On Light-Induced Photoconversion of B800 Bacteriochlorophylls in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum.

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On Light-Induced Photoconversion of B800 Bacteriochlorophylls in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum Adam Kell, Mahboobe Jassas, Kirsty Hacking, Richard J. Cogdell, and Ryszard J Jankowiak J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06185 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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The Journal of Physical Chemistry

On Light-Induced Photoconversion of B800 Bacteriochlorophylls in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum Adam Kell,† Mahboobe Jassas,† Kirsty Hacking,‡ Richard J. Cogdell‡ and Ryszard Jankowiak*,†,§ †

Department of Chemistry and §Department of Physics, Kansas State University, Manhattan,

Kansas 66506, United States ‡

Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life

Sciences, University of Glasgow, Glasgow G12 8TA, Scotland

ABSTRACT The B800-850 LH2 antenna from the photosynthetic purple sulfur bacterium Allochromatium vinosum exhibits an unusual spectral splitting of the B800 absorption band; i.e., two bands are well-resolved at 5 K with maxima at 805 nm (B800R) and 792 nm (B800B). To provide more insight into the nature of the B800 Bacteriochlorophyll (BChl) a molecules, highresolution hole-burning (HB) spectroscopy is employed. Both white light illumination and selective laser excitations into B800R or B800B lead to B800R → B800B phototransformation. Selective excitation into B800B leads to uncorrelated excitation energy transfer (EET) to B800R and subsequent B800R → B800B phototransformation. The B800B → B800R EET time is 0.9 ±

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0.1 ps. Excitation at 808.4 nm (into the low-energy side of B800R) shows that the lower limit of B800R → B850 EET is about 2 ps, as the B800R → B800B phototransformation process could contribute to the corresponding zero-phonon hole width. The phototransformation of B800R leads to a ~200 cm-1 average blue-shift of transition energies, i.e., B800R changes into B800B. We argue that it is unlikely that B800-B850 excitonic interactions give rise to a splitting of the B800 band. We propose that the latter is caused by different protein conformations that can lead to both strong or weak hydrogen bond(s) between B800 pigments and the protein scaffolding. Temperature-dependent absorption spectra of B800, which revealed a well-defined isosbestic point, supports a two-site model, likely with strongly and weakly hydrogen-bonded B800 BChls. Thus, BChls contributing to B800R and B800B could differ in the position of the proton in the BChl carbonyl-protein hydrogen bond, i.e., proton dynamics along the hydrogen bond may well be the major mechanism of this phototransformation. However, the effective tunneling mass is likely larger than the proton mass.

1. INTRODUCTION In contrast to LH2 complexes from Rhodobacter (Rb.) sphaeroides,1 Rhodoblastus (Rh.) acidophilus (formerly known as Rhodopseudomonas acidophila),2 and Phaeospirillum (Ph.) molischianum (formerly Rhodospirillum molischianum),3 where single B800 peaks are observed, a spectral splitting of B800 was observed in LH2 from Allochromatium (Alc.) vinosum and Thermochromatium tepidum, both formerly known as Chromatium species.4-8 The origin of the unusual B800 peak observed in the LH2 complexes of Alc. vinosum is currently unknown, but several recent studies attempted to narrow the possibilities. Recent transient absorption spectroscopy measurements showed that intra-B800 energy transfer, from the blue-shifted B800B


