Biochemical and Spectroscopic Characterizations of a Hybrid Light

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Biochemical and Spectroscopic Characterizations of a Hybrid Light Harvesting Reaction Center Core Complex Yukihiro Kimura, Kanako Hashimoto, Seiji Akimoto, Mizuki Takenouchi, Kengo Suzuki, Rikako Kishi, Michie Imanishi, Shinji Takenaka, Michael T. Madigan, Kenji V. P. Nagashima, and Wang-Otomo Zheng-Yu Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00644 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Biochemistry

Biochemical and Spectroscopic Characterizations of a Hybrid Light Harvesting Reaction Center Core Complex

Yukihiro Kimura1,*, Kanako Hashimoto1, Seiji Akimoto2, Mizuki Takenouchi3, Kengo Suzuki4, Rikako Kishi1, Michie Imanishi1, Shinji Takenaka1, Michael T. Madigan5, Kenji V. P. Nagashima6, and Zheng-Yu Wang-Otomo3,*

1

Department of Agrobioscience, Graduate School of Agriculture, Kobe University, Nada, Kobe 6578501, Japan. 2Department of Science, Graduate School of Science, Kobe University, Nada, Kobe 657-8501, Japan. 3Faculty of Science, Ibaraki University, Bunkyo, Mito 310-8512, Japan. 4

Hamamatsu Photonics K. K., Joko-cho, Hamamatsu 431-3196, Japan.

5

Department of

6

Microbiology, Southern Illinois University, Carbondale, IL 62901 USA. Research Institute for Photobiological Hydrogen Production, Kanagawa University, Tsuchiya, Hiratsuka 259-1293, Japan.

*To whom correspondence should be addressed: Dr. Yukihiro Kimura, Tel. & Fax: +81-78-803-5819; E-mail: [email protected] Dr. Zheng-Yu Wang-Otomo, Tel. & Fax: +81-29-228-8352; E-mail: [email protected]

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ABSTRACT The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium (Tch.) tepidum exhibits a largely red-shifted LH1 Qy absorption at 915 nm due to binding of Ca2+, resulting in an “uphill” energy transfer from LH1 to RC. In a recent study, we developed a heterologous expression system (strain TS2) to construct a functional hybrid LH1-RC with the LH1 from Tch. tepidum and the RC from Rhodobacter (Rba.) sphaeroides (Proc. Natl. Acad. Sci. USA 114, 10906; 2017). Here, we present detailed characterizations of the hybrid LH1-RC from strain TS2. Effects of metal cations on phototrophic growth of strain TS2 revealed that Ca2+ is an indispensable element for its growth, which is also true for Tch. tepidum but not for Rba. sphaeroides. Thermal stability of the TS2 LH1-RC was strongly dependent on Ca2+ in a manner similar to that of the native Tch. tepidum, but interactions between the heterologous LH1 and RC became relatively weaker in the strain TS2. An FTIR analysis demonstrated that the Ca2+-binding site of the TS2 LH1 was similar but not identical to that of Tch. tepidum. Steady-state and time-resolved fluorescence measurements revealed that the uphill energy transfer rate from the LH1 to RC was related to the energy gap in an order of Rba. sphaeroides, Tch. tepidum, and strain TS2, however, the quantum yields of LH1 fluorescence did not exhibit such a correlation. Based on these findings, we discuss the roles of Ca2+, interactions between the LH1 and RC from different species, and the uphill energy transfer mechanisms.


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Biochemistry

INTRODUCTION Anoxygenic phototrophic bacteria have long been employed as model organisms for elucidating the basic principles of solar energy conversion. Early events in photosynthesis are carried out by two distinct systems: the antenna, for light-harvesting (LH), and the reaction center (RC), for subsequent charge separation. In purple phototrophic bacteria, the two membrane-integrated pigment protein complexes are physically distinct but fine-tuned to facilitate highly efficient energy transfer and electron/proton transport. In particular, the core antenna (LH1) and RC form a supramolecular complex (the LH1-RC), with the ring-like LH1 encircling the RC. The LH1-RC core complex represents the simplest natural system to perform the entire primary function of photosynthesis. So far, the highest-resolution structure has been reported for the LH1-RC complex from a thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum,1,2 first isolated from a hot spring in Yellowstone National Park by Madigan.3 The native LH1 complex of this organism incorporates Ca2+ as cofactors to enhance thermostability and exhibits a characteristic Qy absorption band at 915 nm.4-6 Moreover, the LH1-Qy transition is tunable to 875-890 nm, a characteristic of the LH1s from most purple bacteria, by removing or exchanging the Ca2+ with other divalent metal ions.5,7,8 Since publication of the crystal structure of the native LH1-RC core complex from Tch. tepidum,1,2 photosynthesis researchers, especially spectroscopists, computationalists, structural biologists and theoreticians, are keen to resolve two important questions: (i) what is the structural basis for the significantly different absorption properties of LH1-RC complexes from different phototrophic bacteria,9-12 and (ii) how does “uphill” energy transfer proceed in a natural photosynthetic system.1315

