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Photophysical Properties of the Excited States of Bacteriochlorophyll f in Solvents and in Chlorosomes Dariusz M. Niedzwiedzki,† Gregory S. Orf,†,‡ Marcus Tank,§ Kajetan Vogl,§ Donald A. Bryant,§,∥ and Robert E. Blankenship*,†,‡ †

Photosynthetic Antenna Research Center, ‡Departments of Biology and Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States § Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ∥ Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States S Supporting Information *

ABSTRACT: Bacteriochlorophyll f (BChl f) is a photosynthetic pigment predicted nearly 40 years ago as a fourth potential member of the Chlorobium chlorophyll family (BChl c, d, and e). However, this pigment still has not been found in a naturally occurring organism. BChl c, d, and e are utilized by anoxygenic green photosynthetic bacteria for assembly of chlorosomeslarge light-harvesting complexes that allow those organisms to survive in habitats with extremely low light intensities. Recently, using genetic methods on two different strains of Chlorobaculum limnaeum that naturally produce BChl e, two research groups produced mutants that synthesize BChl f and assemble it into chlorosomes. In this study, we present detailed investigations on spectral and dynamic characteristics of singlet excited and triplet states of BChl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The studies were performed on isolated BChl f in various solvents, at different temperatures, and on BChl f-containing chlorosomes in order to uncover any unusual or unfavorable properties that stand behind the lack of appearance of this pigment in natural environments.

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INTRODUCTION Chlorophylls and bacteriochlorophylls ((B)Chls) are the most abundant natural photosynthetic pigments employed in several important roles in the process of photosynthesis, including light absorption, primary charge separation in reaction centers, and excitation transfer.1,2 The basic spectroscopic properties of (B)Chl’s can be deduced from the “four-orbital” model of Gouterman.3,4 The model assumes that the major bands visible in the spectrum of electronic absorption are associated with π → π* electronic transitions that derive from electron promotions involving the two highest occupied molecular orbitals (HOMOs) and the two lowest unoccupied molecular orbitals (LUMOs). The resulting electronic transitions are polarized along either the x- or y-axis of the macrocycle (Figure 1) and are commonly referred to as the Qy, Qx, By, and Bx bands.3−6 The B transitions appearing in the UV (350−470 nm) range typically strongly overlap and are usually described as the Soret band. The Qy band can span the spectral region from 600 to 800 nm, depending on the degree of macrocycle saturation and the presence of specific side chain modifications. The Qx band appears in the 500−600 nm range, is the least intense of all of the transitions, and in some cases can be very © 2014 American Chemical Society

difficult to distinguish from Qy vibronic overtones. (B)Chl’s are divided into three subgroups characterized by the degree of unsaturation of the macrocycle: porphyrin-type (fully unsaturated macrocycle), chlorin-type (single bond between C-17 and C-18 carbons in the macrocycle), and bacteriochlorin-like (single bonds between C-7 and C-8 as well as C-17 and C-18 carbons in the macrocycle). The three representatives of the chlorin-like bacteriochlorophyll (BChl) subfamily called BChl c, d, and e (Figure 1) are employed by green sulfur photosynthetic bacteria (Chlorobiaceae) as primary photosynthetic pigments and assembled into large antenna complexes called chlorosomes. An individual chlorosome is usually composed of 100 000 to 250 000 selfassembled but non-covalently bound BChl molecules enclosed in a lipid monolayer envelope. The chlorosome also contains carotenoids that enhance spectral coverage and quench BChl triplet states, quinones to control environmental redox state and CsmA baseplate protein, as well as other proteins all Received: September 23, 2013 Revised: December 6, 2013 Published: January 10, 2014 2295

dx.doi.org/10.1021/jp409495m | J. Phys. Chem. B 2014, 118, 2295−2305

The Journal of Physical Chemistry B

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A change in the −R20 substituent from −CH3 in BChl e to −H in BChl f leads to distinct changes in ground-state electronic absorption. As can be seen in pyridine (Figure 1), excited states of BChl f are destabilized and shifted toward higher energy by 340 cm−1 with respect to BChl e. This corresponds to a Qy band shift from 660 nm (BChl e) to 645 nm (BChl f) and a Soret band shift from 476 nm (BChl e) to 469 nm (BChl f). Practically the same situation is observed if both BChl’s are assembled into chlorosomes. The Qy band of BChl e-containing chlorosomes appears at 721 nm, while in BChl f-containing chlorosomes Qy peaks at 705 nm15 corresponding to an energy difference of 310 cm−1 between them. Preliminary studies of BChl f-containing chlorosomes demonstrated lower energy transfer efficiency, and the bchU mutant grew slower compared to the parent strain utilizing BChl e.15 Thus, from an energetic point of view, production of BChl f and assembly into chlorosomal macro-aggregates seemed to be unfavorable, which suggested that BChl f may not occur naturally because it has been selected against during evolution.15 Subsequent spectroscopic studies on BChl e and f chlorosomes performed at room temperature and at 77 K led to the proposal that the main reason for the decreased performance of BChl f chlorosomes may be related to a significant decrease (2×) of spectral overlap between donor fluorescence (BChl f aggregate) and acceptor absorption (baseplate-bound BChl a), which corresponds to ∼40% decrease in energy transfer compared to the BChl e−BChl a pair.17 Although the observed differences between chlorosomes containing BChl e and f may account for the slow growth rate of BChl f-containing organisms, it does not give a definitive answer to why BChl f has apparently been eliminated from the pool of naturally occurring (B)Chl’s. We have performed more advanced and detailed spectroscopic studies on BChl f in various environments (solvents, chlorosome) and temperatures in order to reveal any unique properties that may differentiate it from other (B)Chl’s. We present studies on BChl f at room and cryogenic temperatures in hexa- and penta-coordinating organic solvents and in isolated chlorosomes. These include measurements of the spectral and temporal characteristics of singlet and triplet excited states by use of femtosecond and nanosecond time-resolved absorption and picosecond time-resolved fluorescence spectroscopies.

