Article pubs.acs.org/JPCB
Effects of Calcium Ions on the Thermostability and Spectroscopic Properties of the LH1-RC Complex from a New Thermophilic Purple Bacterium Allochromatium tepidum Yukihiro Kimura,*,† Shuwen Lyu,† Akira Okoshi,‡ Koudai Okazaki,‡ Natsuki Nakamura,‡ Akira Ohashi,‡ Takashi Ohno,† Manami Kobayashi,† Michie Imanishi,† Shinichi Takaichi,§,∥ Michael T. Madigan,⊥ and Zheng-Yu Wang-Otomo*,‡ †
Department of Agrobioscience, Graduate School of Agriculture, Kobe University, Nada, Kobe 657-8501, Japan Faculty of Science, Ibaraki University, Mito 310-8512, Japan § Department of Biology, Nippon Medical School, Kyonan-cho, Musashino, Tokyo 180-0023, Japan ⊥ Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901, United States ‡
ABSTRACT: The light harvesting-reaction center (LH1-RC) complex from a new thermophilic purple sulfur bacterium Allochromatium (Alc.) tepidum was isolated and characterized by spectroscopic and thermodynamic analyses. The purified Alc. tepidum LH1-RC complex showed a high thermostability comparable to that of another thermophilic purple sulfur bacterium Thermochromatium tepidum, and spectroscopic characteristics similar to those of a mesophilic bacterium Alc. vinosum. Approximately 4−5 Ca2+ per LH1-RC were detected by inductively coupled plasma atomic emission spectroscopy and isothermal titration calorimetry. Upon removal of Ca2+, the denaturing temperature of the Alc. tepidum LH1-RC complex dropped accompanied by a blue-shift of the LH1 Qy absorption band. The effect of Ca2+ was also observed in the resonance Raman shift of the C3-acetyl νCO band of bacteriochlorophyll-a, indicating changes in the hydrogen-bonding interactions between the pigment and LH1 polypeptides. Thermodynamic parameters for the Ca2+-binding to the Alc. tepidum LH1-RC complex indicated that this reaction is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. Our data support a hypothesis that Alc. tepidum may be a transitional organism between mesophilic and thermophilic purple bacteria and that Ca2+ is one of the major keys to the thermostability of LH1-RC complexes in purple bacteria.
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INTRODUCTION Allochromatium (Alc.) tepidum strain NZ is a new species of thermophilic purple sulfur bacteria isolated from a sulfidic microbial mat in a New Zealand hot spring.1 The organism grows optimally near 45 °C, slightly lower than another thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum,2,3 but distinctly higher than that of the mesophilic bacterium Alc. vinosum. Unlike Tch. tepidum, the lightharvesting 1 (LH1) complex of Alc. tepidum exhibits an absorption maximum (Qy transition) at 890 nm in the nearinfrared region, a feature similar to that of Alc. vinosum and the majority of purple photosynthetic bacteria. It has been known that many proteins purified from the thermophilic Tch. tepidum exhibit higher structural and thermal stabilities than their mesophilic counterparts although the corresponding proteins share high sequence homology. These include ribulose-1, 5-bisphosphate carboxylase/oxygenase,4,5 flavocytochrome c,6,7 cytochrome c′,7 high-potential iron− sulfur protein, 8,9 and LH1-reaction center (RC) core complex.10−14 For the water-soluble proteins, the enhanced © XXXX American Chemical Society
stabilities in most cases can be interpreted as a consequence of combined effects of an increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures, and a reduced number of glycine residues.7 Many residues with large side chains in Alc. vinosum proteins were found to be substituted by alanines in the corresponding Tch. tepidum proteins, and the substituted residues are mainly located on the surface and exposed to the solvent.7 On the other hand, the LH1-RC membrane protein complex seems to use an alternative strategy by incorporating Ca2+ ions for enhancing its thermal and structural stabilities.11,14,15 Sixteen Ca ions have been identified in the Tch. tepidum LH1 complex from the crystal structure with each Ca2+ being coordinated by the amino acid residues from both α- and β-polypeptides. As a result, the LH1 complex forms a tight double-ring network of the α- and β-polypeptides connected by the Ca ions, which Received: April 8, 2017 Revised: April 28, 2017 Published: May 1, 2017 A
