Selective Removal of B800 Bacteriochlorophyll a from Light

Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Selective Removal of B800 Bacteriochlorophyll a from LightHarvesting Complex 2 of the Purple Photosynthetic Bacterium Phaeospirillum molischianum Yoshitaka Saga,*,†,‡ Keiya Hirota,† Sayaka Matsui,§ Hitoshi Asakawa,*,‡,§,∥ Hiroshi Ishikita,⊥,# and Keisuke Saito*,⊥,# †

Department of Chemistry, Faculty of Science and Engineering, Kindai University, Higashi-Osaka, Osaka 577-8502, Japan Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan § Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan ∥ Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa 920-1192, Japan ⊥ Department of Applied Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan # Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan ‡

S Supporting Information *

ABSTRACT: The selective removal of B800 bacteriochlorophyll (BChl) a from light-harvesting complex 2 (LH2) in purple photosynthetic bacteria is a clue about elucidation of the mechanism for the transfer of energy from these pigments to B850 BChl a and their roles in the LH2 protein structure. We demonstrated that the kinetics of the removal of B800 BChl a from two representative LH2 proteins derived from Phaeospirillum molischianum and Rhodoblastus acidophilus differed significantly, in contrast to the calculated binding enthalpy. These results may be interpreted as changes in the local structure near B800 BChl a with respect to the geometries of the original crystal structures upon removal of B800 BChl a. Despite the difficulty of removing B800 BChl a from molischianum-LH2, we prepared the molischianumLH2 protein lacking B800 BChl a by combination of two detergents, n-dodecyl β-D-maltoside and n-octyl β-D-glucoside, under acidic conditions. Spectral and atomic force microscopy analyses indicated that the absence of B800 BChl a had little effect on the local structure in the vicinity of B850 BChl a and the circular arrangement in this protein. These results suggest that the hydrophobic domain near B850 BChl a is rigid and plays a major role in the structural formation of molischianum-LH2.

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cally arranged in a circular form in the protein matrix.11−15 The three-dimensional structures of only two LH2 proteins, which are derived from Phaeospirillum molischianum (formerly known as Rhodospirillum molischianum) and Rhodoblastus acidophilus (formerly known as Rhodopseudomonas acidophila), are now available at atomic-level resolutions.13−15 Hereafter, LH2 proteins from Phs. molischianum and Rbl. acidophilus are denoted as molischianum-LH2 and acidophilus-LH2, respectively. Georgakopoulou et al. classified LH2 proteins from various purple bacteria into two groups, acidophilus-like (type 1) and molischianum-like (type 2) LH2, based on their spectral properties.16 Therefore, the two LH2 proteins are recognized to be the representative LH2 of purple photosynthetic bacteria.

ight harvesting is a crucial process for efficient light energy conversion during photosynthesis. Absorption and concentration of light energy in photosynthetic antenna complexes are based on organizations and excitonic interactions of chlorophyll (Chl) and bacteriochlorophyll (BChl) pigments in the protein scaffolds. 1−5 The pigment binding to polypeptides plays important roles in the ordered structural formations and the resultant light-harvesting activities in photosynthetic antenna proteins. The selective removal of (B)Chl pigments from light-harvesting proteins and the subsequent pigment insertion have provided useful information for understanding their roles in the structural formation and the energy transfer mechanisms in light-harvesting pigment− protein complexes.6−10 Light-harvesting complex 2 (LH2), which functions as a peripheral antenna in purple photosynthetic bacteria, is one of the promising proteins for tackling such an investigation. In LH2 proteins, BChl a and carotenoid molecules are symmetri© XXXX American Chemical Society

Received: March 2, 2018 Revised: April 16, 2018

A

DOI: 10.1021/acs.biochem.8b00259 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

corresponds to B850 BChl a. Given the possibility that LH2 and LH1 proteins are derived from a common ancestor protein,39−44 two hypotheses about the evolution of antenna proteins in purple bacteria are assumed; B800-type pigments are acquired by LH1-like proteins or are released from LH2-like proteins. Note that the LH1 protein in Thermochromatium tepidum actually has hydrophobic cavities in the protein matrix, whose positions structurally correspond to the B800-binding sites of LH2.45 The first aim of this study is to demonstrate the differences in the physicochemical features of B800 removal between molischianum-LH2 and acidophilus-LH2 based on kinetic measurements and theoretical analysis. The second aim is the preparation and characterization of molischianum-LH2 in which B800 BChl a is absent (denoted as B800-free LH2) by overcoming the resistance to B800 removal. This study also demonstrates that extrinsic BChl a can be inserted into the B800-binding sites in B800-free molischianum-LH2 (B800-free LH2 that accepts extrinsic BChl a is denoted reconstituted LH2).

BChl a pigments are present as two structural states in the scaffold of α/β-polypeptides and are classified by the positions of the lowest-energy absorption bands (Qy bands) in the nearinfrared region.17−20 One type of BChl a, which is monomeric in the N-terminal region, is called B800 BChl a, because its Qy peak position is around 800 nm. The other type is dimeric, sandwiched between the α- and β-polypeptides, and is called B850 BChl a because of its Qy peak position around 850 nm. B850 BChl a pigments are excitonically coupled in a circular arrangement in the protein matrix. B800 BChl a pigments in LH2 proteins are appropriate for the study of pigment binding, because these pigments are spectroscopically sensitive to the protein structures and have been utilized as intrinsic probes of the LH2 structures and the transfer of energy to B850 BChl a.21−27 In this regard, the removal of B800 BChl a from acidophilus-like (type 1) LH2 proteins and insertion of the pigment into their B800-binding sites have been reported.28−34 In contrast, no report of the removal of B800 from molischianum-like (type 2) LH2 proteins is available. Indeed, Bandilla et al. argued that molischianum-LH2 cannot release B800 BChl a under acidic conditions for acidophilus-like (type 1) LH2.28 There are differences in the binding motif of B800 BChl a between the LH2 proteins, e.g., (i) axial ligands (α-Asp6 for molischianum-LH2 and the N-terminal carboxy group in the αpeptide for acidophilus-LH2) (Figure 1), (ii) hydrogen bond