Figure 1. Chemical structures of BChl c, d, e, and f and their steadystate absorption spectra taken in pyridine at room temperature. The spectra were normalized at the maximum of the Soret band for better comparability. Spectra of BChl c, d, and e were taken from Niedzwiedzki et al.11

localized in the chlorosome envelope. The CsmA baseplate protein is part of a BChl a−protein complex serving as an energy transfer interface and structural bridge between the chlorosome and the FMO protein, which subsequently transfers excitations to the reaction center, which is embedded in the cytoplasmic membrane.7−10 The unusual structure of these antenna complexes allows green phototrophic bacteria to survive in habitats with extremely low light intensities, inaccessible for any other photosynthetic organisms; e.g., some Chlorobiaceae representatives were found to grow phototrophically in the Black Sea at depths ∼100 m below the surface at the astonishingly low light intensity of ∼0.002 μmol of photons m−2 s−1.12 BChl c, d, and e differ only at their C-7 and C-20 positions, which influence their spectroscopic properties (Figure 1).11 On the basis of analogy of the differences of these substituents within this pigment family, it was possible to predict the fourth member of the group, which was named BChl f. However, since predicting BChl f nearly 40 years ago, this pigment has not yet been found in nature,13 even though it was synthesized in vitro by modifying Chl b.14 Recently, two research groups independently constructed bchU mutants of two different strains of Chlorobaculum limnaeum that naturally produce BChl e and thereby constructed strains that synthesize BChl f. Moreover, both bacterial strains assemble BChl f into chlorosomes and are capable of photoautotrophic growth.15,16

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MATERIALS AND METHODS Mutagenesis, Culture Growth, and Chlorosome Isolation. Growth conditions for wild-type (WT) Cba. limnaeum cells (BChl e-containing) and the construction and growth of a bchU mutant strain (BChl f-containing) were previously described.15 Chlorosomes were isolated essentially as previously described.15 Briefly, cells were harvested via centrifugation, resuspended in an isolation buffer (10 mM Tris−HCl, pH 7.5, 2.0 M NaSCN, 5.0 mM EDTA, 1.0 mM PMSF, 2.0 mM DTT, 3 mg of lysozyme/1 mL of solution), and incubated at room temperature for 0.5 h. Afterward, the cells were mechanically disrupted using a chilled French press operated at 138 MPa. Large cell debris and unbroken cells were removed by centrifugation (10 000 × g for 20 min). The membranes and chlorosomes in the resulting supernatant were concentrated by ultracentrifugation (220 000 × g for 2 h) and resuspended in a minimal volume of 10 mM Tris−HCl buffer, pH 7.5. The chlorosomes were separated from membranes on continuous sucrose density gradients (10−53% linear gradients prepared in isolation buffer) by ultracentrifugation (220 000 × 2296



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g for 18 h) at 4 °C. The chlorosome layer was collected, washed twice with 4 volumes of phosphate buffered saline (10 mM potassium phosphate, pH 7.2, 150 mM NaCl), and pelleted by ultracentrifugation (220 00 × g for 1.5 h). The isolated chlorosomes were resuspended in 1−2 mL of the same phosphate buffer containing 1.0 mM PMSF and 2.0 mM DTT and stored at 4 °C until required. Isolation of BChl f . BChl f was isolated from purified chlorosomes essentially as follows: Chlorosomes containing BChl f were pelleted by ultracentrifugation (220 000 × g for 1.5 h). After removal of the supernatant, pigments were extracted via the addition of 500 μL of HPLC-grade 7:2 acetone:methanol (v/v). The pigment solution was transferred to a microcentrifuge tube, vortexed thoroughly, and centrifuged (13 000 × g for 5 min). Aliquots (100 μL) of the clear, green supernatant were directly injected into an Agilent Series 1100 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a reversed-phase Zorbax C-18 column (4.6 × 250 mm2, Agilent Technologies Inc., Santa Clara, CA, USA) regulated at 20 °C. Pigments were eluted with 94:6 methanol:water (v/v) pumped at a rate of 1.5 mL min−1. Because there is no reason to believe that BChl f homologues have different biophysical properties (homologues differ in alkyl length at the variable C-8 and/or C-12 positions), three main BChl f peaks were combined and studies were performed on a homologous mixture.15 The pooled pigment fraction was dried under a stream of nitrogen gas and stored under nitrogen in the dark at −20 °C until needed. Determination of Molar Extinction Coefficient. The molar extinction coefficient of BChl f in acetonitrile (ACN) was determined on the basis of the concentration of Mg in the sample. The BChl f sample was collected from HPLC, using acetonitrile as the mobile phase, and its volume was adjusted to 1 mL having an absorbance of 0.37 at the maximum of the Qy band (636 nm) at 1 cm path and then dried with nitrogen. The concentration of Mg was measured using a PerkinElmer ELAN DRC II inductively coupled plasma mass spectrometer (ICPMS) that was first calibrated with Mg standards of 1, 10, 50, 100, and 200 μg/L. Sample preparation involved adding 200 μL of concentrated aqua regia (1:3 mixture of HNO3 and HCL) into the vial with dried pigment and allowing them to react for 24 h until digestion was completed (solutions were clear and no particle settling). The completely digested samples were diluted up to 0.5 mL and again diluted by a factor of 15 before ICP-MS analysis. The same procedure was applied for the vial that initially contained 1 mL of pure acetonitrile eluted for the HPLC system (also dried before ICP-MS). The concentration of Mg in the BChl f sample was corrected for background coming from the solvent used in HPLC (Table S1, Supporting Information). The molar extinction coefficient, ε, was calculated on the basis of the Beer−Lambert law, ελnm = Absλnm·24.30/CMg (μg/mL) × 1000, where Absλnm is the absorbance measured at a chosen wavelength, 24.30 is the mass of a Mg atom, and CMg is the concentration of Mg atoms obtained from ICP-MS. For acetonitrile, ε636 = 31 000 L mol−1 cm−1 with an uncertainty of 5%. On the basis of comparative measurements of absorption of the same amount for BChl f in ACN, pyridine (Pyr), diethyl ether (DE), and 2-methyltetrahydrofuran (2-MTHF), values of ε (L mol−1 cm−1) in these solvents (at maximum of Qy band) are as follows: Pyr, 36 500; DE, 40 100; 2-MTHF, 45 600. Sample Preparation for Spectroscopic Measurements. Immediately before spectroscopic measurements,

