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Spectrally Selective Spectroscopy of Native Ca-Containing and BaSubstituted LH1-RC Core Complexes from Thermochromatium tepidum Margus Rätsep, Kõu Timpmann, Tomoaki Kawakami, Zheng-Yu Wang-Otomo, and Arvi Freiberg J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07841 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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The Journal of Physical Chemistry

Spectrally Selective Spectroscopy of Native Ca-containing and Basubstituted LH1-RC Core Complexes from Thermochromatium tepidum

Margus Rätsep,a Kõu Timpmann,a Tomoaki Kawakami,b Zheng-Yu Wang-Otomo,b and Arvi Freiberg *,a,c a

Institute of Physics, University of Tartu, W. Ostwald Str. 1, 50411 Tartu, Estonia

b

Faculty of Science, Ibaraki University, Mito 310-8512, Japan

c

Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia

Abstract The LH1-RC core complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted interest of many researches because of several unique properties such as an increased robustness against environmental hardships and the much red-shifted near-infrared absorption spectrum of the LH1 antenna exciton polarons. The known near-atomic-resolution crystal structure of the complex well supported this attention. Yet several mechanistic aspects of the complex prominence remained to be understood. In this work, the samples of the native, Ca2+-containing core complexes were investigated along with those destabilized by Ba2+ substitution, using various spectrally selective steady-state and picosecond time-resolved spectroscopic techniques at physiological and cryogenic temperatures. As a result, the current interpretation of exciton spectra of the complex was significantly clarified. 1 ACS Paragon Plus Environment


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Specifically, by evaluating the homogeneous and inhomogeneous compositions of the spectra we showed that there is little to no effect of cation substitution on the dynamic or kinetic properties of antenna excitons. Reasons of the extra red-shift of absorption/fluorescence spectra observed in the Ca-LH1-RC and not in the Ba-LH1-RC complex should thus be searched in subtle structural differences following the inclusion of different cations into the core complex scaffold.

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INTRODUCTION Thermochromatium (Tch.) tepidum is a photosynthetic purple sulfur bacterium that grows in the optimal temperature range of 48–50°C with an upper limit of 58°C.1 The LH1-RC core complex from this bacterium has recently attracted interest of many researches because of several unique properties such as notably higher thermal stability and much red-shifted nearinfrared absorption compared to complexes from its mesophilic non-sulfur counterparts like Rhodobacter (Rba.) sphaeroides.2-7 The absorption spectrum under discussion, traditionally called the B875 band, is related to the Qy singlet electronic transition in individual bacteriochlorophyll a (BChl) pigment chromophores these complexes contain. Structural bases of the above remarkable changes were revealed in Refs,2,8 where the near-atomic structure of the LH1-RC core complex was reported. It was shown that differently from the complexes from non-sulfur purple bacteria the core complex from Tch. tepidum includes Ca2+ ions. In total 16 Ca2+ ions bind LH1 α-inner ring and β-outer ring tightly at their C-terminal domains in a manner that each ion is coordinated by α-Trp46, α-Asp49, α-Ile51, and β-Trp45 in the adjacent protomer along with two water molecules. A more intuitive picture of the structure can be obtained from recent specialized publications (see, e.g. Fig. 5 of Ref. 2). Kimura et al.9,10 have further demonstrated that depletion of the Ca2+ ions caused significant, almost 35 nm at room temperature blue shift of the B875 band from about 915 nm to ~880 nm. The latter position is not far from the absorption maximum position for LH1 of Rba. sphaeroides.11 Replacement of native Ca2+ ions with other divalent cations resulted in much smaller (up to 8 nm) red shift relative to the above 880-nm reference wavelength. Hence, the specific to Tch. tepidum red shift of the absorption spectrum was mainly attributed to the presence of Ca2+ ions.9,12 Yet another surprising property of Tch. tepidum is that despite the 3 ACS Paragon Plus Environment


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strong red shift of the core antenna absorption spectrum, its excitation energy transfer and light harvesting functionalities seem to be maintained over the wide temperature range of 77– 329 K.3,13,14 In the present work, we systematically studied and interpreted the stationary and timeresolved spectral properties of native Ca-containing and Ba-substituted LH1-RC core complexes of Tch. tepidum at physiological and liquid He temperatures. Wide selection of spectroscopic techniques in visible optical range have been applied, including common broadband absorption– fluorescence and fluorescence excitation, spectrally selective hole-burning and fluorescence linenarrowing, and selectively excited picosecond time-resolved fluorescence. As a result, we reassessed the existing in the literature interpretation of minor blue-shifted absorption and fluorescence components systematically found in the spectra of core complexes from Tch. Tepidum. We also established significant variances in the inhomogeneous broadening of the LH1 antenna exciton spectra upon the substitution of Ca2+ ions with Ba2+ ions, at almost no change in their homogenous (dynamic) characteristics.