To further address these questions, we have developed a system for producing a chimeric LH1-

RC complex in which the LH1 is from Tch. tepidum and the RC is from a mesophilic purple nonsulfur bacterium Rhodobacter (Rba.) sphaeroides. The design of this system has been described16 and the system deployed to explore the Ca-binding site and structure-function relationships in this unique chimeric LH1-RC complex. The full-length Tch. tepidum LH1 was also 3 ACS Paragon Plus Environment

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heterologously expressed using a similar system, however the expression product appeared to be in an LH1-only form.17 Here we characterize this complex in more detail and explore the roles of Ca2+ in photosynthetic growth, light harvesting, thermal and structural stability, interactions between the LH1 and RC, and uphill energy transfer. The present results advance our understanding of the structure and function of LH complexes from a thermophilic phototroph and provide new insight on how the LH1 complex has been functionally optimized during the evolutionary process.

MATERIALS AND METHODS Phototrophic growth of strains TS2 and DP2. Cells of strain TS2 harboring the LH1 from Tch. tepidum and the RC from an LH2-deficient Rba. sphaeroides strain (DP2)16 were cultured in a mineral salts-malate medium (M6) with no addition of Ca2+ or addition of 0.20 mM CaCl2 or SrCl2 at 25˚C under illumination of incandescent lamp (60 W) for 14 days. The M6 medium was prepared by dissolving special grade reagents in deionized and distilled water, of which the contents of Ca2+ were under the detectable limitation by atomic absorption spectrometry. The growth curves of the phototrophs were evaluated by monitoring the absorbance of the culture at each LH1 Qy maximum.

Purification of LH1-RC Complexes. Cells of strains TS2 and DP2 cultured at 25˚C for 10 days were harvested and sonicated (Sonopuls HD3200, Bandelin) at 0˚C in a buffer containing 20 mM Tris-HCl (pH 8.5) followed by ultracentrifugation at 195,000×g to obtain chromatophores. The resulting pellets were suspended in the same buffer and treated with 0.25% (w/v) lauryldimethylamine

N-oxide

(LDAO, Anatrace) at

25°C

for

60

min, followed

by

ultracentrifugation at 19,5000×g to remove misassembled pigment-protein components. The pellets were further treated with 1.25% (w/v) n-octyl-β-D-glucopyranoside (OG, Anatrace) at 25°C for 60 min to extract the crude LH1-RC. All purifications for strain TS2 were performed in the presence of 4 ACS Paragon Plus Environment

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Biochemistry

2 mM CaCl2 to avoid degradation of the LH1-RC component. The supernatants after ultracentrifugation were loaded onto a DEAE anion-exchange column (Toyopearl 650S, TOSOH) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 0.10% (w/v) LDAO and 10 mM CaCl2 for strain TS2 or 0.10% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) for Rba. sphaeroides strain DP2 at 4°C. The LH1-RC fractions were eluted by a linear gradient of CaCl2 from 10 mM to 30 mM for strain TS2 or NaCl from 0 mM to 200 mM for Rba. sphaeroides strain DP2. Purification of the native Tch. tepidum LH1-RC complexes was conducted as described previously.18 LH1-only complexes from TS2 and DP2 were prepared as follows. The chromatophores of TS2 and DP2 were treated with 0.23 – 0.25% LDAO in 20 mM Tris-HCl buffer at pH7.5 and pH8.5, respectively, for 60 min, followed by ultracentrifugation at 4˚C and 196,000×g for 60 min. The extracts were loaded onto DEAE anion-exchange column (Toyopearl 650S, TOSOH) equilibrated with Tris-HCl (pH7.5) containing 0.1% DDM for TS2 or 0.8% OG at 4˚C. LH1-only fractions were eluted by linear gradient of CaCl2 from 0 mM to 200 mM for TS2 and NaCl from 0 mM to 400 mM. The LH1-only complex from Tch. tepidum was prepared as reported previously.8 For sucrose density gradient centrifugation analyses, purified LH1-RC complexes were loaded onto a continuous gradient of sucrose from 10% to 40% (w/v) in a buffer containing 20 mM TrisHCl (pH 7.5) and 0.08% (w/v) DDPC and 20 mM CaCl2. For ATR-FTIR measurements, the LH1-RC fractions were concentrated with Amicon Ultra 100K (Millipore), and diluted with 10-fold of buffer A (20 mM Tris-HCl, 20 mM CaCl2, pH7.5), followed by ultracentrifugation at 195,000×g for 10 min. The resulting pellet was suspended in buffer A containing 0.008% DDPC to be ~100 µM of LH1-RC complex, and used for ATR-FTIR measurements.8