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to the red-shifted B800R, proceeds within ~2 ps at 77 K.9 This fast excitation energy transfer (EET) rate suggested that both peaks originate from within a single LH2 complex, likely from two spectral forms of the monomeric B800 bacteriochlorophyll (BChl) a molecules. Carey et al.10 established that there is no additional BChl per heterodimer subunit as the BChl:carotenoid (Car) ratio is maintained at the standard ratio of 3 BChl:1 Car, as previously observed in many LH2 complexes of purple bacteria.2,3 Similarly to other purple photosynthetic bacteria and Alc. vinosum LH1 complexes,11 the different LH2 complex types of Alc. vinosum (B800-850, B800840 and B800-820) have all been found to contain multiple α and β polypeptides.10 As shown in Figure S1 of the Supporting Information, B800 is not only split at 5 K in the B800-850 complex (discussed in this work), but also in B800-840 and B800-820 complexes of Alc. vinosum. The heterogeneity mentioned above could explain the origin of the split B800 as the heterodimer subunits that form each LH2 complex are non-identical and, therefore, the binding environment of each B800 BChl a to the protein could vary; potentially producing two distinct B800 peaks. An alternative explanation suggested that the B800 molecules in the LH2 complexes from Alc. vinosum are excitonically coupled (V = 19–25 cm-1),12,13 and, in addition to B800-B850 excitonic interactions, give rise to a splitting of B800.14 Even though the X-ray structure of the Alc. vinosum LH2 protein is unknown, in ref 14 it was concluded that the B800 BChls in Alc. vinosum have a different organization than Rh. acidophilus based on room-temperature circular dichroism (CD) spectra. Under this assumption the 1.2 K fluorescence excitation spectrum was calculated with a dimerized B800 model including interactions between B800 and B850 BChls.14 However, the fluorescence excitation spectrum (obtained in a PVA film) was significantly altered by high-intensity probing light.15 Additionally, the LH2 complex from Ph. molischianum exhibits a similar room-temperature CD spectrum which was simulated by combined MD/QC


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calculations16 using the known X-ray structure,3 i.e., no dimerization of B800 BChls. A split B800 has not been observed for Ph. molischianum, so a connection between the roomtemperature CD and spectral split of B800 seems premature. Thus, the origin of these two bands is not clear and hole burning (HB) spectroscopy seems to be an ideal tool to test whether there is exciton coupling between B800 pigments and between B800 and B850 BChl a molecules via the shape of the resulting HB spectra and the presence or absence of correlated bleaching.17 In addition, HB spectroscopy can provide insight into dynamics and phototransformation processes if present. In both nonphotochemical (NPHB) and photochemical HB (PHB) spectra the product is reached via a tunneling process in the excited state, although in NPHB and PHB the local environment experiences rearrangement or the chromophore–protein system undergoes a chemical change, respectively.17 Thus, this work focuses on the possibility of proton tunneling and argues that BChls contributing to B800R and B800B might differ in the position of the proton in the BChl carbonyl-protein hydrogen bond. 2. MATERIALS AND METHODS 2.1. Isolation and Purification of LH2 Complexes from Alc. vinosum. Alc. vinosum strain D was grown anaerobically in high light (above 60-80 µmol s-1 m2) at 40 °C (HL40) in media containing either sodium sulfide or sodium thiosulfate as a reduced sulfur source. The growth of cultures and extraction/purification of LH2 complexes, as well as the experimental setup, are described in ref 10. Samples were then concentrated to 400 µL in order to obtain an optical density of ~1 for a final volume of 1 mL. 2.2. Experimental Setup. A Bruker HR125 Fourier transform spectrometer was used to measure low-temperature absorption and HB spectra. The white light intensity and spectral range were restricted for some measurements by adding colored and gray filters to the beam path


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before the sample holder. The tunable wavelengths came from a Coherent CR899-21 Ti:sapphire laser (line width of 0.07 cm-1) pumped by a Millenia 10s (Spectra-Physics) diode-pumped, solidstate laser at 532 nm. Power from the laser output was stabilized with a Laser Power Controller (Brockton Electro-Optics Corp.) and precisely set by a continuously adjustable neutral density filter. All experiments were performed inside an Oxford Instruments Optistat CF2 liquid helium cryostat. The glass-forming solution was 55:45 (v/v) glycerol:ethylene glycol. Samples were diluted 40:60 (v/v) with a buffer:glass solution. Sample temperature was read and controlled with a Mercury iTC temperature controller. 3. RESULTS 3.1. B800 Region Absorption and Persistent HB Spectra. This work focuses on highresolution, low-temperature B800 absorption and HB spectra. We emphasize that the main difference between Alc. vinosum LH2 and related LH2 antennas, measured at 5 K in a buffer/glycerol matrix under similar experimental conditions, is that the low-temperature B800 absorption of Alc. vinosum is split into two well-resolved peaks with maxima at 804.2 and 791.6 nm, referred to below as B800R and B800B, respectively. Curves a in both frames of Figure 1 are the 5 K absorption spectra in the B800 spectral region, while curves b are resonant HB spectra obtained at burn wavelengths (λB) of 808.4 (I = 100 mW cm-2, t = 70 s, f = 7 J cm-2) and 785.2 nm (I = 100 mW cm-2, t = 370 s, f = 37 J cm-2) for frames A and B, respectively. The laser fluence (f) is calculated as f = I · t, where I and t are the laser intensity and burn time, respectively. The experimental zero-phonon hole (ZPH) burned at 785.2 nm (blue) and its Lorentzian fit are shown in the inset of frame B. The ZPH width is independent of fluence up to 37 J cm-2. The ZPH homogeneous line width (i.e., ΓZPH/2) corresponds to a lower limit of 0.9 ± 0.1 ps for the B800B → B800R EET time. Note that