MATERIALS AND METHODS Sample preparation. The native Ca-LH1-RC and Ba-substituted Ba-LH1-RC complexes from Tch. tepidum were prepared as described earlier.2,8 The concentrated LH1-RC complexes were stored at –78°C in deep freezer. Prior the use the samples were diluted with 20 mM TrisHCl buffer containing a specific detergent n-dodecyl β-D-maltopyranoside (β-DDM) to prevent aggregation. In case of the native complex 0.05% of β-DDM was added at room temperature, in the Ba-substituted complex slightly higher concentration of 0.08% was used. In order to keep stable saturated Ba concentration, 50–100 mM BaCl2 was added to the Ba-substituted complex. 4 ACS Paragon Plus Environment

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The samples for low temperature measurements included glycerol with a 2:1 volume ratio in the buffer solution for securing transparent glassy samples. In most cases, the concentration of detergent was 0.1% and 0.2% in native and Ba-substituted sample, respectively. In separate measurements detergent concentrations between 0.05 and 0.5% were used. Spectroscopy. The steady state absorption and fluorescence spectra were recorded with spectral resolution of 1.5 nm using a 0.3 m spectrograph Shamrock SR-303i, equipped with a thermo-electrically cooled CCD camera DV420A-OE (both Andor Technology). For absorption measurements a high-stability tungsten light source BPS100 (BWTek) was employed. Spectrally non-selective florescence excitation was provided by a 5 mW diode laser at 407 nm. The fluorescence spectra were corrected for spectral sensitivity of the experimental set-up. Holeburning and fluorescence line-narrowing (FLN) spectra were measured using a model 3900S Ti: sapphire laser of 0.5 cm–1 linewidth pumped by a Millennia Prime solid state laser (both Spectra Physics). In the fluorescence kinetics measurements the samples were excited at several selected wavelengths between 760 and 850 nm with ~100 fs pulses of a femtosecond mode-locked Ti:sapphire laser (Coherent Mira-900) at 76 MHz repetition rate. Spectral bandwidth of the pulses was ~15 nm. To achieve convenient signal-to-noise ratio, relatively high excitation intensity of 0.5 W/cm2 was applied that corresponded to saturated photosynthesis with most of the RCs in photo-chemically inactive (photo-oxidized) state. At the same time, care was taken to keep the excitation intensity sufficiently low in order to avoid any non-linear excitation quenching effects, see Refs 15, 16 for more detail. The fluorescence decay was recorded with a time-correlated single photon counting system (SPC-150, Becker & Hickl) using a hybrid photomultiplier detector (HPM-100-50, Becker & Hickl). The proper wavelength of detected 5 ACS Paragon Plus Environment


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emission was selected by a double subtractive dispersion monochromator (DTMc300, Bentham) with varied spectral resolution between 2 to 4 nm in different measurements. The experimental fluorescence decay curves were deconvoluted with the temporal response function (~140 ps full width at half maximum, fwhm) of the detector. The estimated uncertainty of the lifetimes is ≤10% relative to their mean value. Plastic cuvettes (Brand) of 10 mm path length or gelatine capsules (Torpac) of 4 mm diameter were used as sample containers. For low-temperature measurements the sample was placed in a helium bath cryostat (Utreks), where the temperature was controlled with the precision of ±0.5 K. Data evaluation. The effective electron-phonon coupling strength of antenna exciton polarons was evaluated according to Eq. (1) from the ratio of integral intensities IZPL and IPSB of, respectively, zero-phonon line (ZPL) and phonon sideband (PSB), see, e.g.:17 exp( − S ph ) =

୍ౖౌై ୍ౖౌై ା୍ౌ౏ా

,

(1)

where Sph is the effective Huang–Rhys factor of matrix phonons. The fluorescence decay curves were generally evaluated in double-exponential approximation, recovering the decay times τ1, and τ2, and respective amplitudes A1, and A2, of two individual components.