chlorosomes were pelleted again by ultracentrifugation (220 000 × g for 1.5 h) and gently resuspended in 1−2 mL of 20 mM Tris−HCl buffer, pH 8.0. Chlorosome samples were chemically reduced via the addition of sodium dithionite to a final concentration of 10 mM, sealed into cuvettes, and incubated in the dark at 4 °C for 0.5 h. For chlorosome samples measured at cryogenic temperatures, glycerol was added to a final concentration of 60% (v/v). Purified BChl f was dissolved in either pyridine (Pyr) (Mg hexa-coordinated), diethyl ether (DE) (Mg penta-coordinated), or 2-methyltetrahydrofuran (2MTHF) (Mg hexa-coordinated) prior to spectroscopic measurements. BChl f measurements at cryogenic temperatures were taken only in the 2-MTHF solvent. All cryogenic experiments at 77 K were taken by cooling the sample with liquid nitrogen in a Janis SVP-100 Cryostat (Janis, Woburn, MA, USA). Femtosecond time-resolved absorption and picosecond time-resolved fluorescence measurements were performed on air-saturated samples. For triplet dynamics measurements with BChl f, samples were prepared as follows. In order to remove molecular oxygen, a pump−freeze−thaw procedure was applied as described earlier.11 The procedure was repeated 10 times, until the influence of residual oxygen was negligible. The pump−freeze− thaw procedure was applied under vacuum quality better than 1 mTorr compared to the typical atmospheric pressure of 760 Torr, and samples were kept in the vacuum during the spectroscopic measurements. Femtosecond Time-Resolved Transient Absorption Spectroscopy. Time-resolved pump−probe absorption experiments of BChl f in solvents at room temperature and at 77 K were carried out using Helios, a femtosecond transient absorption spectrometer (Ultrafast Systems LCC, Sarasota, FL, USA) coupled to a femtosecond laser system previously described in detail.18 The samples were excited in the Qy band with a pump beam energy of 500 nJ in a spot size of 1 mm diameter corresponding to an intensity of ∼2 × 1014 photons/ cm2. Picosecond Time-Resolved Fluorescence Spectroscopy. Time-resolved fluorescence experiments were carried out using a Hamamatsu universal streak camera consisting of a cooled N51716-04 streak tube, C5680 blanking unit, digital CCD camera (Orca2), slow speed M5677 unit, C10647 and C1097-05 delay generators, and a 250IS imaging spectrograph from Bruker. The emitted light was dispersed on a 150 g/mm grading blazed at 800 nm. For focusing of the excitation beam on the sample and emitted light on the spectrograph, a standard optics setup A8110-01 from Hamamatsu was used. The slit at the spectrograph was set to 100 μm corresponding to 2 nm resolution. Excitation pulses were produced by Inspire100, an ultrafast optical parametric oscillator (OPO; Radiantis-SpectraPhysics, CA, USA) pumped with Mai-Tai, an ultrafast Ti:sapphire laser, generating ∼90 fs laser pulses at 820 nm with a frequency of 80 MHz. After the OPO, the pulse frequency of the excitation beam was lowered to 8 MHz (125 ns between subsequent excitations) by a 3980 Pulse Selector from Spectra-Physics equipped with a model 3986 controller. We assumed that for BChl f dissolved in solvents this time interval between concurrent excitations does not cause accumulation of a pool of BChl f being in triplet state that will overwhelm the pool of the molecules in the singlet excited state (due to competitive oxygen sensitization) over the time of the experiment and will lead to fast disappearance of the fluorescence signal. For chlorosome samples, accumulation of 2297



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resolved fluorescence data of BChl f taken in solvents were first cleared from random noise by recomposing the data from the dominant principal components using singular value decomposition (SVD)19 and then fitted globally with monoexponentially decaying spectral-kinetic components convoluted by Gaussians with full width at half-maxima (fwhm) mimicking the instrument response function (IRF). We called the resulting spectrum the decay associated fluorescence spectrum (DAFS), which in the case of isolated BChl f is similar to the steady-state fluorescence spectrum. The IRF of the streak camera tube was obtained by recording a profile of the scattered excitation laser beam in an appropriate streak camera time window.