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excitation into B800B at λB = 785.2 nm bleaches the entire B800R via uncorrelated EET, as indicated by the pink curve, which perfectly fits B800R. The pink curve is the inverted and expanded fragment of the broad hole (curve b). Curves c (shown in both frames of Figure 1 for easy comparison) represent a typical nonresonant bleach (obtained with λB = 496.5 nm) whose broad shape is independent of laser excitation (and fluence). Such a bleach was also observed using white light illumination (I ~ 225 µW cm-2).15 Therefore, this bleach is referred to as a nonresonant bleach. The difference between measured curves b and c (in both frames) is shown at the bottom in both frames of Figure 1 as red curves (b – c). That is, curves b are contributed to by resonant and nonresonant photochemical holes. Resonant burning at λB = 808.4 nm with f = 7 J cm-2 (curve b in frame A) shows a narrower and blue shifted anti-hole in contrast to that observed in curve b (for λB = 785.2 nm) of frame B. This is due to partial photoselection of B800R pigments. However, the broad nonresonant contributions (described by curve c) observed in both curves b are very similar. These spectral characteristics are very different than those observed in LH2 complexes of non-sulfur purple bacteria.1 To display similar behavior to Alc. vinosum, the photoproduct of non-sulfur purple bacteria LH2 would lie near 12600 cm-1 as illustrated in Figure S2 in the Supporting Information (see the broad red arrow). Apparently, this is not the case, as in the LH2 complex of Rh. acidophilus the anti-hole lies in vicinity of λB clearly indicating a NPHB process.18 Thus, it appears that upon resonant excitation B800R BChls are photoconverted to B800B BChls by two independent processes, i.e., resonant and nonresonant HB. The entire photoproduct of B800R is distributed outside the inhomogeneous distribution of the B800R pigments, i.e., the photoproduct is identical to B800B, suggesting the presence of a PHB process. While resonant HB at λB = 808.4 nm is clearly selective, it appears that the resonant hole is superimposed on the


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broad nonresonant bleach (similar to the shape of curve c in Figure 1); vide infra. Note that the maximum of the site-distribution function for B800R chromophores is near 12400 cm-1 (806.5 nm) with a fwhm of 140 cm-1.15 3.2. Isosbestic Point and Thermal Hole Refilling. Frame A of Figure 2 shows the Qy absorption (curve a) and three persistent holes (curves b-d) burned near the B800R maximum (λB = 804.3 nm/12433 cm-1) read out with low-intensity probing white light (~50 µW cm-2), i.e., no probing white light-induced bleaching was observed at these experimental conditions. Curves bd in Figure 2 are obtained with increasing f = 1 (I = 40 mW cm-2; t = 25 s), 7 (I = 40 mW cm-2; t = 175 s), and 31 J cm-2 (I = 40 mW cm-2; t = 775 s), respectively. This excitation (vB = 12433 cm1

) also suggests that burning has two contributions, in agreement with data shown in Figure 1.

The latter is reflected via uncorrelated narrow and broad bleaches. Therefore, we suggest that selective excitation into B800R not only leads to B800R → B850 EET, but also to B800R → B800B photoconversion and holes within B800 are contributed to by both resonant and nonresonant bleaches. Interestingly, probing by low-intensity white light in a spectral range of 667-1000 nm leads to very small bleaching of B850 BChls (see arrow in left frame near 11500 cm-1/870 nm). Laser excitation of B800R BChls (under our experimental conditions) also leads to a very weak bleach of B850 BChls (see ref 15 for more details). The inset of frame B in Figure 2 shows the experimental ZPH from curve b (νB = 12433 cm1

) and its Lorentzian fit with fwhm of 7 ± 1 cm-1, corresponding to a 2.3 ± 0.5 ps lifetime (when

corrected for spectral resolution). The latter lifetime of ~2 ps is in good agreement with the B800R → B850 EET time obtained from recent time-resolved pump-probe data.9 Thus, the B800R → B800B phototransformation time has a lower limit of ~2 ps.