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RESULTS AND DISCUSSION Overview absorption and fluorescence spectra. The absorption and fluorescence spectra of the wild type core complex (Ca-LH1-RC) from Tch. tepidum measured at temperatures 4.5 K and 295 K are shown in Fig. 1. The main absorption band with a peak at 916 nm at ambient temperature (938 nm at cryogenic temperature) and a spectral width (here and henceforth defined as fwhm) of 535 cm–1 (218 cm–1) corresponds to the lowest singlet electronic transition of tightly packed BChls in the B875 ring. The 32 BChl pigment chromophores forming the B875 ring are slightly paired. Strong excitonic couplings within and between the BChl dimers are considered to be the main reason of the large red shift of the B875 band relative to the Qy singlet absorption band of individual BChl pigments.18-24 As it was already mentioned in Introduction, an additional, specific to Tch. tepidum red shift of the B875 absorption spectrum is attributed to the binding of Ca2+ ions to the C-terminus of LH1 polypeptides.9,12 The weak absorption bands seen at 800 nm and 758 nm are usually associated with the pigment cofactors located in the RC complex - two accessory BChls and two bacteriopheophytins, respectively.11 We will see below that in our case this spectral region is also contributed by absorption of trace amounts of peripheral LH2 antenna complexes. The LH1 antenna fluorescence in the native Ca-LH1-RC complex, which originates from the lowest energy exciton polaron states,25,26 appears to peak at 932 nm at room temperature and at 957 nm at low temperature. A double structure of the fluorescence spectrum is clearly seen at low temperatures. Apart from the main “red” emission band there also is a weak “blue” band that peaks at 904 nm. At room temperature this band appears at 868 nm. As follows, we will provide



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multiple evidence that this fluorescence signal is related to impurity LH2 complexes, rather than being an intrinsic part of the LH1-RC core emission, as suggested in Ref. 3. Exchange of Ca2+ ions by Ba2+ ions results in blue-shifting of the B875 absorption band relative to its position in wild type core complexes.2 According to Kimura et al.9 this substitution is a slow process. In our experience, the extent of the blue shift depended on both the concentration of Ba2+ ions in the buffer and on the incubation time before freezing of the sample.

Figure 1. Absorption (blue) and fluorescence (red, excited at 407 nm) spectra of the native (Ca-LH1-RC) core complex from Tch. tepidum recorded at 4.5 (solid lines) and 295 K (dashed lines). The numbers label spectral positions in nanometers. Bold numbers 868 and 904 distinguish the positions of the impurity LH2 fluorescence bands at 4.5 and 295 K, respectively; bold B850 indicates the position of the weak B850 absorption band of LH2 complexes at low temperature. Inset shows expanded view of the Qy origin region of absorption and fluorescence spectra together with a Gaussian approximation (shaded) of the experimental distribution of the lowest energy exciton-polaron states (IDF, open rings) obtained by holeburning action spectroscopy at constant fluence of 1.2 J/cm2. The IDF distribution peaks at 951.3±0.4 nm and has a width (fwhm) of 117±20 cm–1.

As an example, demonstrated in Fig. 2 are the absorption spectra of two differently prepared Ba-LH1-RC samples. Sample (a) was incubated for ∼15 min in a buffer containing 100 8 ACS Paragon Plus Environment

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mM BaCl2, before adding glycerol and cooling down to 4.5 K. Sample (b) was treated similarly but in a twice less concentrated (50 mM BaCl2) buffer. The former treatment resulted in the B875 absorption band peaking at 907 nm and having fwhm of 354 cm–1. The latter, apparently less complete handling resulted in only slightly more red-shifted (peak at ~909 nm) but significantly broader (427 cm–1) absorption band. Comparison with the literature data9 suggests incomplete replacement of the cations in both samples.

Figure 2. Absorption (blue) and fluorescence (red, excited at 407 nm) spectra of two Ba-substituted (BaLH1-RC) core complexes (sample (a) solid line, sample (b) dashed line) recorded at 4.5 K. IDF shows the distribution of the lowest energy emitting states for sample (a). Experimental IDF (open rings) approximated by a Gaussian curve (shaded) is centered at 924.1±0.6 nm and has a fwhm of 198±30 cm–1.