triplet states of either BChl f or BChl e in chlorosomes is negligible due to very competitive sub-nanosecond transfer of excited state energy to BChl a in the CsmA baseplate. The excitation beam with power of ∼120 μW was focused on the sample in a circular spot of 1 mm diameter, corresponding to a photon intensity of ∼(6−7) × 109 photons/cm2 per pulse (depending on the excitation wavelength). To ensure isotropic excitation of the sample, the excitation laser beam was depolarized (polarization was randomized) before the sample using an achromatic depolarizer (DPU-25, Thorlabs). The samples were adjusted to OD ∼0.3 in the Qy band in a 1 cm cuvette, corresponding to a BChl f concentration between 6.6 and 8.2 μM/L depending on the solvent; however, the excitation beam focus point was adjusted to be very close to the cuvette wall that was used to measure emission (at right angles) and this procedure assured that self-absorption was negligible in all cases. Microsecond Flash Photolysis Spectroscopy. Experiments were performed on samples in a 1 cm path length cuvette using LP920-K/S, a flash photolysis spectrometer from Edinburgh Instruments equipped with a Tektronix digital oscilloscope (TDS 3012C). Excitation pulses were delivered from an Opotek Vibrant 355 tunable laser system equipped with a 10 Hz Nd:YAG laser (Quantel), second and third harmonic generators, and OPO (Opotek Inc., CA, USA). The energy of the excitation beam (355 nm) was set to ∼10 mJ consistent with a photon density of ∼1016 photons/cm2. A 450 W xenon arc lamp was used as a probe light source. The excitation (laser) and probe (white light) beams were configured in standard cross-beam geometry. The probe light was dispersed in a symmetrical Czerny−Turner monochromator (TMS300) and focused onto a Hamamatsu R2658 photomultiplier (kinetic mode) or an IStar DH720-18H-13 Andor ICCD camera (spectral mode). In order to obtain an adequate signal-to-noise ratio, the T−S spectra and triplet decay kinetics traces were averaged 5−10 times. Steady-state absorption spectra were taken before and after every experiment to assess the integrity of the samples. Transient Data Processing and Global Fitting. Group velocity dispersion of the transient absorption (TA) spectra was corrected using Surface Xplorer (Ultrafast Systems LCC, Sarasota, FL, USA) by building a dispersion correction curve from a set of initial times of transient signals obtained from single wavelength fits of the representative kinetics. Global fitting of the data sets was performed using a modified version of ASUfit, a program kindly provided by Dr. Evaldas Katilius at Arizona State University (http://www.public.asu.edu/ ∼laserweb/asufit/asufit.html). The full width at half-maximum of the instrument response function was obtained as one of the global analysis parameters and was in the 150−200 fs range. Global analysis was done using an unbranched, unidirectional decay path model (A → B → C → D → ...) that assumes that the energy losses are large enough that the reverse reaction rates are negligible. The spectral profiles obtained from this fitting of the TA data sets are termed evolution associated difference spectra (EADS).19 Analogously, we used evolution associated fluorescence spectra (EAFS) terms for fitting results of time-resolved fluorescence data. The sequential model quite accurately represents the excitation decay path of individual BChl f molecules (internal relaxation and subsequent decay of the excited state). However, it is oversimplified for chlorosomes, in which the enormous structural complexity allows much more complicated excitation decay paths. Time-

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RESULTS BChl f in Solvents. Steady-state absorption and fluorescence emission spectra of BChl f in the hexa-coordinating solvent pyridine at room temperature (RT) are shown in Figure 2A. The major absorption bands Soret and Qy appear at 472

Figure 2. Spectroscopic properties of BChl f in pyridine at RT: (A) steady-state absorption and fluorescence spectra; (B) transient absorption spectra taken at various delay times after the excitation; (C) results of global fitting of the TA data sets obtained using a sequential decay path; (D) representative decay traces accompanied with fits from global fitting. TA, transient absorption; EADS, evolution associated difference spectra.

and 645 nm, respectively. The main band in the fluorescence emission spectrum (excitation: 472 nm) appears at 655 nm with a Stokes shift of 237 cm−1 with respect to the maximum of the Qy band. The (0−1) vibronic band is clearly resolved at 711 nm. Figure 2B shows transient absorption spectra recorded in the VIS range at various delay times after excitation into the Qy band. The negative bands originate from bleaching of the ground state absorption and are the mirror image of the steadystate absorption slightly distorted by a broad, positive excitedstate absorption band. At early delay times, the position of the Qy band in the TA spectra is shifted by a few nanometers to longer wavelengths. The observed shift results from super2298



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imposing of the true Qy bleaching and stimulated emission (SE) being indistinguishable by the detector. The TA spectrum taken at long delay times (7.2 ns) shows diminished SE with an almost correct position of the Qy band and also a more pronounced ESA band at ∼495 nm. This 7.2 ns spectrum is primarily a combination of triplet−triplet transient absorption and bleaching of the ground state absorption, and is usually called the triplet-minus-singlet (T−S) spectrum.20 Global fitting results of the TA data set (EADS) are shown in Figure 2C. Four kinetic components were required to obtain an acceptable fit. The spectral-kinetic components (EADS) with lifetimes of 7 ps, 130 ps, and 3.4 ns are practically the same in line shape, meaning that all are associated with the same excited state, S1. The presence of the fast-decaying components (7 and 130 ps) is probably associated with a rapid, two-phase energetic relaxation of the S1 excited state. The 3.4 ns EADS component is associated with the decay of a relaxed singlet excited state, and the lifetime obtained matches perfectly to that acquired previously from time-resolved fluorescence in the same solvent.17 Figure 2D shows representative kinetic traces accompanied with fits obtained from global fitting. The kinetic traces taken at 470 and 647 nm represent recovery of the ground state absorption (Soret and Qy bands, respectively). The 490 nm trace is taken at the point at which spectral features associated with the singlet excited state cross zero; thus, it represents the initial time evolution of the triplet population. Steady-state absorption and fluorescence spectra of BChl f in the penta-coordinating solvent diethyl ether are shown in Figure 3A. The Soret and Qy bands appear at 450 and 633 nm, respectively. The main band in the fluorescence spectrum (excitation: 450 nm) is maximal at 643 nm (Stokes shift of 246 cm−1) with a (0−1) vibronic band at 699 nm. Figure 3B shows the TA spectra recorded in the VIS range at various delay times after excitation into the Qy band. The TA spectra taken at 0.5 and 100 ps are practically identical but differ from that taken at 7.2 ns, which shows a noticeable ESA band at ∼480 nm associated with triplet−triplet ESA. Figure 3C shows global fitting results of the TA data set. For this solvent, the fit required only three kinetic components. The identical (with BChl f in pyridine) singlet excited state lifetime (3.4 ns) demonstrates that BChl f does not aggregate in DE at low concentrations and the lifetime shortening of the excited state commonly observed in aggregates and associated with excited state annihilation processes is not observed.21−23 Figure 3D shows a two-dimensional contour of time-resolved fluorescence measured in a 20 ns time window. Global fitting (Figure 3E) resulted in one spectral-kinetic component, the decay associated fluorescence spectrum (DAFS), with a lifetime of 3.4 ns and a spectral line shape identical to the steady-state fluorescence spectrum (Figure 3A). Figure 3F shows a representative kinetic trace of fluorescence decay extracted from the TRF data set and accompanied by the fit obtained from global fitting. The kinetic traces extracted from the TA data set and taken at 450 nm (Soret), 472 nm (T1 → Tn ESA), and 636 nm (Qy) are shown in Figure 3G. Spectroscopic properties of BChl f at 77 K in the hexacoordinating solvent 2-MTHF are shown in Figure 4. Steady-state absorption and fluorescence spectra (Figure 4A) demonstrate enhanced vibronic resolution. The Soret band that is visible as a smooth peak at RT shows some distortion on the long wavelength edge at 77 K, which may be indicative of partial resolution of the Bx and By bands. The Qy band appears