DOI: 10.1021/acs.jpcb.7b03341 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B
contents of Ca, Mg and Fe atoms in the Alc. tepidum LH1RC complex were carried out on an ICP-AES spectrophotometer (ICPS-7510, Shimadzu). The detection wavelengths were set to 393.366 nm for Ca, 279.553 nm for Mg, and 259.940 nm for Fe. The highly purified LH1-RC fractions eluted from the DEAE column with NaCl salt were adjusted to concentrations of OD890 = 10−15 and were treated with 5% (w/v) HNO3 solution. Multi-element Standard Solution W−II (Wako Pure Chemical) containing 1000 mg/L of Ca, 100 mg/ L of Fe, and 1000 mg/L of Mg in 1 M HNO3 solutions was diluted to appropriate concentrations and used for calibrations. The ICP-AES measurements were also used, in combination with absorption data, to evaluate molar extinction coefficient of the LH1-RC complex by utilizing the nature of invariant mole ratio of Mg to Fe atoms within the LH1-RC complex. Resonance Raman Spectroscopy. NIR and visible Raman spectra of the purified LH1-RC complexes were recorded on an NRS-7100 Raman microscope (JASCO Corporation). An aliquot (∼30 μL) of the LH1-RC solution (OD = 150−200 at the Qy maximum) was deposited onto a stainless plate. Excitation beam provided from the fundamental (1064 nm) or frequency-doubled (532 nm) Nd3+: YAG laser was focused onto the sample solution through a ×50 or ×20 objective lens. The laser intensity at the sample surface was adjusted to ∼10 mW for 1064 nm or ∼6.0 mW for 532 nm excitation to alleviate sample decomposition. Under these conditions, neither decrease in band intensities nor elevation of baselines due to degradation was detected during the measurements. The backscattering from the sample solution was collected at 25 °C for 30−60 s with a liquid N2-cooled InGaAs detector for 1064 nm or a CCD detector for 532 nm. Five to ten spectra were averaged to improve the signal-to-noise ratio. Isothermal Titration Calorimetry (ITC). ITC profiles were recorded on a MicroCal iTC200 microcalorimeter. A solution of Ca2+-depleted LH1-RC complexes (OD514 = ∼ 90) was titrated 20 times with 25 mM CaCl2 solution at 25 °C and intervals of 2 min. The accumulated molar heat of the injectant was plotted as a function of time and analyzed by one site model of Origin software to yield the thermodynamic parameters of the Ca2+-binding to the LH1-RC complexes. Differential Scanning Calorimetry (DSC). DSC thermograms were recorded on a Microcal VP-DSC calorimeter.11 The sample concentration of the LH1-RC was adjusted to OD514 = 25−35 in a buffer containing 20 mM Tris-HCl (pH 7.5) and 0.08% (w/v) DDPC. Thermal degradation of the LH1-RC complex was monitored in the range of 25−100 °C at a heating rate of 1 °C/min.
significantly stabilizes the whole LH1 structure. Since the Cabinding sites are located in close vicinity to the bacteriochlorophyll (BChl)-binding sites in the LH1 C-terminal region, the spectroscopic property of Tch. tepidum LH1 is highly sensitive to Ca2+. Removal of Ca2+ resulted in a 35 nm blue shift of the LH1 Qy band accompanied by a reduced thermal stability.11,16 Therefore, Ca2+ can be used as a sensitive dualprobe for monitoring the subtle changes in both spectroscopic and thermodynamic properties.13,17−20 Alc. tepidum is the second thermophilic species discovered in the purple sulfur photosynthetic bacteria, and thus it is of interest to investigate whether Ca2+ also plays a role in modulating the properties of its LH1-RC core complex. In this work, we describe purification process of the LH1-RC complex from Alc. tepidum and examine the effects of Ca2+ on its thermodynamic and spectroscopic properties. The purified Alc. tepidum LH1-RC complex is characterized using inductively coupled plasma atomic emission spectroscopy (ICP-AES), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and resonance Raman spectroscopy. The results are compared between the three purple sulfur bacteria Alc. tepidum, Tch. tepidum, and Alc. vinosum.