ATR-FTIR Measurement. Perfusion-induced ATR-FTIR difference spectra were recorded on an FTIR spectrophotometer (Prestige-21, Shimadzu) equipped with a three-bounce Si/ZnSe ATR prism 5 ACS Paragon Plus Environment

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(DuraSamplIR II, Smiths Detection), a mercury-cadmium-telluride detector (Shimadzu), and a Si long-pass filter as described previously.8 A dehydrated LH1-RC biofilm on the ATR prism was covered with a flow-attachment connected to a peristaltic pump (MP-1000-H, EYELA). Background spectra were measured after a perfusion of Ca2+-buffer (20 mM Tris-HCl, 25 mM NaCl, 20 mM CaCl2, pH 7.5) at a flow rate of 1 ml/min for 30 min. The buffer was switched to Sr2+-buffer (20 mM Tris-HCl, 25 mM NaCl, 20 mM SrCl2, pH 7.5) at a flow rate of 1 ml/min for 20 min, and then, sample spectra were recorded to yield an Sr2+/Ca2+ ATR-FTIR difference spectrum. A Ca2+/Sr2+ ATR-FTIR difference spectrum was obtained by switching Sr2+-buffer to Ca2+-buffer. All the spectra were collected at 25˚C and accumulated 150 scans. To improve the S/N ratio, 30 – 40 spectra from different samples were averaged.

Fluorescence Measurement. Relative fluorescence quantum yields of the LH1 complexes from Rba. sphaeroides strain DP2, strain TS2, and Tch. tepidum were recorded on a JASCO FP-8600DS spectrofluorometer with an emission band-passes of 10 nm.16 Sample concentration of the LH1-RC and LH1 complexes were adjusted to ~0.1 OD at their LH1-Qy maxima and the fluorescence spectra were obtained by excitations at their LH1 Qx bands. Absolute fluorescence quantum yields were measured with an absolute photoluminescence quantum yield measurement system (Hamamatsu, Quantaurus-QY Plus, C13534).19 An excitation light from a 150 W xenon lamp (Hamamatsu, L13685-01) was guided into an integrating sphere with an optical light guide after passing through a band-pass filter (center wavelength, 400 nm or 600 nm; FWHM, 50 nm) and an IR cut filter. The integrating sphere was used as a sample chamber, which can mount a quartz cuvette with a 1 mm path length. A photonic multichannel analyzer was used as the multichannel detector. This employed a back-thinned CCD (BT-CCD) linear image sensor, making it possible to measure photoluminescence within a wide range from 350 nm to 1100 nm.


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Biochemistry

Time-Resolved Fluorescence Measurement. Fluorescence decay curves were measured by a time-correlated single counting system (SPC-630, Becker & Hickl GmbH, Germany). The excitation source was the second harmonic of a Ti:Sapphire laser (Tsunami, Spectra-Physics, U. S. A.) or a picosecond diode laser (PiL040X, Advanced Laser Diode Systems, Germany), by which the excitation wavelength was set at 400 nm or 408 nm, respectively. The trapping time of the RCs from TS2, DP2, and Tch. tepidum was measured in the presence of 2 mM sodium ascorbate and 0.1 mM N-methylphenazonium methosulphate (PMS), typically used as exogenous electron donors.

RESULTS AND DISCUSSION Effects of Ca2+ on phototrophic growth of strain TS2 Figure 1 compares the effects of Ca2+ on spectral properties and phototrophic growth rate of Rba. sphaeroides strain DP2 (A and B) and strain TS2 (C and D). In panel A, the LH1 Qy peak position of Rba. sphaeroides strain DP2 was insensitive to the depletion of Ca2+ or replacement of Ca2+ with Sr2+. This indicates that these metal cations have little influence on the spectral properties of the Rba. sphaeroides LH1-RC complex, although the growth rates of the Ca2+-free and Sr2+-substituted cells were slightly decreased compared with that of the Ca2+-containing cells (B). In strain TS2 (C and D), the LH1 Qy band appeared at 917 nm in the presence of Ca2+ but no clear Qy band was detected in cells in the absence of Ca2+ during cultivation. The requirement of Ca2+ for phototrophic growth of strain TS2 is consistent with that of native Tch. tepidum,12 and this strongly implies that Ca2+ is an indispensable cofactor for the growth of strain TS2 as well as for the assembly of a functional LH1 complex. When Ca2+ was replaced with Sr2+, the growth rate was largely reduced and showed a modified LH1 Qy maximum near 890 nm, a characteristic of the biosynthetically 7 ACS Paragon Plus Environment

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Sr2+-substituted LH-RC complex from Tch. tepidum.18 An additional band at 863 nm was also present that likely originates from RC-only complexes that were not assembled into LH1-RC complexes. These results suggest that Sr2+ can be incorporated into the LH1-RC complexes of strain TS2 in place of Ca2+, although the population of functional Sr2+-substituted complexes was much lower than that observed in biosynthetically Sr2+-substituted LH1-RC complexes from Tch. tepidum.18 Therefore, in contrast to Tch. tepidum, Sr2+ is not a functional replacement for Ca2+ in strain TS2 and is unable to support normal phototrophic growth of this chimeric strain.