Figure 3. Spectroscopic properties of BChl f in diethyl ether at RT: (A) steady-state absorption and fluorescence spectra; (B) transient absorption spectra taken at various delay times after the excitation; (C) results of global fitting of TA data sets obtained using a sequential decay path; (D) contour map of TRF shown for 15 ns after excitation; (E) spectral line shape lifetime of DAFS obtained from global fitting of the TRF data set; (F) representative TRF decay trace accompanied with fit from global fitting and IRF; (G) representative TA decay traces accompanied with fits from global fitting. TA, transient absorption; EADS, evolution associated difference spectra; DAFS, fluorescence decay associated spectra; TRF, time-resolved fluorescence; IRF, instrument response function.

at 645 nm, and the main band in the fluorescence spectrum (excitation: 475 nm) is shifted by 2 nm to 647 nm (Stokes shift of 48 cm−1). Figure 4B shows the TA spectra recorded in the VIS range at various delay times after excitation into the Qy band. The TA spectra taken at 0.5 ps, 100 ps, and 7.4 ns show similar characters as their counterparts taken at RT. Global fitting (Figure 4C) revealed kinetic components with lifetimes of 17 ps, 5.1 ns, and “inf”, i.e., a nondecaying component. The excited singlet state lifetime of 5.1 ns demonstrates that low temperature reduces the coupling between ground and excited states by causing a slight elongation of its lifetime. The temperature-induced elongation of the lifetime was also confirmed by TRF (Figure 5D−F), for which the fitting of the fluorescence decay (Figure 4E and F) revealed essentially the same lifetime. Kinetic traces extracted from the TA data set and taken at the Soret band (470 nm), the T1 → Tn ESA band (490 nm), and the Qy band (648 nm) are shown in Figure 5G. The raw data traces are accompanied by fits obtained from global fitting. 2299



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Figure 5. Triplet state properties of BChl f in pyridine at RT: (A) Evolution of the concentration of BChl f triplets accompanied with a simulated curve obtained by applying eq 3. The values of known parameters are marked as fixed. See main text for parameter descriptions. (B) Representative triplet-minus-singlet (T−S) spectrum taken at 1 μs after excitation at 355 nm. (C) Representative triplet state decay−recovery of the ground state absorption recorded at 470 nm. (D) Observed triplet decay rates as a function of BChl f concentration nominated as absorbance of the Qy band. The fit (solid line) was done according to kTobs = kTint = k[BChl f ].

Figure 4. Spectroscopic properties of BChl f in 2-MTHF at 77 K: (A) steady-state absorption and fluorescence spectra; (B) transient absorption spectra taken at various delay times after the excitation; (C) results of global fitting of the TA data sets obtained using a sequential decay path; (D) contour map of TRF shown for 15 ns time after excitation; (E) spectral line shape and lifetime of DAFS obtained from global fitting of the TRF data set; (F) representative TRF decay trace accompanied with a fit from global fitting and IRF; (G) representative TA decay traces accompanied with fits from global fitting. TA, transient absorption; EADS, evolution associated difference spectra; DAFS, decay associated fluorescence spectra; TRF, timeresolved fluorescence; IRF, instrument response function.

d[3BChl f ] = k isc·[1BChl* f ] − k T·[3BChl f ] dt d[1BChl* f ] = k S·[1BChl* f ] dt

(2)

Because the initial population of BChl f molecules 1BChl0* f being in the singlet excited state is instantaneously populated via laser excitation, [1BChl* f ] = 1BChl*0 f·e−kSt and [3BChl f ]t=0 = 0 (no triplets at time of excitation)

Photophysical properties of the triplet state of BChl f in pyridine are demonstrated in Figure 5. The temporally wellresolved rise of the triplet state seen in the TA data set provides an occasion to calculate the rate of the intersystem crossing (ISC) (kisc) by simulation of the rise of the T1 → Tn transition at wavelengths on which it does not coincide with the simultaneous decay of the excited singlet state. In pyridine, such a kinetic trace is visible at 490 nm (Figure 2D). In order to obtain the rate, the following kinetic scheme should be considered:

BChl*0 f ·k isc ⎛ 1 ⎞ ⎜1 − k ·t ⎟ ⎝ kS eS ⎠

1

[3BChl f ] =

(3)