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Figure 3 shows thermal hole refilling of the HB spectrum (λB = 804.3 nm, I = 40 mW cm-2, t = 775 s, f = 31 J cm-2) as the temperature is raised from 5 to 80 K. After the HB spectrum is measured the temperature is raised while recording the absorption at 10 K intervals. By 70 K the narrow ZPH has refilled and the B800B anti-hole (photoproduct) has depleted. The absorption changes at 70-80 K are largely due to temperature broadening of B800R and B800B bands, although we cannot exclude that the contribution from the broad (nonresonant) PHB has been totally eliminated. Nevertheless, we suggest that the barrier height between B800R and B800B configurations is on the order of ~50 cm-1. For completeness, the temperature-dependent absorption spectra for B800R and B800B are shown in Figure S3 of the Supporting Information. Consistent with previous results for LH2 from various purple bacteria,19,20 B850 and B800 shift blue and red, respectively, with increasing temperature. 3.3. Two Types of PHB spectra. Curve a in Figure 4 is B800 absorption of the Alc. vinosum LH2 complex as shown above. Curve b corresponds to the experimental persistent hole (λB = 804.3 nm), which is also made up from two different bleaches. Namely, spectrum d (blue) in Figure 4 corresponds to the broad nonresonant contribution to curve b, while spectrum c (with a very narrow ZPH and ~200 cm-1 blue-shifted photoproduct) represents a selective resonant bleach. The latter was obtained by subtracting curve d from curve b. That is, also at this excitation wavelength the measured hole (curve b) has two contributions represented by curves c and d. Though both holes are assigned to PHB spectra, the broad bleach (curve d) appears to be nonresonant in nature (vide infra). The inset shows the ZPH with fwhm of ~7 ± 1 cm-1 and it Lorentzian fit. The resolution-corrected fwhm of 5 ± 1 cm-1 corresponds to a lifetime of 2.3 ± 0.5 ps. 4. DISCUSSION


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4.1. B800 Absorption and Persistent HB Spectra. Data shown above suggest that proton dynamics is likely responsible for the shapes of HB spectra, as indicated by the large (~200 cm-1) blue-shifted photoproduct in both resonant and nonresonant bleaches. The large photoproduct shift suggests that the major HB mechanism in Alc. vinosum is photochemical in nature. Recall that PHB means that light affects the chromophore itself, whereas in NPHB the matrix surrounding the chromophore is affected.17 HB spectra excited at λB = 808.4 nm (i.e., resonant with B800R) reveal a selective photoconversion for which a narrower photoproduct is distributed within B800B for the resonant contribution (compare curve b and the b – c difference curve in Figure 1A). The blue-shifted anti-hole in curve c (Figure 1A) is due to additional broad nonresonant bleach, whose origin is discussed below. The same broad bleach, i.e., phototransformation, is observed by high-intensity white light (I > 225 µW cm-2) illumination and is independent of laser excitation wavelength.15 Thus, the B800 pigments in Alc. vinosum have a very different HB mechanism(s) than other LH2 complexes, including Rb. sphaeroides1 and Rh. acidophilus.18 The width of the ZPH (Figures 2 and 4) is 7 ± 1 cm-1 and corresponds to a (resolution corrected) lifetime of 2.3 ± 0.5 ps. We note that the ~2 ps time may not be solely due to B800R → B850 EET, as a small contribution to this ZPH from the B800R → B800B phototransformation time cannot be excluded. Thus, the ~2 ps time reflects the lower limit of the B800R → B800B phototransformation process. However, bleaching of B850 chromophores under the experimental conditions of this work is very small, i.e., the B850 bleach shows very shallow hole depth (~0.1%; see Figure 2A). The latter is in part due to a restricted spectral range of probing white light which does not cover the Car absorption region. We have shown previously that the bleach of B850 is large when: i) much larger white light intensity is used to measure absorption spectra