The “blue” emission peaking at 904 nm appears significantly enhanced in the Basubstituted core complex. Still, the expected B850 absorption band, origin band of the “blue” fluorescence, appears only as an unspecified shoulder of the broad B875 absorption band. Absorption spectrum at around 800 nm shows two separate peaks, consistent with higher contamination level of the Ba-substituted sample with LH2 complexes, see below.



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The fluorescence spectra presented in Figs. 1 and 2 were obtained using non-selective excitation at 407 nm. This light directly hits carotenoid pigments, which along with BChls are present in the antenna complexes and rather effectively but non-selectively pass their excitations to the lower-energy LH1 and LH2 excitons. To better reveal the suspected sample heterogeneity, LH1 and LH2 antenna excitons should be more directly excited. This was achieved using 760 nm excitation for LH1 and 800 nm excitation for LH2. Typical results obtained at room and low temperatures for native and Ba-substituted core complexes are demonstrated in Fig. 3. It is well seen from Fig. 3 that relative contribution of the “blue” fluorescence varies significantly with the excitation wavelength. In agreement with the greater heterogeneity of the Ba-substituted samples, selectivity is better in native sample. Despite large variations of spectral intensity the position of the “blue” band (~ 868 nm at room temperature and ~904 nm at 6 K) are similar in both samples. This supports our provisional association of the “blue” fluorescence with contaminating LH2 complexes, an issue which will be further elaborated in two subsequent chapters.

Assessment of the “blue” and “red” emitting species by fluorescence excitation spectroscopy. The fluorescence excitation spectra allow revealing the heterogeneous content of the samples. Here we applied this technique for decisive proof of the LH2 origin of the “blue” emission bands. The measurements were performed both at room and low temperatures. However, because of better spectral resolution, only the results obtained at 6 K for two different samples are explicitly presented. The excitation spectra demonstrated in Fig. 4 were measured by recording the fluorescence successively in “blue” and “red” bands (corresponding spectral windows are denoted by crossed boxes). Recording in the former range allows identifying the origin of the “blue” emission, while that in the latter area, the source of the “red” emission. 10 ACS Paragon Plus Environment

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Figure 3. Fluorescence spectra of Ca-LH1-RC (left) and Ba-LH1-RC (right) complexes measured at 0.5 nm resolution either at ambient (RT, top panels) or cryogenic (6 K, bottom panels) temperatures using 760- (blue curves) and 800-nm (red curves) excitation.

The accordingly colored excitation spectra in Fig. 4 yield explicit answers. The red spectra with a single maximum around 940 or 908 nm in native or Ba-substituted samples, respectively, and characteristic long tail extending toward high energies can undoubtedly be assigned to LH1 complexes by similarity with main parts of the absorption spectra shown in Figs. 1 and 2. Missing contribution of the RC emission as well as of recombination luminescence under the present experimental conditions explains some of the discrepancies observed between the absorption and excitation spectra in the spectral range of 757 - 800 nm.11,15,27,28



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Figure 4. Normalized fluorescence excitation spectra of Ca-LH1-RC (A) and Ba-LH1-RC (B) complexes from Tch. tepidum at 6 K. The numbers label spectral positions of the excitation spectra maxima in nanometers. The fluorescence spectra non-resonantly excited at 804 nm are shown by shaded grey areas. Colored crossed boxes within the “blue” and “red” fluorescence bands specify spectral intervals where the colored excitation spectra (connected beads) were recorded. Colors of the boxes and excitation spectra are inter-related.

The excitation spectra recorded in the “blue” emission range appear very similar in case of native/Ba-substituted samples. There is an intense single band at 875/874 nm and a weaker split band around 800 nm with its two spectral components peaking at 792/793 and 805/806 nm. Similar spectra at cryogenic temperatures have been measured earlier for the LH2 complex from Tch. tepidum.29-31 This leaves no doubt that responsible for the “blue” fluorescence of core complexes are contaminating LH2 complexes.


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At room temperature (data not shown) when the fluorescence is detected at “blue” fluorescence range the fluorescence excitation peaks of both complexes appear at around 800 (no splitting) and 855 nm. Similar spectra have been observed in Refs.13,30 At “red” range registration, again, a LH1-like excitation spectrum is obtained that peaks at 914 nm or 895 nm in native or Ba-substituted samples, respectively.