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MATERIALS AND METHODS Apparatus. Visible absorption spectra were measured with a Shimadzu UV-2450 spectrophotometer. Circular dichroism (CD) spectra were measured with a JASCO J-820 spectropolarimeter. High-performance liquid chromatography (HPLC) was performed with a Shimadzu LC-20AT pump and an SPDM20A detector. Size exclusion chromatography (SEC) was performed with a GE Healthcare Ä KTAprime plus system. Fluorescence emission spectra of LH2 proteins in 20 mM Tris buffer containing 0.1% n-dodecyl β-D-maltoside (DDM) (pH 8.0) were measured by excitation at 800 nm at room temperature with a Hamamatsu model C9920-03G fluorescence measurement system. Materials. LH2 proteins were isolated from the purple photosynthetic bacteria Phs. molischianum DSM120 and Rbl. acidophilus DSM145 as reported elsewhere.33,46 BChl a was extracted from a purple photosynthetic bacterium Rhodobacter sphaeroides 2.4.1 reported elsewhere47,48 and purified on a reverse-phase Cosmosil 5C18-AR-II column (10 mmϕ × 250 mm) with methanol at a flow rate of 1 mL min−1. Kinetic Measurements of B800 Removal. LH2 proteins (0.2 μM) were incubated in two kinds of buffer solutions: 20 mM Tris buffer containing 0.1% DDM and 0.05% Triton X-100 (pH 4.0) and 20 mM Tris buffer containing 0.5% DDM and 0.75% n-octyl β-D-glucoside (OG) (pH 3.5). Absorbance changes at the B800 and B850 bands of LH2 proteins in these buffers were monitored under the control of reaction temperature. The buffer pH was checked after the reactions. Preparation of B800-Free LH2 from Phs. molischianum under Optimal Conditions. LH2 proteins isolated from Phs. molischianum were solubilized in 20 mM Tris buffer containing 0.5% DDM (pH 8.0), in which the B850 absorbance was adjusted to 0.5. An aliquot of a buffer solution containing OG was added to this solution (the final concentration of OG was 0.75%), and the pH of this solution was adjusted to 3.5 with a 10% aqueous solution of acetic acid, followed by incubation at 35 °C for 96 h in the dark. B800-free molischianum-LH2 was purified via cation-exchange column chromatography using a Whatman CM52 resin. B800-free molischianum-LH2 was eluted with 20 mM Tris buffer containing 0.02% DDM and 100 mM NaCl (pH 4.5). The collected B800-free molischianum-LH2 was desalted by ultra-

Figure 1. Differences in the structures in the proximity of B800 BChl a in (A) molischianum-LH2 and (B) acidophilus-LH2.

patterns of the 3-acetyl group (β′-Thr23 for molischianum-LH2 and β-Arg20 for acidophilus-LH2) (Figure 1), and (iii) location of the phytyl chains (which are located on the protein surface in molischianum-LH2 and in the protein interior in acidophilusLH2) (Figure S1). However, the effects of these structural differences on the contrast in the B800 removal between molischianum-LH2 and acidophilus-LH2 mentioned above are still unclear. The development of selective release of B800 BChl a from molischianum-LH2 will also contribute to elucidation of the energy transfer mechanism in LH2 proteins. Comparison of the intracomplex energy transfer between molischianum-LH2 and acidophilus-LH2 has attracted attention,35−38 but substitution of B800 BChl a with other chlorophyllous pigments in LH2 complexes with known atomic-resolution structures has been applied to only acidophilus-LH2 to investigate the energy transfer mechanism.31 The removal and insertion of B800 BChl a of molischianumLH2 will also be of interest from the viewpoint of the evolution of antenna proteins in photosynthetic bacteria, because this protein is closely related to the core antenna proteins (LH1) in phylogenetic analyses.39−41 Generally, LH2 proteins have B800 and B850 BChl a, but LH1 has only one kind of BChl a that B


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Biochemistry

ΔHcorr = ΔHQM H‐bond − ΔHMM H‐bond + ΔHQM ligand − ΔHMM ligand

centrifugation using Amicon centricon concentrators (30 kDa cutoff). Reconstitution of BChl a into B800-Free LH2 from Phs. molischianum. An acetone solution of BChl a (0.6 mM) was mixed with a solution of B800-free LH2 (0.23 μM) in 20 mM Tris buffer containing 0.1% DDM (pH 4.5): the volume ratio of the BChl a solution and the B800-free LH2 solution was 1:19. The pH of the mixed solution was adjusted to 8.0, followed by incubation at 35 °C for 2 h in the dark. The sample was concentrated by ultracentrifugation with Amicon centricon concentrators (30 kDa cutoff) and loaded onto a SephacrylS200 column in 20 mM Tris buffer containing 0.1% DDM and 150 mM NaCl (pH 8.0). The collected reconstituted LH2 proteins were desalted by ultracentrifugation using Amicon centricon concentrators (30 kDa cutoff). Atomic Force Microscopy (AFM) Measurements. A 100 μL aliquot of an LH2 solution in 20 mM Tris buffer containing 0.1% DDM and 150 mM NaCl (pH 8.0 and 4.0 for native/ reconstituted LH2 and B800-free LH2, respectively) was placed onto cleaved mica with a diameter of 12 mm (SPI Supplies, West Chester, PA). After standing for 30 min at room temperature in the dark, the mica surface was rigorously rinsed with 20 mM Tris buffer containing 150 mM NaCl at the corresponding pH. The sample was then observed in 20 mM Tris buffer containing 150 mM NaCl at the corresponding pH by a home-built frequency modulation AFM instrument (FMAFM)49,50 with a silicone cantilever (PPP-NCHAuD, Nanoworld), which had a nominal spring constant of 42 N/m. The typical resonance frequency and Q factor in an aqueous buffer solution are 150 kHz and 7, respectively.49,50 A commercially available AFM controller (ARC2, Asylum Research, Santa Barbara, CA) was employed for the control of the FM-AFM system.49,50 Size Exclusion Chromatography. LH2 proteins were analyzed by SEC on a HiPrep 16/60 Sephacryl S-300 HR column with a mixed buffer of 20 mM Tris and 10 mM succinate containing 0.05% DDM and 150 mM NaCl (pH 8.0 and 5.5 for native/reconstituted and B800-free LH2, respectively) at a flow rate of 0.4 mL min−1 at room temperature. Binding Enthalpy Calculations. Binding energy ΔG is represented by a sum of enthalpy term ΔH and entropy term TΔS: ΔG = ΔH − T ΔS