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EXPERIMENTAL SECTION Sample Preparation. LH1-RC complexes from Tch. tepidum strain MC and Alc. vinosum strain D were purified as described previously.18 Alc. tepidum strain NZ cells were cultivated anaerobically under an incandescent lamp at 43−45 °C for 7−10 days. Universally 13C-labeled Alc. tepidum cells were cultured in a medium containing 13CH313COOH and NaH13CO3 (Masstrace Inc., 99 atom % 13C) as the carbon source. The harvested cells were sonicated in 20 mM Tris-HCl buffer (pH 8.5) to obtain chromatophores. A large portion of LH2 complexes were removed by solubilization with 0.33% (w/ v) lauryldimethylamine N-oxide (Anatrace) at 25 °C for 60 min, followed by ultracentrifugation at 4 °C and 195 000 × g for 60 min. The pellets were further treated with 1.0% (w/v) noctyl-β-D-glucopyranoside (OG, Anatrace) and ultracentrifuged to solubilize LH1-RC components. The supernatant containing crude LH1-RC was loaded onto a DEAE anion-exchange column (Toyopearl 650S, TOSOH) equilibrated at 4 °C with 20 mM Tris-HCl (pH 7.5) buffer containing 0.08% (w/v) dodecylphosphocholine (DDPC, Anatrace). The LH1-RC fraction was eluted by a linear gradient of CaCl2 from 10 mM to 45 mM or NaCl from 20 mM to 80 mM, and the peak fractions with an A890/A280 > 2.0 were collected. Ca2+-depleted LH1-RC complexes were prepared as described previously.20 The purified LH1-RC complexes were first passed through a size-exclusion column (Sephadex G25 M PD10, GE Healthcare) to remove excess of salts in the solution. Then, the filtrates were incubated at 0 °C for 60 min under darkness in the presence of 1 mM EDTA to remove the tightly bound Ca2+, followed by extensive washing with a buffer containing 20 mM Tris-HCl (pH 7.5) and 0.08% (w/v) DDPC to remove residual EDTA and then concentrated with Amicon Ultra 100 K filters (Millipore). The resulting Ca2+-depleted LH1-RC complex from Alc. tepidum exhibited the LH1 Qy maximum at 882 nm. Carotenoid compositions were measured with the C18-HPLC system as described elsewhere10 except that the UV monitor was replaced by a photodiode-array detector (SPD-M10A vp, Shimadzu). Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). Simultaneous determinations of the
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RESULTS AND DISCUSSION Contents of Metal Atoms. The X-ray crystallographic structure of the LH1-RC complex from Tch. tepidum demonstrated that 16 and one Ca2+ were bound to the LH1 and RC complexes, respectively.14 The presence of 17 Ca2+ per LH1-RC complex has been further confirmed by the direct detection of 18 ± 1 Ca2+ using atomic absorption spectroscopy.20 Similar to Tch. tepidum, Alc. tepidum is also a moderate thermophile and it is natural to speculate that Ca2+ might also exist in the Alc. tepidum LH1-RC complex and modulate its properties. To verify this hypothesis, we applied the ICP-AES analysis to the Alc. tepidum LH1-RC complex. This technique has advantages of high sensitivity and multidetection, which enables us to use Mg and Fe contents in the LH1-RC complex B
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Figure 1. (A) Absorption spectra of purified LH1-RC complexes from Tch. tepidum (black), Alc. tepidum (magenta), and Alc. vinosum (gray). (B) An expanded view of the absorption spectra in the 750−1000 nm region for the LH1-RC complexes of Tch. tepidum (a), Alc. tepidum (b), and Alc. vinosum (c) in the presence of Ca2+ (solid lines) or those after removal of Ca2+ (dashed lines).