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(i.e., white light induced bleach of the B850 BChls), and ii) when probing white light (or selective laser excitation) excite Cars.15 This indicates that Car → B850 EET is very efficient. Before we discuss the nature of B800 molecules in more detail, we emphasize that the intensity ratio of B800R/B800B in absorption spectra reported in this work is significantly larger in comparison with the ratio observed in the fluorescence excitation spectra reported in ref 14, as the fluorescence excitation spectra were measured with significantly larger intensity white light.15 The latter led to a large B800R → B800B phototransformation. The shapes of HB spectra shown in Figures 1, 2 and 4 are not consistent with a model of B800 excitonic dimers.14 If such a scenario existed, then HB spectra would exhibit an excitonic response (bleach) of B800B when B800R is bleached. However, analysis of the data shown in this work and ref 15 does not indicate any such excitonic response. The phototransformation observed in this work suggests that B800R BChl a molecules have a strong hydrogen bond(s) with the surrounding protein, which could explain the ~200 cm-1 B800R red shift in comparison with B800B. This assertion is based on the apparent photochemical nature of the HB mechanism revealed by the large blue-shifted photoproduct. That is, a BChl a Qy transition energy shift of 200 cm-1 is consistent with previously reported data on the effects of hydrogen bonding.21-23 Similar shifts are observed in both resonant and nonresonant bleaches. Since the structure of this LH2 protein complex is still unknown one cannot entirely exclude that variation in BChl-protein interactions also contributes (at least in part) to the above-observed spectral shift. However, we will argue below that a twosite model with strongly and weakly hydrogen-bonded B800 BChls provides a reasonable explanation of our data. 4.2. Phototransformation. The question is whether the very narrow resonant bleaches and the broad bleaches (with corresponding broad holes and broad photoproduct distributions


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assigned above to nonresonant bleaching) in Figures 1, 2 and 4 originate from the same process. Data shown in Figure 4 reveal that the narrow ZPH and the broad bleaches have similar blueshifts of the corresponding photoproducts. However, only the nonresonant broad symmetric bleach and its corresponding photoproduct are independent of laser excitation (vide supra). Thus, these two types of bleaches must correspond to two different processes. The first, i.e., the narrow bleaches in Figure 1A (curve b-c) and Figure 4 (curve c) are assigned to the selective resonant PHB spectrum while the second, i.e., the broad bleaches (see curves c and d in Figures 1A and 4, respectively), must be induced by “nonresonant” changes whose origin is not well understood at this time. Likely, the latter is due to dissipation of a fraction of the absorbed energy leading to nonresonant B800R → B800B phototransformation. This is supported by data demonstrating that a phototransformation of B800 molecules occurs by both blue and red excitations as well as high-intensity white light illumination.15 Thus, it is feasible that this behavior is due to a low fluorescence quantum yield, possibly caused by the presence of a dark state (e.g., charge-transfer states) within B850 and its mixing with low-energy Frenkel exciton states.24,25 Such a scenario could lead to a dissipation of a large fraction of absorbed energy, resulting in excitation of nuclear motions of the protein matrix. Such motions (on relaxation back to thermal equilibrium) could induce conformational changes that in turn could induce the observed proton dynamics. The latter could change transition energies of the B800R BChls, i.e., lead to nonselective B800R → B800B phototransformation. The shapes of the holes shown in Figures 1 and 4 support the above assignment, indicating that the two bleaches discussed above are independent. For example, curve c (c = b – d) in Figure 4 reveals the shape of the selective resonant PHB spectrum. Finally, the difference spectrum (b – c) in Figure 1B clearly illustrates the uncorrelated B800B →B800R EET and subsequent B800R → B800B phototransformation.