(4)

where ΔHQM is the quantum-chemically calculated interaction for the hydrogen bond, ΔH QMligand is the quantum-chemically calculated interaction for the axial ligand, ΔHMMH‑bond is the interaction for the hydrogen bond calculated using the MM force field, and ΔHMMligand is the interaction calculated using the MM force field for axial ligand. Molecular Structure and Computational Details. ΔH was calculated for two pairs of α-β protein subunits, one B800 BChl a, four B850 BChl a molecules (i.e., two B850 dimers), and two lycopenes in molischianum-LH2 or four rhodopin β-Dglucosides in acidophilus-LH2 (Figure S2). Atomic coordinates were obtained from the LH2 crystal structures of Phs. molischianum and Rbl. acidophilus [Protein Data Bank (PDB) entries 1LGH and 1NKZ, respectively].13,15 After addition of hydrogen atoms, to refine the molecular structures in the proximity of B800 BChl a, we optimized geometries of amino acids and cofactors located within 3 Å of the B800 BChl a as well as all hydrogen atoms with the MM force field. Because the phytyl chain of B800 BChl a is absent in the molischianum-LH2 crystal structure (Figure S1),13 we removed the phytyl chain of B800 BChl a by replacing the C175 atom (Figure 1) with a methyl group and evaluated ΔH of the bacteriochlorin ring of B800 BChl a. To calculate ΔHQMH‑bond and ΔHMMH‑bond, we optimized geometries of BChl a and the hydrogen bond partner (β′Thr23 for molischianum-LH2 or β-Arg20 for acidophilus-LH2) in vacuum. To calculate ΔHQMligand and ΔHMMligand, we optimized geometries of BChl a and the axial ligand (α-Asp6 for molischianum-LH2 or the carboxy group at the N-terminus of the α-peptide for acidophilus-LH2) in vacuum (Figure 1). After geometry optimizations, we evaluated ΔHQM and ΔHMM using eq 2. To perform calculations based on the MM force field, the AMBER 14 force field51 with NAMD52 was employed. To perform quantum-mechanical calculations, the density functional theory (RB3LYP/LACVP* level) with JAGUAR53 was used. The BSSE (basis set superposition error) correction54 was considered for calculations of ΔHQM. H‑bond

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RESULTS AND DISCUSSION Differences in B800 Removal. The acidophilus-LH2 protein rapidly released B800 BChl a within 120 min under an acidic condition (pH 4.0) in the presence of two detergents, DDM and Triton X-100,33 at 35 °C (Figure 2A). In contrast, the Qy absorbance of B800 BChl a in molischianum-LH2 barely decreased under this condition, indicating that B800 BChl a in molischianum-LH2 was much more tolerant of acid-induced removal than that in acidophilus-LH2 (Figure 2A). When LH2 proteins were incubated at 35 °C in 20 mM Tris buffer containing 0.5% DDM and 0.75% OG (pH 3.5), molischianumLH2 gradually released B800 BChl a and acidophilus-LH2 released B800 BChl a very quickly (Figure 2B). Note that the removal of B800 from molischianum-LH2 at 1200 min (20 h) in Figure 2B was part of the reaction and B800-free molischianumLH2 was obtained after incubation for 96 h (see Materials and Methods). The removal of B800 BChl a from molischianum-LH2 was slower than that from acidophilus-LH2 at 25 and 15 °C (Figure S3). B800 removal in the initial phase was analyzed as a pseudo-first-order reaction, and the relationships between the

(1)

where T is the temperature and ΔS is the binding entropy. Binding enthalpy ΔH(AB) for complex AB can be described by ΔH(AB) = E(AB) − E(A) − E(B)

(2)

where E(A) is the energy for molecule A, E(B) is the energy for molecule B, and E(AB) is the energy for the AB complex. To calculate ΔH for the pigment−protein complex (B800 BChl a-LH2), we used the following equation: ΔH = ΔHMM + ΔHcorr

(3)

where ΔHMM represents ΔH between B800 BChl a and the LH2 protein obtained using the molecular mechanical (MM) force field and ΔHcorr is the quantum mechanical (QM) correlation. To describe interactions for axial ligands and hydrogen bonds quantum-mechanically, ΔHcorr was introduced by C