as the inner references to determine the stoichiometry of Ca2+. The metal contents of Mg, Fe, and Ca in the highly purified Alc. tepidum LH1-RC complex were simultaneously determined to be 37 ± 2, 5.3 ± 1.0, and 4.2 ± 1.0 per LH1-RC, respectively. In view of the phylogenetic similarity between Alc. tepidum and Tch. tepidum,1 each Alc. tepidum LH1-RC complex is assumed to contain 36 Mg atoms (4 BChls in RC and 32 BChls in LH1), and 5 Fe atoms (4 hemes in C-subunit of RC and one nonheme iron in RC). The results from ICP-AES analysis were in good agreement with the calculated stoichiometries for these metal atoms. As a result, the number of Ca2+ was determined to be approximately 4 per LH1-RC complex in the native Alc. tepidum. Intriguingly, this value is much lower than the 18 ± 1 Ca2+ in the Tch. tepidum LH1-RC complex as detected by atomic absorption spectroscopy20 and 17 Ca2+ in the crystallographic structure of the Tch. tepidum LH1-RC complex.14 Based on the contents of Mg and Fe atoms and the Qy band intensity of the Alc. tepidum LH1-RC solution, the molar extinction coefficient was estimated to be 3815 mM−1cm−1 for the LH1 Qy band, which was used to calculate sample concentrations for other analyses. Effects of Ca2+ on the LH1 Absorption Property. Since the result of ICP-AES analysis indicated that Ca2+ are present in the Alc. tepidum LH1-RC complex, the effects of Ca2+ on the LH1 Qy absorption were examined. Figure 1A shows a comparison of the absorption spectra of purified LH1-RC complexes from Tch. tepidum, Alc. tepidum, and Alc. vinosum. The LH1 Qy band of Alc. tepidum was observed at 890 nm, which was red-shifted by 6 nm than that of Alc. vinosum but largely blue-shifted by 25 nm from that of Tch. tepidum. Upon removal of Ca2+ from the LH1-RC complexes (Figure 1B), the Qy peaks were blue-shifted from 890 to 882 nm for Alc. tepidum and from 915 to 880 nm for Tch. tepidum with decreased intensities. In contrast, the Qy band of Alc. vinosum was not affected at all upon Ca2+ depletion in both peak position and band intensity. The shape of the Alc. vinosum LH1-RC complex spectrum was quite similar to that of the Ca2+-depleted Alc. tepidum LH1-RC complex. These results indicate that Ca2+ are also involved in the spectral properties of the Alc. tepidum LH1RC complex and affect the conformation of the LH1 BChl-a molecules in a manner similar to the Tch. tepidum LH1-RC complex.16
The carotenoid absorption bands of Alc. tepidum LH1-RC complex appeared at 487, 514, and 550 nm (Figure 1A), almost identical with those of the Tch. tepidum and Alc. vinosum LH1RC complexes. However, the relative band intensities were in an order of Tch. tepidum > Alc. tepidum ≈ Alc. vinosum when the spectra were normalized at 280 nm. Carotenoid analysis revealed that the major carotenoid in the Alc. vinosum LH1-RC complex is spirilloxanthin (Table 1). The proportion of Table 1. Carotenoid Composition (mol% of Total Carotenoids) in Whole Cell, LH1-RC, and LH2 of Alc. tepidum carotenoid
cell
LH1-RC
LH2
lycopene rhodopin dihydoxylycopene anhydrorhodovibrin rhodovibrin spirilloxanthin
4.3 57.4 5.9 13.7 2.2 16.5
0.4 7.4 8.1 6.1 4.7 73.2
5.8 71.0 2.6 17.3 0.6 2.7
