

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