Figure 1. Absorption spectra of the matured cells from Rba. sphaeroides strain DP2 (A) and strain TS2 (C) cultivated in the presence of Ca2+ (orange), Sr2+ (light blue), or in the absence of both cations (black). Phototrophic growth curves for strain DP2 (B) and strain TS2 (D) in the presence of Ca2+ (orange), Sr2+ (light blue), or in the absence of these cations (black) monitored with each Qy band intensity.


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Biochemistry

Absorption and aggregation of the LH1-RC complex from strain TS2 Figure 2 shows absorption spectra of the purified LH1-RC complexes from Tch. tepidum (a), strain TS2 (b), and Rba. sphaeroides strain DP2 (c). The TS2 LH1-RC complex exhibited an LH1 Qy maximum at 918 nm, markedly red-shifted from that of Rba. sphaeroides (875 nm) and comparable to that of native Tch. tepidum (915 nm). Similar tendency was observed for their Qx bands appeared at 593 nm (TS2), 590 nm (Rba. sphaeroides), and 592 nm (Tch. tepidum) although the degree of the shift was much smaller than those of Qx bands. These results demonstrate that the conformation of BChl-a molecules and their interactions in the TS2 LH1 complex differ from those in the Rba. sphaeroides LH1 complex but closely resemble those in the Tch. tepidum LH1 complex. As for carotenoid molecules, the 505, 472, and 446 nm bands in the Rba. sphaeroides spectrum were assigned to spheroidene. Similar bands were observed in the TS2 spectrum at 501, 469, and 442 nm, indicating that the carotenoid molecules in the LH1 of strain TS2 contained spheroidene in place of the spirilloxanthin present in Tch. tepidum LH1. The small blue-shifts relative to those of Rba. sphaeroides strain DP2 are due to slightly modified interactions of the spheroidenes in a nonnative LH1 environment, which is in consistent with the result of a previous resonance Raman study.16 Based on biochemical and structural analyses,1,4 the Tch. tepidum LH1-RC complex exists as a monomer in the purified state. In contrast, the Rba. sphaeroides LH1-RC complexes show a monomer/dimer distribution, which is closely tied to the expression of the PufX protein.20,21 To examine the aggregation state of the chimeric LH1-RC complexes, we conducted a sucrose density gradient ultracentrifugation analysis (inset of Figure 2). A single band of the TS2 LH1-RC complex was observed at a slightly higher position than that of the Tch. tepidum LH1-RC complex. This difference in band position is attributed to a lack of the C-subunit in the RC complex of strain TS2 and is supported by the absence of an absorption band at 410 nm in the TS2 spectrum originating from the RC C-subunit of Tch. tepidum.22 These data indicate that the strain TS2 LH1-RC complex 9 ACS Paragon Plus Environment

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was purified in a monomeric state in the current study in agreement with our previous finding that PufX is absent from the TS2 LH1-RC complex.16

Figure 2. Absorption spectra of purified LH1-RC complexes from Tch. tepidum (black), strain TS2 (magenta), and Rba. sphaeroides strain DP2 (gray). The inset figure shows sedimentation patterns of the LH1-RC complexes from Tch. tepidum (left) and strain TS2 (right) after a continuous 10-40% (w/v) sucrose density gradient ultracentrifugation at 195,000 ×g for 7 h.

Thermal stability of the LH1-RC complex from strain TS2 After documenting the Ca2+ requirement of the chimeric LH1-RC complex of strain TS2, one of the most important further considerations was whether the complex retains the thermal stability of the native Tch. tepidum complex. We therefore examined the effect of Ca2+ on the thermal stability of the TS2 LH1-RC complex by monitoring absorption spectra during incubation at 50˚C in the presence of Ca2+ or after addition of EDTA (Figure 3). In the presence of Ca2+, the LH1 Qy band intensity at 918 nm gradually decreased to about 40% with the appearance of a broad band at ~770 nm originating from BChl a bound to LH1 α- or β-apoproteins (Figure 3A). Upon addition of EDTA, the LH1 Qy peak was immediately blue-shifted to 863 nm, and the band intensity was almost completely decomposed following incubation at 50˚C for 4 min. These results demonstrate 10 ACS Paragon Plus Environment