spirilloxanthin (73%) is somewhat lower than that in Tch. tepidum LH1-RC complex (92%).10 Therefore, the slight differences in the intensities of carotenoid bands are presumably attributable to the contents of spirilloxanthin. It is noted that the carotenoid absorption bands were not influenced by Ca2+ depletion (data not shown), indicating the lack of interaction between Ca2+ and carotenoid molecules. Carotenoid−Protein Interaction. To investigate carotenoid-protein interactions, resonance Raman spectra of unlabeled and universally 13C-labeled LH1-RC complexes were measured upon 532 nm excitation (Figure 2A). In Alc. tepidum (spectrum b, solid line), characteristic carotenoid bands were observed at 1507, 1151, and 1001 cm−1, which can be ascribed to CC stretching mode, C−C stretching/CH bending modes, and CH3 rocking mode of all-trans spirilloxanthin, respectively.18,21 Upon universal 13C-labeling (spectrum b, dashed line), the carotenoid bands were clearly downshifted by 32, 30, and 15 cm−1 for νCC, νC−C/δCH, and δCH3 modes, respectively. The unlabeled and universally 13C-labeled carotenoid bands were also detected for the Alc. vinosum LH1RC complexes (spectra c) at almost identical positions, strongly C
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Figure 2. (A) Resonance Raman spectra of the unlabeled (solid lines) and universally 13C-labeled (dashed lines) LH1-RC complexes from Tch. tepidum (a), Alc. tepidum (b), and Alc. vinosum (c) obtained by the excitation at 532 nm. (B) Interaction between spirilloxanthin and His residue ligating BChl-a in the LH1 αβ-polypeptides of Tch. tepidum.
Figure 3. (A) Resonance Raman spectra of the LH1-RC complexes from Tch. tepidum (a),11 Alc. tepidum (b), and Alc. vinosum (c) in the presence of Ca2+ (solid lines) or those after removal of Ca2+ (dotted lines) obtained by the excitation at 1064 nm. (B) Hydrogen-bonding interactions between BChls-a and Trp residues residing in the vicinity of the LH1 Ca2+-binding site of Tch. tepidum. (C) Plots of the C3-acetyl νCO band peak against the LH1 Qy maximum of the Tch. tepidum, Alc. tepidum, and Alc. vinosum LH1-RC complexes.
indicating that the composition and conformation of carotenoids are highly similar between Alc. tepidum and Alc. vinosum LH1-RC complexes. In contrast, νCC and νC−C bands of the Tch. tepidum LH1-RC complex (spectrum a, solid line) were slightly downshifted by 3−5 cm−1 compared with those of Allochromatium species. This is further confirmed in the universally 13C-labeled spectra in which the carotenoid bands of Tch. tepidum (spectrum a, dashed line) exhibited 7−8 cm−1 downshifts from those of Allochromatium species (spectra
b and c, dashed lines). Generally, elongation of the conjugation system results in the low-frequency shift of νCC bands.22 Spirilloxanthin has the most elongated conjugation system (N = 13) compared to other minor carotenoids (N = 11−12) detected in the Alc. tepidum LH1-RC complex. Thus, the differences in the Raman spectra may be attributable to the differences in the proportion of spirilloxanthin in the LH1-RC complexes between these species. Alternatively, some specific interaction between carotenoid molecules and proteins may D
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Figure 4. ITC profiles for the binding of Ca2+ to Ca2+-depleted LH1-RC complexes from Alc. tepidum (A) and Tch. tepidum (B). ITC titrations of 25 mM CaCl2 into the Ca2+-depleted LH1-RC complexes at 25 °C (upper panel), and binding isotherms of the titrations (lower panel) 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 for the Binding of Ca2+ to Ca2+ -Depleted LH1-RC Complexes from Alc. tepidum or Tch. tepidum20 Evaluated by ITC Analysisa species
N
K (104 M−1)
ΔH (kcal/mol)
TΔS (kcal/mol)
ΔG (kcal/mol)
Alc. tepidum Tch. tepidum
0.33 ± 0.04 1.0 ± 0.1
0.96 ± 0.11 3.7 ± 0.7
−7.6 ± 0.5 −3.4 ± 0.1
−2.1 ± 0.6 2.8 ± 0.1
−5.5 ± 0.1 −6.2 ± 0.2
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