Verification of the heterogeneous sample structure by spectrally resolved picosecond fluorescence decay. Fluorescence decay kinetics is a sensitive probe of sample heterogeneity. Therefore, we next survey the results of comparative wavelength-selective fluorescence decay measurements of native Ca-containing and Ba-substituted core complexes. The data collected over the conventional spectral ranges of “blue” and “red” fluorescence at ambient and cryogenic temperatures are summarized in Fig. 5 and Table 1. We once again specify that due to saturating excitation intensity the fluorescence quenching in this work is mainly due to photosynthetically inactive (photo-oxidized) RCs. The subject of excitation intensity dependent trapping will be deliberated in a separate publication. We begin with presenting data for the native sample at 6 K (Fig. 5A) because of better spectral selectivity obtained at low temperatures. In the red part of the spectrum (950–990 nm) that corresponds to emission of LH1 complexes the kinetics is well approximated by double exponential decay with the decay constants (lifetimes) of τ1 ≈ 278 ps (shown by red filled rings) and τ2 ≈ 760 ps (blue filled rings). Both the lifetimes and respective amplitudes show flat values over most part of the LH1 fluorescence band. The dominating shorter component (red open rings) with >80% relative amplitude supposedly reflects quenching of the antenna excitons by photo-oxidized RCs. Comparable lifetimes have been detected in various other purple bacterial core complexes.11,27,32-34 On similar basis (see Ref. 35 for an involved discussion of this issue), 13 ACS Paragon Plus Environment


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the longer lifetime component may be related to the core complexes that miss RCs. It is known11,25 that in isolated LH1 antenna complexes from Rba. spaeroides devoid of RCs the decay lifetime at low temperatures is ~1 ns. By separate measurements (data not shown) we verified that contribution of this component depended on the detergent concentration used. Increased detergent concentration led to the growth of the 1-ns component in the decay kinetics. At detergent concentration of 0.5% the contributions of long and short components practically equaled. The lifetime values of the components did not change during this exchange. It is thus likely that increasing number of core complexes became inactivated by detergent-induced removal of RCs. In the blue range of 890–920 nm, where mostly LH2 complexes emit, in contrast, a long (>1 ns) lifetime component prevails (~80%) over a short (~335 ps) lifetime component (red symbols). While the amplitude of the nanosecond component (see blue open rings) is practically constant, its decay time (blue filled rings) persistently increases toward shorter wavelengths, a tendency which is characteristic to exciton polarons in LH2 complexes.36,37 Origin of the short lifetime component of the “blue” fluorescence is not quite clear at this point. We only notice that its amplitude was extremely weak and also that the amplitude ratio of the long- and shortlifetime components did not change with increasing detergent concentration (up to 0.5%). It is thus unlikely that this component is due to some degradation product of either the LH2 or LH1RC complex.


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Figure 5. Spectral dependence of fluorescence lifetimes (τ1/2) and corresponding amplitudes (A1/2) in native Ca-LH1-RC (A, C) and Ba-substituted Ba-LH1-RC (B, D) core complexes recorded at 6 K (A, B) and at ambient temperature (C, D). The excitation wavelengths used are as follows: 850 nm (A), 820 nm (B), 830 nm (C) and 760 nm (D). Lines connecting experimental points are for leading the eye only. Shown on the background are grey-shaded experimental fluorescence spectra. In panels B-D, the spectra are deconvoluted to their LH2 (blue) and LH1 (red) antenna constituents based on the distribution of the respective A1 and A2 kinetic amplitudes.

Qualitatively very similar pattern of low-temperature kinetics was followed in case of Basubstituted core complexes (Fig. 5B). Yet the data were much more scrambled due to greater inhomogeneous broadening (see below), larger relative contribution of the LH2 emission as well as blue-shifted LH1 emission. The resulting, significantly enlarged spectral overlap between the “blue” and “red” spectra complicated their independent study. Sill, similar to native samples, in the “red” range just two lifetime components were revealed: a major (ca 80%) one with short lifetime of ~270 ps (indicated by red symbols) and a minor one with long lifetime of ~1 ns (blue 15 ACS Paragon Plus Environment


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symbols). In the “blue” range the nanosecond component with almost constant amplitude of ~90% (blue symbols) totally dominated the signal. Its value increased toward shorter wavelengths from 1.3 to 1.6 ns, again as in the native complex. Decay time of the minor component (~520 ps, red symbols) was somewhat longer than that found in native sample.