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Biochemistry

that the thermal stability of the TS2 LH1-RC complex is strongly dependent on the Ca2+ in a manner similar to that of the Tch. tepidum LH1-RC complex.6 However, the stabilities of the chimeric LH1-RC complexes were lower than those of the Tch. tepidum LH1-RC complexes in both the presence and absence of Ca2+ (Figure 3C). In addition, Ca2+-dependence differed between the two species. For example, upon incubation for 4 min, the relative LH1 Qy intensity for the Ca2+depleted TS2 LH1-RC complex decreased sharply in contrast to only a modest decrease in Ca2+depleted Tch. tepidum LH1-RC complex, although both Ca2+-bound forms began the experiment at nearly identical relative Qy intensities (Figure 3C). This indicates that the chimeric LH1-RC complex has much stronger Ca2+ dependence for its thermal stability than that does that of Tch. tepidum. Moreover, the rapid thermal decomposition observed for the Ca2+-depleted TS2 LH1-RC complex was also confirmed in the LH1-only complex from Tch. tepidum (Figure 3C), possibly due to the lack of specific interactions between the LH1 and RC complexes. In strain TS2, the LH1 and RC complexes from different species were assembled, which may lead to a reduced interaction between the two complexes. Taken together, thermal stability of the hybrid LH1-RC complex is much lower than that of native Tch. tepidum in both the presence and absence of Ca2+, and the result highlights the importance of interactions between LH1 and RC complexes for its thermal stability.


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Figure 3. Changes of absorption spectra for the strain TS2 LH1-RC complexes upon incubation at 50˚C in the presence of 20 mM Ca2+ (A) or 2 mM EDTA (B). (C) Plots of relative Qy band intensities versus incubation time for the LH1-RC complexes from strain TS2 (magenta) and Tch. tepidum (black) in the presence of 20 mM Ca2+ (circle) or 2 mM EDTA (triangle). The gray triangle plots exhibit the thermal stability of the Tch. tepidum LH1-only complex in the presence of 2 mM EDTA presented for comparison.

Conformational changes of Ca2+ binding site in the TS2 LH1-RC complex Figure 4 shows perfusion-induced Sr2+/Ca2+ ATR-FTIR difference spectra of the purified LH1-RC complexes from strain TS2 and Tch. tepidum. Upon metal substitution from Ca2+ to Sr2+ (Sr2+/Ca2+), characteristic difference bands were clearly observed in the TS2 LH1-RC complex. 12 ACS Paragon Plus Environment

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Biochemistry

These bands reflect structural and/or conformational changes in main chains of the LH1 polypeptide (amide I, 1700−1600 cm-1; amide II, 1600−1500 cm-1) and the amino acid side chains involved in the metal binding sites. The difference bands upon switching back from Sr2+ to Ca2+ (Ca2+/Sr2+) appeared in reverse sign but with almost identical intensities, reflecting a reversible structural change upon the metal substitution. The characteristic features in the difference spectra of the TS2 LH1-RC complex closely resembled those of the Tch. tepidum LH1-RC complex in both peak position and band intensity.8 These results provide evidence that strain TS2 possesses Ca2+ binding sites in the LH1 C-terminal region as is true of Tch. tepidum. However, close inspection of the difference spectra from these species revealed that there are slight but distinct differences in several bands at the 1650 – 1500 cm-1 and 1050 – 850 cm-1 region as clearly seen in the double difference spectra for the Ca2+/Sr2+ and Sr2+/Ca2+ differences (c). These bands reflect conformational differences in the LH1 Ca2+ binding sites and its surroundings in strain TS2 and Tch. tepidum due to the different major carotenoid present. In strain TS2, spheroidenes were biosynthetically incorporated in the LH1 complex instead of spirilloxanthins, the major carotenoid in the Tch. tepidum LH1 complex. In addition, the crystallographic structure of Tch. tepidum LH1-RC complex revealed that the spirilloxanthins in the LH1 complex interact with BChls-a through one of the methoxy groups located in close proximity to the imidazole group of the BChl-coordinating His residues and the central Mg atom of BChl-a.1 Based on isotope-edited ATR-FTIR analyses (unpublished results), the 887 cm-1 band of the Tch. tepidum was assigned to the CH out of plane bending mode of aromatic rings. Thus, it is possible that the double difference band at 887 cm-1 originates from differences in BChl–carotenoid interactions between the TS2 and Tch. tepidum LH1 complexes. Such interactions may also be responsible for the slightly red-shifted LH1 Qy band of strain TS2 compared with that of Tch. tepidum as well as the blue-shifted carotenoid bands of strain TS2 relative to those of Rba. sphaeroides strain DP2 (Figure 2). This conclusion is also consistent with the fact that selective removal of the carotenoids from LH1 complexes results in a blue shift of ~10 nm for the LH1-Qy band.23,24 13 ACS Paragon Plus Environment