cm−1 red-shift, respectively. Consistent with previous studies for Rba. sphaeroides,23 the peak positions of the C3-acetyl C O stretching bands were linearly correlated with the LH1 Qy maxima in Tch. tepidum, Alc. tepidum, and Alc. vinosum (Figure 3C), suggesting that the hydrogen-bonding interaction between BChl−Trp influences the LH1 Qy transition energy of these species. Upon Ca2+ depletion, the C3-acetyl and C13-keto CO stretching bands of Tch. tepidum LH1-RC complex were blueand red-shifted to 1645 and 1670 cm−1, respectively, indicating that hydrogen-bonding interactions between BChl-a and LH1polypeptides were significantly modified.18 In Alc. tepidum, the C3-acetyl νCO band was slightly blue-shifted by 2 cm−1 in the Ca2+-depleted form, whereas the C13-keto νCO band was not affected. The former may be partly responsible for the 8 nm upshift of the LH1 Qy absorption in Alc. tepidum (Figure 1B). These results support our contention that the spectral properties of the Alc. tepidum are dependent on Ca2+, but not as strongly as for the Tch. tepidum LH1-RC complex. Metal−Protein Interaction. The spectroscopic results above indicate that Ca2+ is involved in the LH1-RC complex from Alc. tepidum. However, the effects were relatively smaller compared to those of the Tch. tepidum LH1-RC complex (Figures 1 and 3). To understand the role of Ca2+ in Alc. tepidum, interactions between Ca2+ and LH1-RC proteins were examined thermodynamically. Figure 4 shows the ITC profiles
also be responsible for the differences in the Raman spectra. Based on the structural information on Tch. tepidum LH1-RC complex,14 one of the methoxy groups of spirilloxanthin locates in a close proximity to the imidazole group of the α-His36 coordinating to the LH1 BChl-a (Figure 2B). It is likely that such interaction could be responsible for the formation of a different conformation of the spirilloxanthin that shows the downshifts of νCC and νC−C bands in the Tch. tepidum LH1-RC complex. BChl−Protein Interaction. The effects of Ca2+ on the interaction between BChl a and LH1-polypeptides were examined by near-infrared resonance Raman spectroscopy. Figure 3A shows the Raman spectra of the Tch. tepidum, Alc. tepidum, and Alc. vinosum LH1-RC complexes in the Ca2+bound and Ca2+-depleted forms. In Tch. tepidum (spectrum a), CO stretching bands of C3 acetyl and C13 keto groups appeared at 1637 and 1675 cm−1, respectively, for the Ca2+bound form.18 The former band is most red-shifted among the LH1-RC complexes of purple bacteria reported, representing a strong hydrogen-bonding between the C3 acetyl CO groups and Trp residues of the LH1 polypeptides (Figure 3B). In Alc. tepidum (spectrum b), the C3-acetyl and C13-keto νCO modes appeared at 1644 and 1667 cm−1, which were largely similar to those of Alc. vinosum (spectrum c), but significantly different from those of Tch. tepidum; that is, the C3-acetyl and C13-keto νCO bands exhibited a 7 cm−1 blue-shift and 8 E
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Figure 5. (A) DSC curves of the LH1-RC complexes from Alc. tepidum (magenta) and Tch. tepidum (black)11 in the presence of Ca2+ (a) or those after removal of Ca2+ (b) obtained with a heating rate of 1 °C/min. (B) Changes in the relative LH1 Qy band intensities of Alc. tepidum (magenta) and Tch. tepidum (black) in the presence of Ca2+ (closed circle) or those after removal of Ca2+ (open circle) under the incubation at 50 °C for 96 min. Effects of Ca2+ on the growth of Alc. tepidum (C) and Tch. tepidum (D) cells cultivated at 45 °C. (E) The relative LH1 Qy band intensities of Alc. tepidum or Tch. tepidum cells cultivated in the presence (gray bars) or absence of Ca2+ (blue bars) at 45 °C for 10 days.