Table 1. Dominating fluorescence decay times (±10%) in the “blue” (LH2) and “red” (LH1) spectral ranges, as recorded at 6 K or at ambient temperature (in parenthesis). τ1 (ps) Ca-LH1-RC

τ2 (ps) Ba-LH1-RC

“Blue” (LH2) “Red” (LH1)

278 (234)

Ca-LH1-RC

Ba-LH1-RC

1170-1340 (1110)

~1250-1660 (1140)

268 (229)

The room-temperature kinetics presented in Figs. 5C and 5D is generally consistent with low-temperature data. In the native sample (Fig. 5C), the “red” fluorescence decays practically mono-exponentially with a short lifetime of ~230 ps (red symbols). This decay is ca 20% faster than observed at 6 K, a tendency found in other purple bacterial antenna complexes as well.11,37 The amplitude of the long (~1.1 ns) decay component (blue symbols) in this spectral range is less than 3%. Similar kinetic components were found in the “blue” emission range. However, now the nanosecond component (blue symbols) prevails over the short lifetime one, although less strongly than at low temperature (amplitude ~ 55%). Also its spectral dependence has vanished. This latter effect for LH2 complexes is long known.36,37 In the Ba-substituted complexes at room temperature (Fig. 5D) the overlap of LH2 and LH1 spectra is still greater and so is the 16 ACS Paragon Plus Environment

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contribution of the LH2 fluorescence in the “red” part of the spectrum. The kinetics in native and Ba-substituted complexes is otherwise rather similar. Apparent lack at room temperature of the lifetime component(s) corresponding to lone LH1 complexes (as found in low temperature spectra) awaits a short comment. Either this complex is indeed missing or its absence is an artefact of our data analysis. Although freezing is known to harm antenna complexes, in this particular case, we are inclined to believe the latter explanation. This is because a small but systematic decline of long-lifetime values in the LH1 part of the spectrum observed, see blue symbols in Figs. 5C and D. It appears possible that in this spectral range the kinetics may involve more than two decay components, as applied in current modelling.

Characterization of the native and Ca-substituted core complexes in terms of disorder broadening of the spectra and electron-phonon coupling strength. The position and inhomogeneous broadening of the fluorescence origin states can be determined by spectral holeburning action spectroscopy.18 In this spectroscopy, feasible only at very low temperatures, spectral holes are burned across the lowest-energy absorption band using constant burn fluence. The zero-phonon hole depths plotted as a function of burn wavelength/frequency provides an inhomogeneous distribution of the states, IDF. IDF for the lowest-energy absorption band of Ca-LH1-RC complexes is depicted by green rings in the inset of Fig. 1. The spectrum peaks at 951.3±0.4 nm and shows a width of 117±20 cm–1. It is worth noticing that in all core complexes this spectrum relates to just LH1 component of the complex, because hole-burning of RCs is relatively very inefficient process.38,39 Similar IDF widths between 108 and 128 cm–1 were observed for LH1 and LH1-RC complexes from other purple bacteria such as Rba. sphaeroides11,25,26 (see Table 2 for details). 17 ACS Paragon Plus Environment


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This shows that at least native Ca2+ ions do not impose additional disorder into the core antenna complex of Tch. tepidum. IDF for the Ba- substituted sample (a) is shown in Fig. 2. Centered at 924.1 nm it has a width of 198 cm–1, i.e., much larger than found for native complexes. In sample (b) (data not shown) it is greater still. We thus may conclude that random replacement of native Ca2+ ions with Ba2+ ions not only leads to a blue shift of the core complex spectra but also to their extra broadening. Careful reader might have noticed different apparent Stokes shifts between the maxima of IDF and fluorescence spectra in native and Ba-substituted core complexes. Specifically, the Stokes shifts are as follows: 5.7 nm in native sample; 11.9 nm in the Ba-substituted sample (a); 10.0 nm in the Ba-substituted sample (b). These different values may simplistically be taken as a sign for varying shapes of the underlying homogeneous spectra. To clarify this issue, difference FLN (∆FLN) spectra of the samples were studied at several wavelengths around the IDF peak. ∆FLN provides nearly homogeneously broadened fluorescence spectrum when recorded under low-fluence burning light.40-42 Figure 6 describes the measuring procedure as well as demonstrates the resulting ∆FLN spectra of the Ca-LH1-RC complex. Two different excitation wavelengths were applied, one close to the maximum of IDF and other at red side of the distribution, see the IDF in Fig. 7. The ∆FLN spectra shown by red color comprise a narrow ZPL and an accompanying broad PSB from the low energy side. These features of the homogeneously broadened spectrum allow evaluation of the effective electron-phonon coupling strength, as described in Materials and Methods part.