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4.3. Proton Dynamics. We cannot reliably calculate the tunneling rate as we do not know the tunneling parameter λ (and its distribution) nor the effective tunneling mass. However, λ ~ 10 (as suggested in previous work18,26) and a barrier of 50 cm-1 yields md2 = 2.2 × 10-44 kg m2. For a reaction coordinate displacement of ~1 Å the effective tunneling mass is 34 times larger than the mass of a single proton. Still, one cannot exclude the possibility that a larger number of particles are connected with the hydrogen bond rearrangement. Figure 5 shows potential B800 BChl-protein hydrogen bonds for Ph. molischianum. There is a likely hydrogen bond between the 2-acetyl-carbonyl of the BChl and the hydroxyl group of threonine 23. The mass of two oxygen molecules and one proton is approximately 33 times the proton mass, and is consistent with the effective mass found above. Larger effective tunneling masses (due to concerted motion of several groups of atoms) were also suggested to be present in NPHB of the CP43 antenna complex.26 Therefore, further work will be needed to sort out what this means in terms of what entities are doing the tunneling during hydrogen bond rearrangement in PHB process in Alc. vinosum. Nevertheless, we suggest that the large shift of the anti-hole (200 cm-1) supports the assignment of the bleach to a PHB process where proton dynamics leads to a rearrangement of hydrogen bond(s). For example, BChls contributing to B800R and B800B could differ in the proton position in the BChl carbonyl-protein hydrogen bond. If this is the case, then the B800R and B800B BChls in this particular LH2 complex could have strong and weak hydrogen bonds due to different alternate forms of β1/β2 apoproteins.10 The latter is feasible, as it was shown that B800 molecules in LH2 complexes do have hydrogen bonds to the β apoprotein3,27,28 (see Figure 5) and a model including charged amino acid side-chains with hydrogen bonding was able to fit the absorption and CD spectral characteristics of LH2.29 In Rh. acidophilus, it has been shown


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that displacement of the Arg20 proton towards the B800 BChl decreases the excitation energy.30 While this scenario is not energetically favorable for Rh. acidophilus LH2, the potential energy surface for Alc. vinosum likely differs and such effects (displacement of the proton towards BChl) may be more prevalent. Additionally, there is a diffusion pathway for a water molecule into the B800 binding pocket.31 It is well known that the strength of hydrogen bonding, which governs proton dynamics, leads to a double minimum potential along the reaction coordinate.32,33 In the case of strong hydrogen bonding there is a low-energy barrier allowing for efficient proton tunneling. Such tunneling is similar to the NPHB mechanism,17 however, in the case of B800R molecules the photoconversion most likely involves tunneling between alternate configurations of the BChlprotein hydrogen bond(s). Thus, the observed B800R → B800B photoconversion might lead to very complex BChl-protein rearrangement(s) which results in an average change of BChl transition energy of ~200 cm-1. Interestingly, the resonant hole width obtained for laser excitation at 804.3 nm (near the B800R peak) reveals a lifetime of ~2 ps, in agreement with the B800R → B850 EET time from recent time-resolved pump-probe data.9 However, very small bleaching of the B850 pigments (for both λB = 804.3 and 808.4 nm) suggests that the hole width might be also contributed to by laser-induced B800R → B800B phototransformation. The data presented in this work also indicate that B800B molecules have a major deactivation pathway at 5 K via B800B → B800R EET and subsequent B800R → B800B phototransformation, and (at least for 5 K) do not reflect significant bleach caused by efficient B800B → B850 EET, although EET from B800 → B850 definitely occurs.9,15 We reemphasize that Cars transfer excitation energy very efficiently to the B850 pigments at 77 K.9 Thus, the rate of B800 → B850 EET should be measured as a function


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of temperature which, unfortunately, cannot be addressed by HB spectroscopy. The former, however, could be easily assessed by time-resolved spectroscopy since this rate is temperature dependent in other LH2 complexes.34 Temperature could possibly play a major role in the observed dynamics for Alc. vinosum, as the above-mentioned pump-probe data showed that 40% of excitation energy is transferred from B800B to B800R while the remaining 60% is transferred to B850,9 indicating that EET to B850 BChls is more efficient at higher temperatures. 5. CONCLUSIONS We suggest that LH2 of Alc. vinosum contains B800 BChls with strong and weak hydrogen bonds to the protein, with different protein environments near BChls contributing to B800R and B800B. Therefore, both selective laser-induced excitations and high-intensity broad white light illumination may lead to B800R → B800B phototransformation in the excited state. Selective excitation into B800B mostly results in uncorrelated EET to B800R and subsequent B800R → B800B phototransformation. B800B → B800R EET occurs in 0.9 ± 0.1 ps, similar to the 2 ps time observed for 77 K pump-probe data.9 Tunneling of protons along the hydrogen bond is likely the mechanism of photoconversion of pigments contributing to B800R, leading to a ~200 cm-1 blue shift of transition energies. Thus, BChls contributing to B800R and B800B most likely differ only in the position of the proton in the BChl carbonyl-protein hydrogen bond, reflecting structural heterogeneity. Excitation into the low-energy side of B800R, with a small bleach of B850 chromophoes,15 suggests that the observed 2 ps lifetime is contributed to by both B800R → B850 EET and B800R → B800B phototransformation processes. Although we believe that tunneling is responsible for the PHB discussed in this work, the distribution of the tunneling parameter and the effective tunneling mass are unknown at the present time. Although with hydrogen bond rearrangements the tunneling particles can be identified as protons, the effective tunneling mass