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Figure 4. ATR-FTIR difference spectra of the LH1-RC complexes from strain TS2 (a, magenta) and Tch. tepidum (b, black) induced by buffer switching from Ca2+-buffer to Sr2+-buffer (Sr2+/Ca2+ difference spectra, solid lines) and switching from Sr2+-buffer to Ca2+-buffer (Ca2+/Sr2+ difference spectra, dashed lines). Spectra c represent double difference spectra obtained by subtracting Tch. tepidum difference spectra from strain TS2 difference spectra for Ca2+-to-Sr2+ substitution (blue, solid line) and Sr2+-to-Ca2+ substitution (blue, dashed line)

Uphill energy transfer rate To determine the uphill energy transfer rates, we measured fluorescence lifetimes of the RC from the Tch. tepidum, TS2, and DP2 LH1-RC complexes by time-resolved fluorescence measurements. Fluorescence decay curves monitored at 860 nm were analyzed by a triple exponential decay and the results are summarized in Table 1. The first component with the shortest lifetime is assigned to trapping of the excitation energy in the open state of RC. In Rba. sphaeroides strain DP2, the first component was predominant with a lifetime of 60 ps, which is comparable to those reported previously.25-36 The lifetime of 80 ps for Tch. tepidum is also in good agreement with previous results13,15,36,37 although the amplitude was decreased to 58.4%. Interestingly, the first component 14 ACS Paragon Plus Environment

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Biochemistry

for the chimeric LH1-RC in strain TS2 was largely increased to 190 ps with an amplitude of 59.7% and the time constant was consistent with that obtained by time-resolved absorption spectroscopy.36 These trapping rates of the RCs seem to be well correlated with the energy gaps ∆E between LH1 and RC complexes under the present experimental conditions. The trapping rate decreased with increasing the ∆E = 132 cm-1 for Rba. sphaeroides strain DP2, 370 cm-1 for Tch. tepidum, and 667 cm-1 for strain TS2. However, this is not the case for the LH1-RC complexes from Roseospirillum (Rss) parvum strain 930I38 and strain 970,39 both of which exhibit unusual LH1 Qy transition peaking at 909 and 963 nm, respectively. The RC trapping time was reported to be 55 ± 5 ps for Rss. parvum strain 930I with an uphill energy gap of 285 cm-1, and 65 ± 5 ps for strain 970 with a gap of 425 cm-1. The second lifetime component can be attributed to trapping of the excitation energy in the closed state of RC,25,27,28 and/or fluorescence due to the LH1 complexes that are quenched by traps including oxidized antenna BChls.31 The second components seem to be involved in the excited state dynamics of the LH1-RC complexes from Tch. tepidum and strain TS2 although such a contribution was negligible in the Rba. sphaeroides LH1-RC complex. This implies that the Oshaped LH1 ring of Tch. tepidum is related to the increases of fluorescence quenching by traps and/or closed reaction center. Furthermore, the third component was small in amplitude but required to obtain a better fitting to the decay profile27 and was assigned to relaxation of the near equilibrium mixture of excited and charge-separated states.31 A recent study of Stark absorption spectroscopy9 indicated that relatively large changes in the dipole moment and polarizability in Tch. tepidum arise from mixing of a charge-transfer state into the lowest exciton state of the special pair40 and LH141 as a result of closely packed and excitonically coupled BChl-a molecules. Therefore, such an equilibrium mixture of the excited states for the RCs with the Tch. tepidum-type LH1 may contribute to the third component. Although the origins of minor components with the long lifetimes are not clear at present, the rapid uphill energy transfer rate from the LH1 to RC was related to the energy gap ∆E in the order 15 ACS Paragon Plus Environment

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of Rba. sphaeroides, Tch. tepidum, and strain TS2. The energy gap of strain TS2 LH1-RC complex is the largest among purple bacteria ever examined, and therefore, the lowest uphill energy transfer rate seems to be rational. Nevertheless, strain TS2 only slightly slowed phototrophic growth in the presence of Ca2+ (Figure 1D). These results imply that an efficient uphill energy transfer may occur in this chimeric LH1-RC complex as described in the following section.