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Biochemistry

acidophilus-LH2 because of the charges and the number of hydrogen bonds (i.e., one uncharged polar hydrogen bond for molischianum-LH2 and two charged hydrogen bonds in acidophilus-LH2). See ref 55 for the difference in hydrogen bond interactions for charged or uncharged polar groups. Resonance Raman spectroscopic studies30,39 also suggested that the hydrogen bond of the 3-acetyl group in B800 BChl a in acidophilus-LH2 (3-CO stretching vibrational band at 1620 cm−1) was stronger than that in molischianum-LH2 (3-CO stretching vibrational band at 1632 cm−1). In contrast to the large difference in the removal kinetics (Figure 2), the difference in the calculated values of ΔH between molischianum-LH2 and acidophilus-LH2 was sufficiently small in comparison with the absolute value of ΔH. These results imply that the LH2 protein structures were altered from the geometries of the LH2 crystal structures (PDB entries 1LGH13 and 1NKZ15) upon the removal of B800 BChl a in the presence of additional detergents (Triton X-100 or OG). Another important aspect is the effect of the phytyl group in B800 BChl a on the binding to LH2 proteins. It should also be noted that in this study ΔH was calculated in the absence of the phytyl group, because the phytyl group was not fully identified in the molischianum-LH2 crystal structure. Given that the phytyl group plays a crucial role in the binding enthalpy, contributions of residues in van der Waals contact with the phytyl group in B800 BChl a might not be ignored. In addition, the entropy term (TΔS) in the binding energy might be different between the LH2 proteins. Note that the estimation of ΔS in the binding energy is quite difficult in the calculation presented here. The removal of B800 from LH2 proteins is complex, which includes effects of the static structures and dynamic fluctuation of pigment−proteins complexes, transient structures through the reactions, and surrounding detergents. Theoretical analyses allow us to discuss the structural factors for B800 binding in LH2 proteins independently and will improve our understanding of the mechanisms for the complex B800 removal and binding reactions. Spectral and Structural Features of B800-Free molischianum-LH2. The kinetic analysis described above allowed us to identify the conditions for the removal of B800 BChl a from molischianum-LH2. The original conditions for the removal of B800 from acidophilus-LH233 were changed as follows. (i) The additive detergent, which played an active role in B800 removal, was changed from Triton X-100 to OG, because OG was reported to be more harsh than Triton X-100 to integral membrane proteins.56 (ii) The concentration of the main detergent DDM increased from 0.1 to 0.5% to aim for destabilization of the protein. (iii) The protein was incubated under a slightly more acidic condition (pH 4.0 → 3.5). (iv) The incubation time was increased. The effect of the surface charge distribution of LH2 proteins would be negligible with a change in the conditions because non-ionic detergents were used and the change in pH from the original conditions was small. B800free molischianum-LH2 was successfully prepared under the revised conditions and the subsequent purification with cationexchange chromatography. SEC analysis confirmed the purification of B800-free molischianum-LH2 (Figure S5). Native molischianum-LH2 exhibited two Qy absorption bands of B800 and B850 BChl a at 799 and 847 nm, respectively (Figure 3A). In the spectrum of B800-free LH2, the B800 Qy band disappeared and the B850 Qy band was positioned at 848 nm (Figure 3B). The bandwidth of the B850 Qy band in B800-

Figure 2. Time courses of relative Qy absorbance of B800 BChl a in molischianum-LH2 (filled blue circles) and acidophilus-LH2 (empty red circles). Absorbance changes were monitored at 35 °C in (A) 20 mM Tris buffer containing 0.1% DDM and 0.05% Triton X-100 (pH 4.0) and (B) 20 mM Tris buffer containing 0.5% DDM and 0.75% OG (pH 3.5). The Qy absorbance of the B800/B850 bands of molischianumLH2 and acidophilus-LH2 was monitored at 798/845 and 802/859 nm, respectively, and the relative ratios of the Qy absorbance of B800 BChl a against B850 BChl a are normalized by the ratio at the onset of measurements.

apparent rate constants of the removal of B800 from the two LH2 proteins and the reaction temperature were examined (Figure S4). As a result, the apparent activation energies of the removal of B800 from molischianum-LH2 and acidophilus-LH2 were estimated to be 97 and 81 kJ mol−1, respectively. To identify the structural factors that differentiate the observed removal kinetics of B800 BChl a from the LH2 proteins under acidic conditions, binging enthalpy ΔH was calculated using the LH2 crystal structures. ΔH was calculated to be −134.3 kcal mol−1 for molischianum-LH2 and −152.4 kcal mol−1 for acidophilus-LH2. Contributions of the axial ligands and the hydrogen bonds to total binding enthalpy ΔH are summarized in Table 1. The contribution of axial ligand α-Asp6 Table 1. Contributions of the Axial Ligands (ΔHQMligand) and the Hydrogen Bonds (ΔHQMH‑bond) to the Total Binding Enthalpy (ΔH, in kilocalories per mole) molischianum-LH2 acidophilus-LH2

ΔH

ΔHQMligand

ΔHQMH‑bond

−134.3 −152.4

−63.5 −62.7

−5.2 −27.8

in molischianum-LH2 to the total binding enthalpy was the same as that of the N-terminal carboxy group in the α-subunit in acidophilus-LH2. On the contrary, the contribution of the hydrogen bond between the 3-acetyl group of B800 BChl a and neutral β′-Thr23 in molischianum-LH2 differed from that between the 3-acetyl group and positively charged β-Arg20 in D


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Figure 3. Electronic absorption spectra of (A) native, (B) B800-free, and (C) reconstituted molischianum-LH2 in 20 mM Tris buffer containing 0.1% n-dodecyl β-D-maltoside (pH 8.0 and 4.0 for native/ reconstituted and B800-free LH2, respectively).

Figure 4. CD spectra of (A and B) native, (C and D) B800-free, and (E and F) reconstituted molishianum-LH2 in the (A, C, and E) UV and (B, D, and F) Qy regions in 20 mM Tris buffer containing 0.1% ndodecyl β-D-maltoside (pH 8.0 and 4.0 for native/reconstituted and B800-free LH2, respectively). The Qy absorbance values of B850 BChl a in the LH2 samples used for the measurements in the UV and Qy regions were 0.5 and 1, respectively.