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Figure 6. Difference FLN spectra (red curves) of the Ca-LH1-RC complex excited at 951 nm (A) and 958 nm (B) at 4.5 K. Black and blue curves denote the corresponding pre- and post-burn FLN spectra recorded with fluencies of 1.8 mJ/cm2. The post-burn spectra were measured after the intermediate holeburning stage (hole-burning fluence of 47 mJ/cm2) at unchanged laser intensity and wavelength. Insets display expanded views of the ∆FLN spectra in relative wavenumber scale. Note that the width of the ZPL is determined by the experimental resolution of 6 cm–1.

Insets of Fig. 6 clearly demonstrate that ∆FLN spectra obtained at different excitation wavelengths do not generally offer identical results. The effective Huang–Rhys factors, Sph, evaluated according to Eq. (1) for the two spectra of Fig. 6 provided Sph = 1.7 at 951 nm excitation and 2.2 at 958 nm excitation. Not only are absolute values of these numbers similar to the ones found for other purple bacteria, but also is the significant increase of Sph with the excitation wavelength (Fig. 7).26,43-45



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Figure 7. The wavelength dependence of the effective Huang–Rhys factor (open rings) for the Ca-LH1RC complex on the background of the distribution of the lowest-energy exciton-polaron states, IDF (shaded), at 4.5 K. The line indicates quasi-linear increase of S. Arrow points the wavelength λ1/2=956.5 nm of half IDF intensity from the long wavelength side. The corresponding Sph(λ1/2) = 2.1.

The ∆FLN spectrum of Ba-substituted core complexes looks very similar to the spectrum of native complexes, apart from the much higher proportion of scattered laser light. The latter is plausibly caused by saturating the sample solution with BaCl2 salt. Similar also is the excitation wavelength dependent electron-phonon coupling. In the Ba-substituted sample (b), for instance, the effective Huang–Rhys factor changed from 1.9 to 2.4 in between 938 and 952 nm. Displayed in Fig. 8 is expanded view of PSBs in the ∆FLN spectra of selected core complexes of both sulfur (Tch. tepidum) and non-sulfur (Rba. sphaeroides and Rhodoblastus (R.) acidophilus) photosynthetic purple bacterial origin. The shapes of PSB of the Ca- and Bacontaining complexes of Tch. tepidum appear almost indistinguishable. Their spectral fine structure with three prominent frequencies of 27, 59, and 103 cm–1 does not visibly depend on the excitation wavelength (Fig. 6). Compared with complexes from Tch. tepidum, the complexes


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from non-sulfur bacteria either miss one of these frequencies (Rba. sphaeroides) or even have an additional frequency (R. acidophilus).

Figure 8. PSB-normalized ∆FLN spectra in relative wavenumber scale of Ca-LH1-RC, Ba-LH1-RC, LH1 complex from R. acidophilus, and LH1 and LH1-RC-PufX complexes from Rba. sphaeroides. Vertical lines label selected vibrational mode frequencies. The spectra measured at 4.5 K and using the resolution of 6 cm–1 were vertically shifted relative to each other for better visibility. Inset indicates the excitation wavelengths applied.

Origin of the above pseudo-local frequencies remains to be studied. At this point, however, it is quite clear that one cannot relate any one of them with the presence/absence of metal cations in the antenna protein structure. In accordance with the literature data,18 coupling to highfrequency intramolecular vibrational modes of BChl is comparatively very weak in all the species studied, see Table 2.



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Table 2. Characteristics of the LH1 complexes from Tch. tepidum and Rba. sphaeroides inferred from selective spectra.