obtained with titration of Ca2+ to the Ca2+-depleted form of Alc. tepidum (A) and Tch. tepidum (B) LH1-RC complexes. Intensive exothermic signals were observed upon Ca2+-binding to the Ca2+-depleted LH1-RC complexes from Alc. tepidum compared with those from Tch. tepidum. The thermograms were analyzed with a one-site model, and the thermodynamic parameters obtained for the Ca2+-binding reaction are shown in Table 2. The molar ratio N of Ca2+ to LH1 αβ-subunit of Alc. tepidum was determined to be 0.33 ± 0.04, which was significantly lower than that of Tch. tepidum (N = 1.0).16 The stoichiometric ratio of N = 1.0 for the Tch. tepidum LH1-RC complex (meaning 16 Ca2+ per LH1 complex) was confirmed by the recent crystallographic structure of this complex.14 Accordingly, the number of Ca2+ bound to the Alc. tepidum LH1 complex was estimated to be 5.3, indicating that approximately 5 Ca ions are bound to the Alc. tepidum LH1 complex. This value is almost identical to that determined by the ICP-AES analysis. The binding constant K for the Alc. tepidum LH1-RC complex was determined to be 0.96 × 104 M−1 from the ITC analysis (Table 2), which was much lower than that of Tch. tepidum (3.7 × 104 M−1).20 This suggests that the properties of Ca2+-binding sites and the interactions between the complexes and Ca2+ are significantly different in Alc. tepidum and Tch. tepidum. Such differences could be interpreted in terms of the changes in enthalpy and entropy for the Ca2+-binding reactions. In Tch. tepidum, the ΔH and TΔS values were −3.4 and 2.8 kcal/mol, respectively, meaning the Ca2+-binding to be favorable both entropically and enthalpically.20 However, the ΔH and TΔS values for Alc. tepidum were −7.6 and −2.1 kcal/ mol, respectively, indicating that the Ca2+-binding to the LH1 complex is predominantly driven by an unusually negative
enthalpic change that counteracts the positive entropic change unfavorable for the reaction. The largely negative ΔH value for Alc. tepidum suggests that the structural changes induced by Ca2+ binding mostly result from a strong and/or a large number of electrostatic interactions. The decrease in entropic change is interpretable as reduced hydrophobic interactions within the LH1 complex and/or a reduced number of free water molecules associated with the predominant electrostatic interactions.20,24,25 These results suggest that the Ca2+-binding sites and/or the interaction modes are significantly different in Alc. tepidum and Tch. tepidum. To understand more details of the Ca2+-protein interactions, FTIR analyses combined with 15N, 13 C, and 2H isotope labelings, are in progress. Thermal Stability. Both ICP-AES and ITC analyses strongly indicated that the Alc. tepidum LH1-RC complex is stabilized by the predominant electrostatic interactions through the binding of 4−5 Ca2+ per LH1 complex. To examine the effect of Ca2+ on the thermostability of the Alc. tepidum LH1RC complex, its thermal denaturing property was measured by DSC analysis. Figure 5A compares the DSC profiles of the Ca2+-bound (spectra a) and Ca2+-depleted (spectra b) forms between Alc. tepidum and Tch. tepidum. For the Ca2+-bound LH1-RC complex from Alc. tepidum, an intensive peak was observed at 75.7 °C, almost identical to that of the Tch. tepidum LH1-RC complex (75.0 °C). The result indicates that the Alc. tepidum LH1-RC complex has a high thermostability, comparable to the Tch. tepidum LH1-RC complex. Upon removal of Ca2+, the denaturing temperatures of the LH1-RC complexes were markedly decreased to 69.6 °C for Alc. tepidum and 59.9 °C for Tch. tepidum11 with increased band widths and decreased intensities. Surprisingly, the Alc. tepidum LH1-RC complex is even more stable than the Tch. tepidum LH1-RC F
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complex in the Ca2+-depleted forms. This is further supported by the time profile of the Qy band intensities for the Ca2+bound and Ca2+-depleted LH1-RC complexes from Alc. tepidum and Tch. tepidum under the incubation at 50 °C (Figure 5B). The thermal stabilities of LH1-RC complexes from both species were almost identical in their Ca2+-bound forms but largely different in their Ca2+-depleted forms. The difference in Ca2+ requirements was also apparent when Alc. tepidum and Tch. tepidum were cultivated in the presence or absence of added Ca2+. Tch. tepidum showed significant phototrophic growth only in the presence of added Ca2+ (Figure 5C). By contrast, Alc. tepidum grew to approximately 60% of full cell density in the absence of added Ca2+ (Figure 5D). These results support a prediction that Ca2+ starvation should affect proper assembly and functioning of the LH1 complexes of both Tch. tepidum and Alc. tepidum and that once trace levels of Ca2+ in the medium were exhausted, further growth of either species would cease. Hence, the greater requirement for Ca2+ in the LH1 of Tch. tepidum than in the LH1 of Alc. tepidum is fully consistent with the growth yields of both species observed under Ca2+ starvation conditions (Figures 5C−E).