free molischianum-LH2 was almost identical to that in native LH2. The Q x absorption band of BChl a in B800-free molischianum-LH2 (587 nm) was slightly shifted to a wavelength shorter than that of native LH2 (590 nm) (Figure 3), indicating that the Qx peak position of BChl a in B800-free molischianum-LH2 is assigned to B850 BChl a and the B850 Qx band differs from that of B800 BChl a in molischianum-LH2. molischianum-LH2 has licopene as a carotenoid pigment. The absorption bands of licopene in B800-free molischianum-LH2 were positioned at 529, 493, and 466 nm. These peak positions are quite similar to those in native molischianum-LH2 (530, 493, and 466 nm). No shift of the absorption peaks of licopene in molischianum-LH2 caused by B800 removal is in sharp contrast to the blue-shifts of carotenoids in acidophilus-type (type 1) LH2 induced by B800 removal.28,29 CD spectra of native and B800-free molischianum-LH2 proteins are shown in Figure 4. B800 BChl a in native molischianum-LH2 exhibited a reverse S-shaped CD signal around 800 nm. This feature is characteristic of molischianumLH2.37,57 The CD signal of B800 BChl a disappeared in the spectrum of B800-free LH2. The shape and intensity of the negative CD signal of B850 BChl a barely changed after removal of B800 BChl a. The CD signals of B850 BChl a were somewhat noisy because the spectropolarimeter used here had a low sensitivity around this wavelength region. The CD intensity of B800-free molischianum-LH2 around 220 nm resembled that of native LH2, indicating that the contents of the α-helices were unchanged by B800 removal. The overall structure of B800-free molischianum-LH2 was observed by FM-AFM. Native and B800-free LH2 exhibited octameric ring structures (Figure 5). The averaged top-to-top distances of these ring structures in native and B800-free molischianum-LH2, which were estimated by the height profiles in the AFM observation (Figure 5, right), were 4.5 ± 0.5 and 4.1 ± 0.7 nm (the averages and standard deviations of 12 samples), respectively. The diameter of the ring structure of B800-free LH2 was analogous to that of native LH2. Note that the top-to-top distance of the ring in native molischianum-LH2

was 87% of that in acidophilus-LH2 (5.2 nm),33 which is consistent with the relative ratio of the subunit number in molischianum-LH2 to that in acidophilus-LH2 (89%). According to our high-resolution AFM observation, deformation of the ring structure is unlikely to be induced in molischianum-LH2 even if B800 BChl a is released from the native protein. These results indicate that the absence of B800 BChl a has little effect on the local protein structure, especially in the vicinity of B850 BChl a, and on the overall ring structure of molischianum-LH2. These structural features, which are in line with those of acidophilus-LH2,33 indicate that the hydrophobic domains around the B850-binding sites in LH2 proteins are inherently rigid, as suggested by the spectral measurements of LH2 under high pressure.24 In contrast, the B800-binding sites in the N-terminal region of LH2 proteins are more flexible than the B850-binding sites, suggested by the same tendency in the SEC analysis of molischianum-LH2 (Figure S5) as that of acidophilus-LH2.33 The electronic absorption band at 799 nm was recovered when BChl a was incubated with B800-free molischianum-LH2 (Figure 3C). The peak position and the bandwidth of the B800 band in reconstituted LH2 were almost identical to those of native LH2. The reverse S-shaped CD signal of B800 BChl a also recovered around 800 nm (Figure 4F); the shape and intensity were almost the same as those in native LH2. The level of the negative CD intensity around 220 nm of reconstituted LH2 was the same as that of native LH2 (Figure 4E). The averaged top-to-top distance of the ring structure in reconstituted LH2 determined by AFM measurements (Figure 5, right) was 4.3 ± 0.4 nm (the averages and standard deviations of 12 samples). These indicate that reconstituted LH2 had the same local and overall ring structures as native LH2. Fluorescence emission spectra showed that the predominant excitation of B800 BChl a in reconstituted LH2 E


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Figure 5. AFM images of (A) native, (B) B800-free, and (C) reconstituted molischianum-LH2 adsorbed on mica surfaces. The left, center, and right columns show wide images of molischianum-LH2, locally enlarged images of molischianum-LH2, and overlapped height profiles of 12 proteins, respectively.

produced emission from B850 BChl a at ∼875 nm, whose position closely resembled to that in native LH2 (Figure 6), indicating that reconstituted BChl a can transfer the excitation energy to B850 BChl a. Reconstitution of BChl a into the B800-binding sites in B800-free molischianum-LH2 was complete after incubation for 2 h, which was almost the same as that of B800-free acidophilusLH2.33 These imply that the kinetics of insertion of BChl a into B800-free molischianum-LH2 does not differ significantly from that of B800-free acidophilus-LH2. The insertion kinetics, therefore, is in sharp contrast to the marked difference in the kinetics of B800 removal between the LH2 proteins, suggesting the structural changes of LH2 proteins with additional detergents in the B800 removal reaction. Note that the precise kinetics of the insertion of BChl a into B800-free molischianumLH2 cannot be compared with that into B800-free acidophilusLH2 under the reconstitution condition presented here, because the B800 absorbance of B800-free acidophilus-LH2 was gradually recovered without addition of BChl a (Figure S6B). Such a recovery of B800 BChl a in B800-free LH2 under

some conditions was reported previously.7,28 In contrast, the electronic absorption spectrum of B800-free molischianum-LH2 was not changed under this condition (Figure S6A).

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CONCLUDING REMARKS B800 BChl a pigments in molischianum-LH2 were significantly more resistant to their release under acidic conditions than those in acidophilus-LH2. The observed differences in B800 removal between the two LH2 proteins are in contrast to the differences in the calculated binding enthalpy of B800 BChl a in these proteins. The contrast between the experimental and theoretical results suggests that the following factors should be taken into consideration in understanding the removal of B800 from the LH2 proteins: possible alteration of the LH2 structures upon removal of B800 BChl a from the geometries of the crystal structures, calculations of the binding enthalpy, including the effect of the orientations of the phytyl group, and the possible difference in the entropy term. We identified the conditions under which B800 BChl a pigments were released from molischianum-LH2. B800-free F


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Biochemistry Funding

This work was supported by JST PRESTO (JPMJPR1416 to Y.S. and JPMJPR1411 to H.A.), JSPS KAKENHI (JP15H00887, JP17K05940, and JP18H05182 to Y.S, JP26800224 to K.S., JP16H06560 to K.S. and H.I., and JP26105012 and JP18H05155 to H.I.), JST CREST (JPMJCR1656), the Japan Agency for Medical Research and Development (AMED), the Materials Integration for engineering polymers of Cross-ministerial Strategic Innovation Promotion Program (SIP), and the Interdisciplinary Computational Science Program in CCS, University of Tsukuba. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Kanji Miyagi, Ken Kaneda, Madoka Yamashita, Kiyoshiro Kawano, and Kokomi Doi (Kindai University) and Hideki Sudo (The University of Tokyo) for their experimental assistance and Dr. Takeshi Fukuma (Kanazawa University) for his kind advice for building the FM-AFM instrument.