IDF peak (nm)

IDF width (cm–1)

Sph(λ1/2)

Sviba

Reference

Ca-LH1-RC

951.3±0.4

117±20

2.1±0.2

Very weak

This work

b

924.1±0.6

198±30

Not available

Very weak

This work

937.0±0.6

~300

2.2±0.2

LH1 Rba. sphaeroides

885±0.4

119±10

1.8±0.2

0.09±0.02

Ref.43

LH1-RC-PufX Rba. sphaeroides

894.6±0.4

128±8

1.8±0.2

Very weak

Ref.11

Complex

Ba-LH1-RC

a

Total vibronic coupling strength; see Ref.43 for definition.

b

Data vary in dependence on the relative concentration of non-native Ba+2 ions. Presented are data for (top) sample (a) and (bottom) sample (b). See text for further explanations.

The various physical characteristics derived from selective spectra and associated with the LH1 component of core complexes are gathered into Table 2. For generality sake, Table 2 also includes data for the LH1-only mutant and native dimeric core complexes of Rba. sphaeroides. Since the Sph values in all the core complexes studied are very similar, the extra red-shift found in only one of them (i.e., in the Ca-containing core complex) is most likely not connected with dynamical properties of photosynthetic excitons. The data of Table 2 also reveal that the electron-phonon coupling in core complexes from sulfur bacteria such as Tch. tepidum is as strong as it is in core complexes from non-sulfur bacteria like Rba. sphaeroides. For one thing, this lifts a ground from association of the strong red shift of the Qy exciton band observed for the Ca-LH1-RC complex with any extra (compared with other core complexes) mixing of exciton and charge transfer states in this sample, as suggested in Ref. 5.


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SUMMARY AND CONCLUSIONS In the present work, we systematically studied and interpreted the spectral and kinetic properties of the native Ca-containing and laboratory-designed Ba-substituted LH1-RC core complexes of thermophilic photosynthetic purple bacterium Tch. tepidum. Availability of the near-atomic-resolution crystal structure of the complex and controlled substitution of native Ca2+ ions with similar but alien cations opened up the possibility to investigate various physicochemical aspects of the assembly of this unique membrane protein complex. Homogenous and inhomogeneous shapes of the LH1 antenna exciton spectra were evaluated using hole-burning and ∆FLN selective spectroscopy methods. These studies showed that (i) there is little to no effect of cation substitution on the dynamic (or kinetic) properties of the core excitons; (ii) both the effective electron-phonon coupling strength and its dependence on wavelength in the complexes from the sulfur purple bacterium Tch. tepidum is similar to those in the complexes from non-sulfur purple bacteria such as Rba. sphaeroides; (iii) the couplings to high-frequency BChl intramolecular vibrations are very weak in all antennae of photosynthetic purple bacteria. There is thus no reason to believe any special connection between the dynamical properties of excitons and the extra red-sift of the absorption/fluorescence spectra observed in the

Ca-containing

core

complex

of Tch.

tepidum.

Because

the

position

of

the

absorption/fluorescence spectrum of the Ba-LH1-RC complex from Tch. tepidum is rather comparable to this in other purple bacteria, we believe that the reason of the extra red-sift observed in Ca-LH1-RC complexes relies on subtle structural effects yet to be discovered. At this point, one might only speculate that an inclusion of large Ca2+ ions causes greater deformations of the protein lattice than addition of relatively smaller Ba2+ (or alike) ions.



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Heterogeneous composition of the samples due to residual LH2 antenna complexes was resolved using steady-state fluorescence excitation and picosecond time-resolved fluorescence spectroscopy. This allowed correction of some current misinterpretations of the Tch. tepidum core exciton spectra. A reasonable order-of-magnitude estimate for the amount of residual LH2 complexes in the samples studied is 1%. This level of purity is satisfactory for most studies, but as we have seen, not sufficient for the current laser spectroscopy measurements. To avoid the LH2 issue in future experiments, we are planning to employ newly designed core complexes from genetically LH2-deficient Tch. tepidum.46


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AUTHOR INFORMATION Corresponding author *E-mail: [email protected]

ORCID Margus Rätsep: 0000-0002-7052-450X Kõu Timpmann: 0000-0002-1865-0998 Arvi Freiberg: 0000-0003-1902-8444

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was partially supported by the Estonian Research Council (grant IUT02-28) (A.F., M.R. and K.T.) and the Australian Research Council Discovery Project (grant DP150103137) (A.F. and M.R.), and by Grants-in-aid for Scientific Research (B) (16H04174) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Z.-Y.W.-O. and T.K.). The authors credit Dr. Liina Kangur for valuable suggestions related to sample handling.



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