Article
AUTHOR INFORMATION
Corresponding Authors
*Tel. and Fax: +81-78-803-5819; E-mail: ykimura@people. kobe-u.ac.jp. *Tel. and Fax: +81-29-228-8352; E-mail:
[email protected]. jp. ORCID
Yukihiro Kimura: 0000-0003-3747-0367 Present Address ∥
Department of Molecular Microbiology, Faculty of Life Sciences, Tokyo University of Agriculture, Sakuragaoka, Setagaya, Tokyo 156−8502, Japan
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) to Y.K. and (B) (16H04174) to Z.-Y.W.-O. 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 for assistance with ITC measurements, and the Instrument Center of the Institute for Molecular Science for DSC measurements.
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CONCLUSIONS In this study, highly purified LH1-RC complexes from a new thermophilic purple sulfur bacterium Alc. tepidum were investigated by spectroscopic and thermodynamic analyses. The Alc. tepidum LH1-RC complex exhibited a LH1 Qy peak at 890 nm, which was slightly red-shifted from that of Alc. vinosum but largely blue-shifted from that of Tch. tepidum. The Qy peaks were linearly correlated with the frequencies for C3-acetyl νCO Raman bands and slightly blue-shifted upon removal of Ca 2+. The results indicate that the hydrogen-bonding interactions between BChl-a and LH1 Trp residues are partly modulated by Ca2+. Based on both ICP-AES and ITC analyses, the number of bound Ca2+ per LH1-RC complex purified from Alc tepidum was determined to be 4−5, which is a quarter of that of Tch. tepidum.20 The DSC analysis demonstrated that the thermal stability of the LH1-RC complexes was similar between Alc. tepidum and Tch. tepidum in their Ca2+-bound forms but decreased significantly in Tch. tepidum but only moderately in Alc. tepidum upon removal of Ca2+. The thermodynamic parameters evaluated from the ITC analysis indicated that Ca2+binding to the Alc. tepidum LH1-RC complex is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. In contrast, Ca2+-binding in Tch. tepidum was favorable, both entropically and enthalpically.20 Based on these findings, we propose that the enhanced thermal stability of the Alc. tepidum LH1-RC complex is in part due to a Ca2+-dependent mechanism, in which the LH1-RC complex is stabilized by the binding of 4−5 Ca2+ per each LH1 complex, but that the location of Ca2+ binding sites and conformational changes around the sites differ significantly from those of Tch. tepidum. Phylogenetic analyses indicated that Alc. tepidum is more closely related to Alc. vinosum than to Tch. tepidum.1 However, the thermophilic phenotype and presence of Ca2+ in the Alc. tepidum LH1 are properties characteristic of Tch. tepidum but not Alc. vinosum. Thus, it is possible that Alc. tepidum is an evolutionary intermediate between mesophilic and thermophilic chromatia1 and that Ca2+ is one of the major keys to thermostability of LH1-RC complexes in purple bacteria.
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