Figure 6. Fluorescence emission spectra of (A) native and (B) reconstituted molischianum-LH2 recorded with excitation at 800 nm in 20 mM Tris buffer containing 0.1% n-dodecyl β-D-maltoside (pH 8.0). The signals denoted with X’s at 800 nm originated from the excitation light.

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(1) Cheng, Y.-C., and Fleming, G. R. (2009) Dynamics of light harvesting in photosynthesis. Annu. Rev. Phys. Chem. 60, 241−262. (2) Scholes, G. D., Fleming, G. R., Olaya-Castro, A., and van Grondelle, R. (2011) Lessons from nature about solar light harvesting. Nat. Chem. 3, 763−774. (3) Croce, R., and van Amerongen, H. (2014) Natural strategies for photosynthetic light harvesting. Nat. Chem. Biol. 10, 492−501. (4) Hashimoto, H., Sugai, Y., Uragami, C., Gardiner, A. T., and Cogdell, R. J. (2015) Natural and artificial light-harvesting systems utilizing the functions of carotenoids. J. Photochem. Photobiol., C 25, 46−70. (5) Saer, R. G., and Blankenship, R. E. (2017) Light-harvesting in phototrophic bacteria: structure and function. Biochem. J. 474, 2107− 2131. (6) Broglie, R. M., Hunter, C. N., Delepelaire, P., Niederman, R. A., Chua, N.-H., and Clayton, R. K. (1980) Isolation and characterization of the pigment-protein complexes of Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis. Proc. Natl. Acad. Sci. U. S. A. 77, 87−91. (7) Clayton, R. K., and Clayton, B. J. (1981) B850 pigment-protein complex of Rhodopseudomonas sphaeroides: extinction coefficients, circular dichroism, and the reversible binding of bacteriochlorophyll. Proc. Natl. Acad. Sci. U. S. A. 78, 5583−5587. (8) Parkes-Loach, P. S., Sprinkle, J. R., and Loach, P. A. (1988) Reconstitution of the B873 light-harvesting complex of Rhodospirillum rubrum from the separately isolated α- and β-polypeptides and bacteriochlorophyll a. Biochemistry 27, 2718−2727. (9) Mimuro, M., and Tanaka, A. (2004) The in vivo and in vitro reconstitution of pigment-protein complexes, and its implication in acquiring a new system. Photosynth. Res. 81, 129−137. (10) Paulsen, H. (2006) Reconstitution and pigment exchange. In Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications (Grimm, B., Porra, R. J., Rüdiger, W., and Scheer, H., Eds.) pp 375−385, Springer, Dordrecht, The Netherlands. (11) Cogdell, R. J., Fyfe, P. K., Barrett, S. J., Prince, S. M., Freer, A. A., Isaacs, N. W., McGlynn, P., and Hunter, C. N. (1996) The purple bacterial photosynthetic unit. Photosynth. Res. 48, 55−63. (12) Cogdell, R. J., Gall, A., and Köhler, J. (2006) The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes. Q. Rev. Biophys. 39, 227−324. (13) Koepke, J., Hu, X., Muenke, C., Schulten, K., and Michel, H. (1996) The crystal structure of the light-harvesting complex II (B800− 850) from Rhodospirillum molischianum. Structure 4, 581−597.

molischianum-LH2 kept the octameric ring structure as well as the local structure in the vicinity of B850 BChl a and the contents of α-helices. Therefore, B800 BChl a has little effect on the protein structure of molischianum-LH2, and the hydrophobic domain in the vicinity of the B850-binding sites plays a major role in the structural formation of LH2 proteins. The removal of B800 from molischianum-LH2 indicates the difference in the Qx positions of B800 and B850 BChl a as well as no effect of B800 BChl a on the absorption bands of licopene. The preparation and characterization of B800-free molischianum-LH2 will allow us to introduce various pigments into the B800-binding sites to study the structural roles of pigment binding and the energy transfer mechanisms in molischianum-type (type 2) LH2 proteins.

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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.biochem.8b00259. Orientation of the phytyl chains in B800 BChl a in LH2 proteins (Figure S1), structures of LH2 proteins used for ΔH calculations (Figure S2), kinetics of B800 removal at 25 and 15 °C (Figure S3), Arrhenius plots for B800 removal (Figure S4), chromatograms of size exclusion chromatography (Figure S5), and spectral changes of B800-free LH2 under the reconstitution condition (Figure S6) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: hi_asa@staff.kanazawa-u.ac.jp. *E-mail: [email protected]. ORCID