Equation 3 was applied to fit the 490 nm kinetic trace from Figure 2D. In order to perform fitting, the ΔA at 490 nm was converted to the actual molecular concentration of BChl f in the triplet state according to the Beer−Lambert law: ΔAT = εT· C·l (ΔA, change of absorbance at 490 nm; C, molar concentration; l, optical path (length of the cross section of probe and pump beams, assumed to be 0.2 mm); εT, molar extinction coefficient of the triplet band at 490 nm). εT can be deduced from the proportion between the amplitudes of the triplet transient band and the bleaching of the ground state absorption (Qy band) in the T−S spectrum that should be equal to the proportion between the molar extinction coefficients of both transitions. Simple arithmetic calculations give εT 490 = 12 200 L mol−1 cm−1.

where k + kisc = kS is the rate constant of excited singlet state decay, kisc is the rate constant of intersystem crossing, k = kF + kic (rate constants of S1 → S0 spontaneous fluorescence and internal conversion, respectively), and kT is the rate constant of triplet decay. The scheme can be mathematically described as follows: 2300



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Table 1. Photophysical Properties of BChl f Obtained from This Study along with Properties of BChl c, d, and e solvent

a

a

absorption (nm)

fluorescence (nm)

Soret

(0−0)

Qy

(0−1)

pyridine (H)

472

645

655

711

diethyl ether (P) 2-MTHF (H)

450 470

633 645

643 647

699 708

pyridine (H)

445

670

674

n.d.

pyridine (H)

438

657

663

n.d.

pyridine (H)

475

660

670

n.d.

Stoke’s shift (cm−1) BChl f 237 246 48 BChl cd 88 BChl dd 137 BChl ed 225

b

c

τs (ns)

ks (ns−1)

τTint (μs)

Φisc (kisc (ns−1))

3.4

0.294

340 ± 17

3.4 5.1

0.294 0.196

n.d. n.d.

0.70 0.75 0.74 0.61

6.7

0.149

474 ± 39

n.d.

293

6.3

0.159

448 ± 8

n.d.

293

2.9

0.345

461 ± 22

n.d.

293

(0.21)b (0.22)c (0.21)b (0.12)b

T (K) 293 293 77

d

H, Mg hexa-coordinated; P, Mg penta-coordinated. Calculated from TA. Calculated from eq 3. Taken from ref 12. n.d., not determined.

kS was set to 0.29 × 109 s−1 (reciprocal of fluorescence lifetime, 3.4 ns), kT = 0.003 × 109 s−1 (reciprocal of the triplet state lifetime in the air saturated sample, ∼330 ns), and 1BChl0* f = 15 μM. The result of fitting of eq 3 to the 490 nm kinetic trace, expressed as the concentration of BChl f triplets, is shown in Figure 5A. The rate of ISC obtained as an output parameter kisc = 0.22 × 109 s−1 (τisc = 4.5 ns) corresponds to an intersystem crossing yield (triplet yield) of Φisc = 0.75. On the other hand, the value of Φisc can also be obtained independently by simply taking the proportion of amplitudes of the bleaching of the Soret band at a delay time in which it is associated mostly with triplet (e.g., at 7.4 ns) to that associated only with the singlet excited state (maximum bleaching at early delay time). This analysis gives Φisc = 0.70 (kisc = 0.21 × 109 s−1) and demonstrates a good agreement with the simulated value. The value of ISC yield for BChl f is in very good agreement with the average ISC yield of ∼0.7 determined for various synthetic chlorins.24−26 Figure 5B shows a representative T−S spectrum taken at 1 μs after excitation at 355 nm. The spectrum consists of two negative bands associated with bleaching of ground state absorption and a broad positive T1 → Tn ESA band peaking at 505 nm and is analogous to the infinite EADS component obtained from global fitting of the TA of BChl f in pyridine. A representative kinetic trace of triplet decay of a highly concentrated BChl f (OD470 = 0.7) recorded at the maximum of the T1→ Tn ESA band is shown in Figure 5C. The decay can be simulated by a monoexponentially decaying curve with a mean lifetime of 171 μs. Figure 5D shows the dependence of triplet state lifetime on the molecular concentration of BChl f. The points represent observed triplet lifetimes measured for various molecular concentrations denoted as the absorbance at the maximum of the Soret band. The solid line was obtained according to kTobs = k·[BChl f ] + kTint, where kTobs is the observed triplet decay rate (reciprocal of observed lifetime), k is the quenching rate of the triplet state by BChl f molecules in the ground state, [BChl f ] is the concentration of BChl f molecules in the ground state, and kTint is the triplet state intrinsic lifetime. The triplet state intrinsic lifetime obtained by applying the above-mentioned equation is 340 ± 17 μs (kTint = 2.9 × 103 s−1), and the quenching rate k = 4.8 × 103 s−1. The photophysical properties of BChl f as well BChl c, d, and e are summarized in Table 1. BChl f in Chlorosomes. Time-resolved fluorescence of chlorosomes containing BChl e and BChl f is shown in Figure

6. The TRF contour maps are presented in Figure 6A and B. These exhibit two fluorescence bands associated with emission

Figure 6. TRF spectroscopy of chlorosomes containing BChl e and BChl f at 77 K: (A, B) contour maps of the TRF recorded within a 5 ns time window after excitation (time range limited to 1.5 ns); (C, D) results of global analysis of TRF data sets upon assumption of sequential decay (EAFS) of excitation within chlorosome. Note the almost negligible differences in kinetic component lifetimes in both chlorosomes.

from either BChl e or f (750 nm/740 nm) and baseplate-bound BChl a (∼820 nm). Chlorosomes were excited into the short wavelength edge of the Qy band of BChl e or f (700 and 680 nm, respectively), and the time course of fluorescence clearly reveals the funneling of excitations into energetically lowest excitons. The fluorescence band shifts from 746 to 755 nm within the first 300 ps for BChl e chlorosome and from 736 to 743 nm for BChl f chlorosome. This can also be traced in the spectral shapes of EAFS obtained from global analysis (Figure 6C and D). 2301