Table 1. Fluorescence quantum yields of LH1 complexes and lifetimes of RC complexes from strain DP2, Tch. tepidum, and strain TS2. Fluorescence Lifetime of RC quantum yield of a (Percentage) λLH1 λRC ∆E LH1b Species -1 (nm) (nm) (cm ) 1st 2nd 3rd Relative Absolute component component component Strain DP2

875

865

132

41%

40%

60 ps (97.4%)

160 ps (2.3%)

2.1 ns (0.3%)

Tch. tepidum

915

885

370

22%

15%

80 ps (58.4%)

320 ps (23.6%)

1.2 ns (18%)

190 ps 590 ps 2.2 ns (59.7%) (29.4%) (10.9%) a ∆E means energy gap between the LH1 and RC Qy bands. bFluorescence quantum yields of LH1 complexes were calculated from the area of the emission bands of the LH1-RC complexes relative to those of the LH1-only complexes. Strain TS2

918

865

667

44%

48%

Uphill energy transfer efficiency To further investigate the uphill energy transfer mechanism, fluorescence quantum yields were determined for the LH1 complexes from different species. Figure 5 compares steady-state fluorescence and absorption spectra of LH1-RC complexes from strain TS2 and Tch. tepidum (A) and Rba. sphaeroides strain DP2 (B). For strain TS2 and Tch. tepidum, fluorescence spectra obtained by Qx excitations exhibited an emission band at 937 nm originating from the LH1 BChl-a molecules, and the band intensity for strain TS2 was slightly higher than that for Tch. tepidum. 16 ACS Paragon Plus Environment

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Biochemistry

Relative band intensities of the LH1-RCs with respect to that of the LH1-only complex (lacking RC) was 22% and 44% for Tch. tepidum and strain TS2, respectively (Table 1). For Rba. sphaeroides LH1-RC complex, the fluorescence band appeared at 895 nm with an intensity of 41% relative to that of the LH1-only complex. These results indicate that despite a relatively large energy gap, the putative uphill energy transfer efficiency of LH1→RC for the chimeric LH1-RC is much lower than that of native Tch. tepidum and comparable to that of Rba. sphaeroides; this explains why the chimeric LH1-RC supports phototrophic (anoxic/light) growth of the strain TS2.16 Furthermore, the fluorescence quantum yields were verified by absolute fluorometry.19 The absolute fluorescence quantum yield for each LH1 was 5.7% for DP2, 6.0% for Tch. tepidum, and 8.0% for TS2, and that for LH1-RC complex was 2.3% for DP2, 0.90% for Tch. tepidum, and 3.8% for TS2. These values were used for calculation of the absolute quantum yields listed in Table 1. The absolute quantum yields were 40%, 15%, and 48% for Rba. sphaeroides strain DP2, Tch. tepidum, and strain TS2, respectively, showing a good agreement with those of the relative yields (Table 1). These results support the idea that the efficiency of fluorescence is comparable in Rba. sphaeroides strain DP2 and strain TS2 but is significantly lower in Tch. tepidum. The intrinsic decay of the excited antenna from chromatophores of an Rba. sphaeroides lacking RCs was estimated to be ~1 ns.42 Furthermore, the decay time constant of the isolated Rba. sphaeroides LH1 complex was reported to be 650 ± 50 ps.43 If the non-radiative deactivation of each excited LH1 complex is independent of the species, the efficiency of excitation energy transfer from the LH1 to the RC should be in the order of Tch. tepidum > strain DP2 > strain TS2. The efficiency of the uphill energy transfer is not necessarily correlated with the energy gap between the LH1 and RC, indicating that a unique mechanism for the efficient uphill energy transfer is involved in the excited state dynamics of Tch. tepidum. If the non-radiative deactivation depends on the species, it is possible that the Tch. tepidum-type LH1 complex has an efficient non-radiative deactivation path. However, the rate of the non-radiative process can be speculated to be low in the Tch tepidum LH1-RC complex because the low flexibility of the LH1 complex induced by Ca2+17 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

binding is one of factors responsible for the enhanced thermal stability.10,44 An alternative interpretation surrounds the difference in the carotenoids of the LH1 complexes; spirilloxanthin for Tch. tepidum and spheroidene for strains TS2 and DP2. The specific interaction between spirilloxanthin and BChl-a in the LH1 complex was demonstrated in the crystallographic structure of the Tch. tepidum LH1-RC complex1 and also supported by the present spectroscopic data (Figures 2 and 4). Therefore, specific interaction between BChl-a and spirilloxanthin may be a factor for facilitating the efficient non-radiative decay and another possible factor responsible for largely reduced energy transfer efficiency16 from carotenoids to BChl-a of Tch. tepidum (23%) compared with those of DP2 (71%) and TS2 (44%) as well as the conjugation length and number of carotenoids, the protein environment and the energy gap between the absorption maxima of the carotenoid and BChl a molecules.

Figure 5. (A) Steady-state fluorescence (solid lines) and absorption (dashed lines) spectra of the LH1-RC complexes from strain TS2 (A, magenta) and Tch. tepidum (A, black) and Rba. 18 ACS Paragon Plus Environment

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Biochemistry

sphaeroides strain DP2 in the monomeric form (B, orange). The fluorescence and absorption spectra of the LH1 complexes lacking the RCs from Tch. tepidum (A, blue) and strain DP2 (B, blue) are presented as references. All the emission spectra were obtained by the excitation at Qx bands.