Yoshitaka Saga: 0000-0001-7384-9169 Hiroshi Ishikita: 0000-0002-5849-8150 G


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Biochemistry

(29) Fraser, N. J., Dominy, P. J., Ü cker, B., Simonin, I., Scheer, H., and Cogdell, R. J. (1999) Selective release, removal, and reconstitution of bacteriochlorophyll a molecules into the B800 sites of LH2 complexes from Rhodopseudomonas acidophila 10050. Biochemistry 38, 9684−9692. (30) Gall, A., Fraser, N. J., Bellissent-Funel, M.-C., Scheer, H., Robert, B., and Cogdell, R. J. (1999) Bacteriochlorin-protein interactions in native B800-B850, B800 deficient and B800-BchlaPreconstituted complexes from Rhodopseudomonas acidophila, strain 10050. FEBS Lett. 449, 269−272. (31) Herek, J. L., Fraser, N. J., Pullerits, T., Martinsson, P., Polívka, T., Scheer, H., Cogdell, R. J., and Sundström, V. (2000) B800→B850 energy transfer mechanism in bacterial LH2 complexes investigated by B800 pigment exchange. Biophys. J. 78, 2590−2596. (32) Gall, A., Robert, B., Cogdell, R. J., Bellissent-Funel, M.-C., and Fraser, N. J. (2001) Probing the binding sites of exchanged chlorophyll a in LH2 by Raman and site-selection fluorescence spectroscopies. FEBS Lett. 491, 143−147. (33) Saga, Y., Hirota, K., Asakawa, H., Takao, K., and Fukuma, Y. (2017) Reversible changes in the structural features of photosynthetic light-harvesting complex 2 by removal and reconstitution of B800 bacteriochlorophyll a pigments. Biochemistry 56, 3484−3491. (34) Saga, Y., Amari, K., and Miyagi, K. (2018) Insertion of chlorophyll a derivatives into the binding sites of B800 bacteriochlorophyll a in light-harvesting complex 2 from the purple photosynthetic bacterium Rhodoblastus acidophilus. J. Photochem. Photobiol., A 353, 591−596. (35) Wu, H.-M., Reddy, N. R. S., Cogdell, R. J., Muenke, C., Michel, H., and Small, G. J. (1996) A comparison of the LH2 antenna complex of three purple bacteria by hole-burning and absorption spectroscopes. Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. A 291, 163−173. (36) Salverda, J. M., van Mourik, F., van der Zwan, G., and van Grondelle, R. (2000) Energy transfer in the B800 rings of the peripheral bacterial light-harvesting complexes of Rhodopseudomonas acidophila and Rhodospirillum molischianum studied with photon echo techniques. J. Phys. Chem. B 104, 11395−11408. (37) Ihalainen, J. A., Linnanto, J., Myllyperkiö, P., van Stokkum, I. H. M., Ü cker, B., Scheer, H., and Korppi-Tommola, J. E. I. (2001) Energy transfer in LH2 of Rhodospirillum molischianum, studied by subpicosecond spectroscopy and configuration interaction exciton calculations. J. Phys. Chem. B 105, 9849−9856. (38) Wendling, M., van Mourik, F., van Stokkum, I. H. M., Salverda, J. M., Michel, H., and van Grondelle, R. (2003) Low-intensity pumpprobe measurements on the B800 band of Rhodospirillum molischianum. Biophys. J. 84, 440−449. (39) Germeroth, L., Lottspeich, F., Robert, B., and Michel, H. (1993) Unexpected similarities of the B800-B850 light-harvesting complex from Rhodospirillum molischianum to the B870 light-harvesting complexes from other purple photosynthetic bacteria. Biochemistry 32, 5615−5621. (40) Nagashima, K. V. P., Matsuura, K., and Shimada, K. (1996) The nucleotide sequence of the puf operon from the purple photosynthetic bacterium, Rhodospirillum molischianum: comparative analyses of lightharvesting proteins and the cytochrome subunits associated with the reaction centers. Photosynth. Res. 50, 61−70. (41) Lang, A. S., Harwood, C. S., and Beatty, J. T. (2012) Evolutionary relationships among antenna proteins of purple phototrophic bacteria. In Functional Genomics and Evolution of Photosynthetic Systems (Burnap, R. L., and Vermaas, W. F. J., Eds.) pp 253−264, Springer, Dordrecht, The Netherlands. (42) Youvan, D. C., and Ismail, S. (1985) Light-harvesting II (B800− B850 complex) structural genes from Rhodopseudomonas capsulata. Proc. Natl. Acad. Sci. U. S. A. 82, 58−62. (43) Hohmann-Marriott, M. F., and Blankenship, R. B. (2011) Evolution of photosynthesis. Annu. Rev. Plant Biol. 62, 515−548. (44) Henry, S. L., and Cogdell, R. J. (2013) The evolution of the purple photosynthetic bacterial light-harvesting system. In Genome Evolution of Photosynthetic Bacteria (Beatty, J. T., Ed.) pp 205−226, Elsevier, Amsterdam.