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reported.40 On the other hand, time-resolved spectroscopic studies on BChl e-containing chlorosomes that can be used for comparison purposes to the TRF presented here are limited.41,42 On the basis of time-resolved absorption on chlorosomes containing BChl e from Chlorobaculum limnaeum performed at RT under excitation light of ∼1011 photons cm−2 pulse−1, Pšenčik et al. concluded that BChl e excited states decay with three different rates (are transferred to baseplate) with a majority of the population decaying with a lifetime of 80−100 ps.38 Adapting the same model to the TRF data presented here (Figure 6A and C), it can be hypothesized that the 140 and 340 ps kinetic components are associated with decay (energy transfer to baseplate) of two pools of relaxed BChl e aggregate excitons. The initial component is associated with relaxation of BChl e aggregate excitons (also no signature of baseplate fluorescence appears in the initial EAFS spectral line shape). The time constant of that process is known to be much shorter than 40−50 ps obtained here (in time range 1−2 ps);38 however, in this case, it is artificially elongated by the substantially different time resolutions of the streak camera tube and the TA spectrometer. The effect of lower temperature (77 K vs RT) and even lower excitation intensities (109 photons cm−2 pulse−1) should also be taken into consideration. The last kinetic component with a lifetime of 1.2 ns is associated with the decay of the excited state of BChl a in the chlorosome baseplate. One may expect that, because energy transfer between BChl f aggregates and the BChl a baseplate decreases significantly compared to the BChl e−BChl a system, the lifetimes of excitons contributing in the process should be considerably longer. The global analysis results (Figure 6D) show that EAFS components associated with decay of BChl f excitons indeed have longer lifetimes, but the differences are relatively minor. However, there could be differences in the overlap between fluorescence of BChl e and BChl f and absorption of BChl a at 77 K with respect to their counterparts from RT. In addition, we assumed that the experimental conditions are “annihilation free”. However, if the chance of BChl f-to-BChl a excitation transfer decreases, ultimately the chance of exciton−exciton annihilation within the BChl f aggregate increases and the expected exciton lifetime increase may almost be compensated.

Interestingly, the dynamics of both chlorosomal systems are not distinctive and the macroscopic decay rates obtained from data fitting are very similar in both cases, spanning time ranges from tens of picoseconds to around 1 ns.

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DISCUSSION Singlet Excited State Properties of BChl f in Solvents. Despite the fact that BChl c, d, e, and f belong to the same chlorin subfamily, they do not share common singlet excited (S1) state properties. On the basis of the S1 state lifetimes, it is apparent that this group of pigments can be split into two subgroups. In addition, if three other representatives of the chlorin-like family but present in oxygenic phototrophs (Chl a, b, and d) are included for evaluation purposes, it becomes apparent that the longevity of the S1 state lifetime is determined by the substituent at the −R7 position in the macrocycle. If a formyl group is introduced in place of methyl (Chl b, BChl e and f), the S1 state lifetime is essentially shortened 2-fold. The S1 state lifetime shortening is associated also with a decrease of the fluorescence quantum yield (ΦF). For Chl a (R7 = CH3, τs = 6.3 ns11), ΦF in pyridine is ∼0.33,27 but for family representatives with decreased singlet excited state lifetimes like BChl f (τs = 3.4 ns) or Chl b (τs = 3.2 ns11), it decreases to 0.1817 and 0.16,27 respectively. Triplet State Properties of BChl f in Solvents. There are several factors that influence the observed lifetime of the triplet state of BChl f. Among the most important are molecular oxygen, concentration of molecules in the ground state, and character of solution (allows or prevents aggregate formation). These factors substantially quench the triplet state and lead to significant shortening of triplet dynamics of all (B)Chl’s.28 To prevent aggregation, hexa-coordinating pyridine was used as solvent. In order to remove oxygen dissolved in the solution, a series of 10 freeze−pump−thaw cycles was applied (see Materials and Methods). If this factor is eliminated, the observed decay rate kTobs becomes a linear function of BChl f concentration, as demonstrated in Figure 5D, and the intrinsic decay rate (and lifetime) of the triplet state can be simply obtained by extrapolation of a linear fit with concentration (absorbance). The fits of individual kinetic traces obtained for different concentrations of BChl f are provided in Figure S1 (Supporting Information). The same method used to measure the intrinsic lifetime of the triplet of BChl c, d, and e in pyridine demonstrated that the lifetime values span a narrow 450−470 μs range; thus, it might be expected that BChl f will fall into or will be very close to that range too.11 However, the BChl f intrinsic triplet state lifetime (340 μs) obtained here demonstrates a deviation from the expectation and could be underestimated because the effect of excitation intensity has not been considered in this case. Singlet Excited State Properties of BChl f in Chlorosome. The exceptionally large number of aggregated BChl molecules in chlorosome systems make these objects extremely challenging for studies of their photophysical properties due to the onset of excitation annihilation at very low excitation intensities of ∼1011 photons/cm−2 per pulse. The majority of studies of the photodynamics of chlorosomes were performed using pico- or femtosecond time-resolved absorption spectroscopy under annihilation-free conditions and in most cases on BChl c-containing chlorosomes from various bacterial species.29−39 However, recently pioneering studies using two-dimensional electronic spectroscopy have also been