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can be larger than the proton mass, as in PHB the chromophore–protein system undergoes a chemical change. The latter prevents reliable calculation of the tunneling rate. Nevertheless, future high-resolution measurements of spectral diffusion in the dark (hole-broadening), hole refilling and thermal cycling experiments (i.e., studies of back reactions), including measurements of hole-growth kinetics (to provide insight on the tunneling parameters), should allow for the development of a model for the protein energy landscape and protein dynamics in this interesting LH2 complex. Such studies (for example, see refs 35 and 36) should provide more insight into the “intrinsic” (host matrix) and “extrinsic” (guest-host) processes related to the two- and/or multi-level system present in the protein energy landscape for these light harvesting complexes, complementing results obtained by single photosynthetic complex studies.37-41 FIGURES

Figure 1. Frame A: Curve a is the 5 K absorption of Alc. vinosum LH2 in the B800 spectral region. Curve b is a HB spectrum for λB = 808.4 nm (I = 100 mW cm-2, t = 70 s, f = 7 J cm-2). Curve c (λB = 496.5 nm) corresponds to the nonresonant bleach. The difference spectrum (red curve) between curves b and c shows the resonant bleach with the ~200 cm-1 blue-shifted


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photoproduct. Frame B: Curve b is the HB spectrum obtained for λB = 785.2 nm (I = 100 mW cm-2, t = 370 s, f = 37 J cm-2). Curve c is from frame A and is shown here for easy comparison. The difference between curves b and c is shown below (red curve (b – c)) and illustrates the uncorrelated B800B → B800R EET. The pink curve overlapping with B800R of the 5 K absorption (curve a) is a fragment of the inverted (and expanded) curve b. The inset shows a Lorentzian fit (dashed red curve) of the experimental ZPH of curve b obtained with 1 cm-1 spectral resolution and low fluence (hole depth ~ 4%).

Figure 2. Frame A: Qy absorption bands and f-dependent persistent holes (curves b-d) obtained with λB = 804.3 nm (12433 cm-1), I = 40 mW cm-2, and t = 25 s (f = 1 J cm-2), t = 175 s (f = 7 J cm-2), and t = 775 s (f = 31 J cm-2), respectively. Note a small bleach of the B850 BChls (see arrow). Frame B: Expanded HB spectra (curves b-d) clearly reveal an isosbestic point (red arrow). The inset shows the Lorentzian fit (black curve) along with the low-fluence (f = 1 J/cm-2) experimental ZPH of curve b (hole depth ~ 2% with fwhm = 7 ± 1 cm-1).


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Figure 3. 5 K pre-burn absorption (black) spectrum and temperature-dependent (10-80 K) absorption difference spectra (∆ absorption) measured at 10 K intervals. Temperature-dependent spectra are measured after burning at λB = 804.3 nm/12433 cm-1 (I = 40 mW cm-2, t = 775 s, f = 31 J cm-2). All ∆ absorption spectra, in comparison with the absorption spectrum, are multiplied by a factor of ten.

Figure 4. Spectra a and b are the 5 K B800 absorption and HB (λB = 804.3 nm, I = 40 mW cm-2, t = 775 s, f = 31 J cm-2) spectra, respectively, obtained for the sample in protonated glass solution. Curve b (red) is contributed to by the resonant (curve c) and nonresonant (curve d) photochemical holes. The inset shows the ZPH (cyan curve) from curve c and its Lorentzian fit (red curve).