(14) McDermott, G., Prince, S. M., Freer, A. A., HawthornthwaiteLawless, A. M., Papiz, M. Z., Cogdell, R. J., and Isaacs, N. W. (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374, 517−521. (15) Papiz, M. Z., Prince, S. M., Howard, T., Cogdell, R. J., and Isaacs, N. W. (2003) The structure and thermal motion of the B800− B850 LH2 complex from Rps. acidophila at 2.0 Å resolution and 100 K: new structural features and functionally relevant motions. J. Mol. Biol. 326, 1523−1538. (16) Georgakopoulou, S., Frese, R. N., Johnson, E., Koolhaas, C., Cogdell, R. J., van Grondelle, R., and van der Zwan, G. (2002) Absorption and CD spectroscopy and modeling of various LH2 complexes from purple bacteria. Biophys. J. 82, 2184−2197. (17) Sundström, V., Pullerits, T., and van Grondelle, R. (1999) Photosynthetic light-harvesting: reconciling dynamics and structure of purple bacterial LH2 reveals function of photosynthetic unit. J. Phys. Chem. B 103, 2327−2346. (18) Cogdell, R. J., Isaacs, N. W., Freer, A. A., Howard, T. D., Gardiner, A. T., Prince, S. M., and Papiz, M. Z. (2003) The structural basis of light-harvesting in purple bacteria. FEBS Lett. 555, 35−39. (19) Cogdell, R. J., Howard, T. D., Isaacs, N. W., McLuskey, K., and Gardiner, A. T. (2002) Structural factors which control the position of the Qy absorption band of bacteriochlorophyll a in purple bacterial antenna complexes. Photosynth. Res. 74, 135−141. (20) Law, C. J., Roszak, A. W., Southall, J., Gardiner, A. T., Isaacs, N. W., and Cogdell, R. J. (2004) The structure and function of bacterial light-harvesting complexes (Review). Mol. Membr. Biol. 21, 183−191. (21) Visschers, R. W., Crielaard, W., Fowler, G. J. S., Hunter, C. N., and van Grondelle, R. (1994) Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. II. A lowtemperature spectroscopy study of structural aspects of the pigmentprotein conformation. Biochim. Biophys. Acta, Bioenerg. 1183, 483−490. (22) Wu, H.-M., Ratsep, M., Jankowiak, R., Cogdell, R. J., and Small, G. J. (1997) Comparison of the LH2 antenna complexes of Rhodopseudomonas acidophila (strain 10050) and Rhodobacter sphaeroides by high-pressure absorption, high-pressure hole burning, and temperature-dependent absorption spectroscopies. J. Phys. Chem. B 101, 7641−7653. (23) Buche, A., Ellis, G., and Ramirez, J. M. (2001) Probing the binding site of 800-nm bacteriochlorophyll in the membrane-linked LH2 protein of Rhodobacter capsulatus by local unfolding and chemical modification. Evidence for the involvement of a βHis20 residue. Eur. J. Biochem. 268, 2792−2800. (24) Gall, A., Ellervee, A., Sturgis, J. N., Fraser, N. J., Cogdell, R. J., Freiberg, A., and Robert, B. (2003) Membrane protein stability: high pressure effects on the structure and chromophore-binding properties of the light-harvesting complex LH2. Biochemistry 42, 13019−13026. (25) Hofmann, C., Aartsma, T. J., Michel, H., and Köhler, J. (2003) Direct observation of tiers in the energy landscape of a chromoprotein: a single-molecule study. Proc. Natl. Acad. Sci. U. S. A. 100, 15534− 15538. (26) Oikawa, H., Fujiyoshi, S., Dewa, T., Nango, M., and Matsushita, M. (2008) How deep is the potential well confining a protein in a specific conformation? A single-molecule study on temperature dependence of conformational change between 5 and 18 K. J. Am. Chem. Soc. 130, 4580−4581. (27) Baier, J., Gabrielsen, M., Oellerich, S., Michel, H., van Heel, M., Cogdell, R. J., and Köhler, J. (2009) Spectral diffusion and electronphonon coupling of the B800 BChl a molecules in LH2 complexes from three different species of purple bacteria. Biophys. J. 97, 2604− 2612. (28) Bandilla, M., Ü cker, B., Ram, M., Simonin, I., Gelhaye, E., McDermott, G., Cogdell, R. J., and Scheer, H. (1998) Reconstitution of the B800 bacteriochlorophylls in the peripheral light harvesting complex B800−850 of Rhodobacter sphaeroides 2.4.1 with BChl a and modified (bacterio-)chlorophylls. Biochim. Biophys. Acta, Bioenerg. 1364, 390−402. H


Article

Biochemistry (45) Niwa, S., Yu, L.-J., Takeda, K., Hirano, Y., Kawakami, T., WangOtomo, Z.-Y., and Miki, K. (2014) Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å. Nature 508, 228−232. (46) Saga, Y., and Hirota, K. (2016) Determination of the molar extinction coefficients of the B800 and B850 absorption bands in lightharvesting complexes 2 derived from three purple photosynthetic bacteria Rhodoblastus acidophilus, Rhodobacter sphaeroides, and Phaeospirillum molischianum by extraction of bacteriochlorophyll a. Anal. Sci. 32, 801−804. (47) Saga, Y., Miura, R., Sadaoka, K., and Hirai, Y. (2011) Kinetic analysis of demetalation of synthetic zinc cyclic tetrapyrroles possessing an acetyl group at the 3-position: effects of tetrapyrrole structures and peripheral substitution. J. Phys. Chem. B 115, 11757− 11762. (48) Saga, Y., Hirota, K., Harada, J., and Tamiaki, H. (2015) In vitro enzymatic activities of bacteriochlorophyll a synthase derived from the green sulfur photosynthetic bacterium Chlorobaculum tepidum. Biochemistry 54, 4998−5005. (49) Fukuma, T. (2009) Wideband low-noise optical beam deflection sensor with photothermal excitation for liquid-environment atomic force microscopy. Rev. Sci. Instrum. 80, 023707−023714. (50) Asakawa, H., Yoshioka, S., Nishimura, K., and Fukuma, T. (2012) Spatial distribution of lipid headgroups and water molecules at membrane/water interfaces visualized by three-dimensional scanning force microscopy. ACS Nano 6, 9013−9020. (51) Maier, J. A., Martinez, C., Kasavajhala, K., Wickstrom, L., Hauser, K. E., and Simmerling, C. (2015) ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696−3713. (52) Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781− 1802. (53) Jaguar, version 7.5 (2008) Schrödinger, LLC, New York. (54) Boys, S. F., and Bernardi, F. (1970) The calculation of small molecular interactions by the differences of separate total energies. Some procedures with reduced errors. Mol. Phys. 19, 553−566. (55) Schutz, C. N., and Warshel, A. (2001) What are the dielectric “constants” of proteins and how to validate electrostatic models? Proteins: Struct., Funct., Genet. 44, 400−417. (56) Odahara, T. (2004) Stability and solubility of integral membrane proteins from photosynthetic bacteria solubilized in different detergents. Biochim. Biophys. Acta, Biomembr. 1660, 80−92. (57) Todd, J. B., Parkes-Loach, P. S., Leykam, J. F., and Loach, P. A. (1998) In vitro reconstitution of the core and peripheral lightharvesting complexes of Rhodospirillum molischianum from separately isolated components. Biochemistry 37, 17458−17468.

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Selective Removal of B800 Bacteriochlorophyll a from Light

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