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AUTHOR INFORMATION

Corresponding Authors Dr. Yukihiro Kimura, Tel. & Fax: +81-78-803-5819; E-mail: [email protected] Dr. Zheng-Yu Wang-Otomo, Tel. & Fax: +81-29-228-8352; E-mail: [email protected]

Funding Sources This work was supported by Grants-in-aid for Scientific Research (C) (16K07295) to Y.K. and (B) (16H04174) to Z.-Y.W.-O. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We thank Y. Shibuya and A. Yamaguchi for their experimental assistance.

ABBREVIATIONS: BChl, bacteriochlorophyll; LH, light-harvesting; RC, reaction center; DDPC, dodecylphosphocholine; ATR, attenuated total reflection; FTIR, Fourier transform infrared.

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Permentier, H. P., Neerken, S., Overmann, J., and Amesz, J. (2001) A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm, Biochemistry 40, 55735578. Middendorf, T. R., Mazzola, L. T., Lao, K. Q., Steffen, M. A., and Boxer, S. G. (1993) Stark-effect (electroabsorption) spectroscopy of photosynthetic reaction centers at 1.5 K evidence that the special pair has a large excited-state polarizability, Biochim. Biophys. Acta 1143, 223-234. Beekman, L. M. P., Steffen, M., vanStokkum, I. H. M., Olsen, J. D., Hunter, C. N., Boxer, S. G., and vanGrondelle, R. (1997) Characterization of the light-harvesting antennas of photosynthetic purple bacteria by stark spectroscopy .1. LH1 antenna complex and the B820 subunit from Rhodospirillum rubrum, J. Phys. Chem. B 101, 7284-7292. Campillo, A. J., Hyer, R. C., Monger, T. G., Parson, W. W., and Shapiro, S. L. (1977) Light collection and harvesting processes in bacterial photosynthesis investigated on a picosecond time scale, Proc. Natl. Acad. Sci. U. S. A. 74, 1997-2001. Bergstrom, H., Westerhuis, W. H. J., Sundstrom, V., Vangrondelle, R., Niederman, R. A., and Gillbro, T. (1988) Energy-transfer within the isolated B875 light-harvesting pigmentprotein complex of Rhodobacter sphaeroides at 77 K Sstudied by picosecond absorption spectroscopy, FEBS Lett. 233, 12-16. Jakob-Grun, S., Radeck, J., and Braun, P. (2012) Ca2+-binding reduces conformational flexibility of RC-LH1 core complex from thermophile Thermochromatium tepidum, Photosynth. Res. 111, 139-147.


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Biochemistry

Table 1. Fluorescence quantum yields of LH1 complexes and lifetimes of RC complexes from strain DP2, Tch. tepidum, and strain TS2. Fluorescence Lifetime of RC quantum yield of a (Percentage) λLH1 λRC ∆E LH1 Species -1 (nm) (nm) (cm ) 1st 2nd 3rd Relative Absolute component component component Strain DP2

875

865

132

41%

40%

60 ps (97.4%)

160 ps (2.3%)

2.1 ns (0.3%)

Tch. tepidum

915

885

370

22%

15%

80 ps (58.4%)

320 ps (23.6%)

1.2 ns (18%)

Strain TS2

918

865

667

44%

48%

190 ps (59.7%)

590 ps (29.4%)

2.2 ns (10.9%)

a

energy gap between the LH1 and RC Qy bands.


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Figure 1：


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Biochemistry

Figure 2：


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Figure 3：


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Biochemistry

Figure 4：


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Figure 5：


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For Table of Contents Use Only

Manuscript Title: Biochemical and Spectroscopic Characterizations of a Hybrid Light-Harvesting-Reaction Center Core Complex

Authors: Yukihiro Kimura, Kanako Hashimoto, Seiji Akimoto, Mizuki Takenouchi, Kengo Suzuki, Rikako Kishi, Michie Imanishi, Shinji Takenaka, Michael T. Madigan, Kenji V. P. Nagashima, and Zheng-Yu Wang-Otomo

TOC graphic:

LH1

LH1

RC

RC

Rba. sphaeorides

Small uphill Rba. sphaeorides

LH1 RC

Tch. tepidum

Tch. tepidum

RC 865 nm 875 nm

Energy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Strain TS2 RC Large uphill

RC

865 nm

Medium uphill 885 nm

LH1 918 nm

915 nm

LH1

LH1

TS2 Absorbance


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Biochemical and Spectroscopic Characterizations of a Hybrid Light

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