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Figure 5. Dashed lines indicate likely hydrogen bonds for B800 pigments in Ph. molischianum.3,31,37

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. 5 K absorption spectra for B800-850, B800-840 and B800-820 LH2 complexes, HB spectra (λB = 807.5 nm) with various irradiation doses for Rh. acidophilus LH2 adopted from ref 18 and temperature-dependent absorption spectra of Alc. vinosum B800. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: 785-532-6785 ORCID Ryszard Jankowiak: 0000-0003-3302-9322 Author Contributions


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LH2 samples were prepared by K.H. and R.J.C. Experiments were performed by A.K. and M.J. The manuscript was written by R.J. with the help of A.K. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award Number DE-SC-0006678 (to R.J.). R.J.C. and K.H. acknowledge support from the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award Number DE-SC-0001035. The authors also acknowledge Dr. Khem Acharya and Ben Douglas for help in the early stages of this project. R.J. acknowledges useful discussion with Drs. Darius Abramavicius and Olga Rancova (Vilnius University, Lithuania). REFERENCES (1) Reddy, N. R. S.; Wu, H.-M.; Jankowiak, R.; Picorel, R.; Cogdell, R. J.; Small, G. J. High Pressure Studies of Energy Transfer and Strongly Coupled Bacteriochlorophyll Dimers in Photosynthetic Protein Complexes. Photosynth. Res. 1996, 48, 277–289. (2) Cogdell, R. J.; Issacs, N. W.; Freer, A. A.; Arrelano, J.; Howard, T. D.; Papiz, M. Z.; Hawthornthwaite-Lawless, A. M.; Prince, S. The Structure and Function of the LH2 (B800–850) Complex from the Purple Photosynthetic Bacterium Rhodopseudomonas acidophila Strain 10050. Prog. Biophys. Mol. Biol. 1997, 68, 1–27.


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The origin of the split B800 absorption peak in the LH2 complexes from Allochromatium vinosum.

Metabolomic profiling of the purple sulfur bacterium Allochromatium vinosum during growth on different reduced sulfur compounds and malate.

A comparative quantitative proteomic study identifies new proteins relevant for sulfur oxidation in the purple sulfur bacterium Allochromatium vinosum.

Antenna organization in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum.

B800-B850 coherence correlates with energy transfer rates in the LH2 complex of photosynthetic purple bacteria.

Stark absorption spectroscopy on the carotenoids bound to B800-820 and B800-850 type LH2 complexes from a purple photosynthetic bacterium, Phaeospirillum molischianum strain DSM120.

The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis.

Spectral heterogeneity and carotenoid-to-bacteriochlorophyll energy transfer in LH2 light-harvesting complexes from Allochromatium vinosum.

New proteins involved in sulfur trafficking in the cytoplasm of Allochromatium vinosum.

Characterization of the quinones in purple sulfur bacterium Thermochromatium tepidum.

The amino acid sequence of cytochrome c' from the purple sulphur bacterium Chromatium vinosum.

α pairs.

Interaction of HydSL hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina BBS with methyl viologen and positively charged polypeptides.

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c' from the thermophilic purple sulfur bacterium Thermochromatium tepidum.

Revised Genome Sequence of the Purple Photosynthetic Bacterium Blastochloris viridis.

Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

Removal of H2S by the purple sulphur bacterium Ectothiorhodospira shaposhnikovii.

Singlet-triplet fission of carotenoid excitation in light-harvesting LH2 complexes of purple phototrophic bacteria.

Dimeric carotenoid interaction in the light-harvesting antenna of purple phototrophic bacteria.

Superoxide dismutase from an anaerobic photosynthetic bacterium, Chromatium vinosum.

Production, crystallization and preliminary crystallographic analysis of Allochromatium vinosum thiosulfate dehydrogenase TsdA, an unusual acidophilic c-type cytochrome.

Proteolytic modifications of the B800-860 complex of the photosynthetic bacterium Rhodocyclus tenuis: Structural and spectral effects.

Studies on the light-harvesting complexes from the thermotolerant purple bacterium Rhodopseudomonas cryptolactis.

EPR and optical spectroscopic properties of the electron carrier intermediate between the reaction center bacteriochlorophylls and the primary acceptor in Chromatium vinosum.