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CONCLUSIONS Why does nature avoid using BChl f for light harvesting? The excited singlet and triplet state properties of BChl f monomers that were investigated in this study demonstrate that BChl f is not a unique pigment. The reduced singlet excited state lifetime and fluorescence quantum yield should not disrupt its robustness in light harvesting because other (B)Chl’s share similar properties and are abundant in nature. The absence of this pigment in nature can be most likely attributed to the ecophysiology of anoxygenic green sulfur bacteria. Because of their physiological adaptations, these organisms live in sulfiderich, aquatic environments with minimal dissolved oxygen, and they typically occur deep below layers enriched with oxygenic phototrophs which contain Chl a. Under such conditions, they are exposed to light environments that are strongly depleted in 650−700 nm light that is filtered out by the Chl’s utilized by oxygenic phototrophs. Like aggregates formed from BChl e, BChl f aggregates in chlorosomes exhibit a split Soret band with enhanced absorption around 500 nm, which is not observed with aggregates formed from BChl c or BChl d. This absorption band centered around 525 nm for BChl e is believed to be 2302



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Biographies

responsible for most of the light collected by the chlorosomes of brown-colored organisms in their natural habitats, because red photons do not penetrate very deeply in aquatic environments. It is for this reason that organisms producing BChl c and BChl d, for which the Qy absorption is mostly used for light harvesting, predominantly occur in shallower stratified lakes and sediments. However, the ability to harvest blue light does not alter the fact that energy transfer from the Qy band to the BChl a associated with CsmA in the baseplate must span a larger energy gap of nearly 100 nm in the case of BChl f, which results in much poorer donor−acceptor overlap for BChl f than for BChl e.17 An additional potential problem might be that the higher energy levels associated with triplet excitons for the BChl f aggregates might lead to the formation of singlet oxygen under some conditions.43 If oxidation products are thereby formed that quench energy transfer, this might explain the very poor fluorescence emission yields associated with chlorosomes containing BChl f under oxic conditions. Experiments to test this possibility are currently in progress. What does this all mean for whether BChl f can be usedor has ever been usedas a natural light-harvesting pigment? A mutant that synthesizes chlorosomes containing BChl f can grow as fast as the parental strain producing BChl e at high light intensity but grows significantly slower than the wild type at low light intensity.15 This suggests that a mutant synthesizing BChl f could compete favorably with strains producing BChl e as long as the environment was devoid of oxygen and had minimal light filtering by Chl a so that far-red light could be utilized. Such environments might occur if sulfide-enriched water occurred closer to the surface, but as noted above, such environments appear to be rare and are more likely to have higher oxygen concentrations. Although suitable environments may occur, none have yet been identified, and as a consequence, no naturally occurring organism synthesizing BChl f has yet been identified. Such environments may have been more common during periods before full oxygenation of the atmosphere by oxygenic photosynthesis.

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Dariusz M. Niedzwiedzki is a Research Scientist in Photosynthetic Antenna Research Center at Washington University in Saint Louis, MO. He received his Ph.D. in biophysics from Maria CurieSkłodowska University in Lublin, Poland, in 2004. Between 2004 and 2009, he was Postdoctoral Fellow in the Department of Chemistry of University of Connecticut concentrating his research primarily on photophysics of carotenoids. Currently, his research interest focuses on spectroscopy of various photosynthetic pigments and light harvesting proteins.

Gregory S. Orf is a Ph.D. candidate in biophysical chemistry at Washington University in St. Louis, MO, under the advising of Professor Robert Blankenship. He received his B.S. degree in biochemistry from Saint Louis University in 2010. His thesis research focuses on the photophysics and structure−function relationships within the photosynthetic antenna systems of green sulfur bacteria. He also collaborates on several engineering projects, developing methods for constructing bio-hybrid and bio-inspired light-harvesting antennas.

ASSOCIATED CONTENT

S Supporting Information *

Table of Mg concentration determination, the kinetic traces of decay of the T1 → Tn band or Soret bleaching taken for different concentrations of BChl f with corresponding fits done according to monoexponential decay. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1 314 935-7971. Fax +1 314 935-4432. Notes

The authors declare no competing financial interest. 2303



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Marcus Tank studied Ocean Sciences, microbiology, and physical oceanography and received his Ph.D. from the Leibniz Institute of Marine Sciences at Kiel University, Germany, in 2010. Since 2011, he has been a postdoc in the department of Biochemistry and Molecular Biology of the Pennsylvania State University, University Park, PA. His research is focused on the ecology and systems biology of anoxygenic phototrophic bacteria, including physiology and interactions, biochemistry, phylogeny, elucidation of biosynthesis pathways, as well as the photochemistry of their photosynthetic apparatus.

Robert E. Blankenship is the Lucille P. Markey Distinguished Professor in Arts and Sciences at Washington University in St. Louis, MO. Dr. Blankenship has spent his entire scientific career of more than 40 years researching the highly interdisciplinary subject of photosynthesis. This research has used a wide range of techniques including ultrafast optical spectroscopy, magnetic resonance, and mass spectrometry as well as genomics and molecular evolutionary analysis. His research investigates energy transfer and electron transfer processes in photosynthetic antenna and reaction center complexes. One of the hallmarks of his research program is that it emphasizes studying the mechanism of energy storage in the complete range of known organisms that do photosynthesis, with the goal of discovering the essential or irreducible aspects of how light energy is stored.

Kajetan Vogl received his Ph.D. in microbiology from the LudwigMaximilians University of Munich in 2008 under the supervision of Jörg Overmann. His research focused on the analysis of the bacterial symbiosis of the phototrophic consortium “Chlorochromatium aggregatum”. He then joined the group of Donald A. Bryant as a postdoctoral fellow at The Pennsylvania State University, where he worked on the biosynthesis of carotenoids of purple sulfur bacteria and the development of a genetic system in a BChl e producing green sulfur bacterium.

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ACKNOWLEDGMENTS All spectroscopic studies were performed in 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. Mutagenesis and cell cultivation were done in D.A.B. laboratory and were funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the Department of Energy through Grant DE-FG02-94ER20137. We thank Sanmathi Chavalmane Subbenaik for help with Mg concentration determinations that were performed in the Nano Research Facility at Washington University in St. Louis.

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REFERENCES

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