Photosynth Res DOI 10.1007/s11120-014-9981-z
REGULAR PAPER
Excited state properties of chlorophyll f in organic solvents at ambient and cryogenic temperatures Dariusz M. Niedzwiedzki • Haijun Liu Min Chen • Robert E. Blankenship
•
Received: 13 November 2013 / Accepted: 5 February 2014 Springer Science+Business Media Dordrecht 2014
Abstract Chlorophyll f is a photosynthetic pigment that was discovered in 2010. In this study, we present investigations on spectral and dynamic characteristics of singletexcited and triplet states of Chl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The pigment was studied at room temperature in two organic solvents: pyridine and diethyl ether that have different characters of coordination of the chlorophyll magnesium (Mg) atom (hexa- and penta-coordination, respectively). Cryogenic measurements (77 K) were performed in 2-methyltetrahydrofuran (hexa-coordination). The singlet-excited state lifetime was measured to be 5.6 ns at room temperature regardless of Mg coordination and 8.1 ns at 77 K. The fluorescence quantum yield of Chl f was also determined in pyridine to be 0.16. The triplet state lifetime was studied in detail in pyridine at room temperature, and the inherent lifetime was estimated to *150 ls. Selective measurements at 77 K demonstrated that the
D. M. Niedzwiedzki (&) H. Liu R. E. Blankenship Photosynthetic Antenna Research Center, Washington University in St. Louis, Campus Box 1138, St. Louis, MO 63130, USA e-mail: [email protected] H. Liu R. E. Blankenship Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA M. Chen School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia R. E. Blankenship Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA
metastability of the triplet state greatly enhances, and its lifetime increases by a factor of more than three. Keywords Chlorophyll f Transient absorption Singlet-excited state Triplet state Time-resolved fluorescence Halomicronemona hongdechloris Abbreviations 2-MTHF 2-Methyl tetrahydrofuran CCD Charge couple device Chl Chlorophyll DE Diethyl ether EADS Evolution-associated difference spectra ESA Excited state absorption FWHM Full width at half maximum HOMO Highest occupied molecular orbital ICP-MS Inductively coupled plasma-mass spectrometry LUMO Lowest unoccupied molecular orbital OPO Optical parametric oscillator Pyr Pyridine RT Room temperature TA Transient absorption TRF Time-resolved fluorescence T-S Triplet-minus-singlet
Introduction Chlorophylls (Chls) serve several important roles in the process of photosynthesis including light absorption, excitation transfer, and primary charge separation in reaction centers. Basic spectroscopic properties of Chls can be deduced from Gouterman’s ‘‘four-orbital’’ model that assumes that the major bands of electronic absorption are
123
Photosynth Res
Fig. 1 Chemical structures of Chl a, b, d, and f and their room temperature steady-state absorption spectra taken in Pyr. Absorption spectra of Chl a, b, and d were taken from Niedzwiedzki and Blankenship (2010)
associated with p ? p* electronic transitions that derive from electron promotions involving the two HOMOs and two LUMOs. The resulting electronic transitions are polarized along either the x or y-axis of the macrocycle (Fig. 1) and are referred to Qy, Qx, By, and Bx bands (Gouterman 1959, 1961, 1978; Weiss 1972). In Chls, B transitions appear between 400 and 500 nm and are collectively called the Soret band. The Qy band can span the spectral region from 620 to 700 nm, depending on the degree of macrocycle saturation and the presence of specific side chain modifications. The Qx band is greatly reduced in amplitude but is expected to appear between 500 and 600 nm. Chls can be divided into two subgroups characterized by the degree of unsaturation of the macrocycle: porphyrin-type (fully unsaturated macrocycle) containing group of Chl c pigments (c1, c2, c3, and numerous clike forms), and chlorin-type (single bond between C-17 and C-18 carbons in the macrocycle) that comprises Chl a, b, d, and f. Chlorophylls: a and b (Fig. 1) were discovered by two French chemists, Caventou and Pelletier in 1818 (Pelletier and Caventou 1818) who also introduced the name ‘‘chlorophyll’’ from a combination of the Greek words, chloros (green) and phyllos (leaves). Chl c was discovered in the early 1900s in aquatic algae, and currently, the name
123
embraces a group of many sub-types (Chl c1, c2, etc.) (Zapata et al. 2006). The next representative, Chl d (Fig. 1), was first reported in the 1940s in red algae (Manning and Strain 1943) but for a long time was considered to be an extraction artifact. In 1996, a marine cyanobacterium named Acaryochloris marina, containing almost exclusively Chl, d was discovered (Miyashita et al. 1996). Chl e was reported in 1943 as a minor pigment in algae Tribonema bombycinum (Strain 1958) with absorption bands at 415 and 654 nm in methanol; however, its structure and properties have never been elucidated, and until now its status remains uncertain. The latest member of the Chls family, Chl f (Fig. 1), was discovered in 2010 in cyanobacteria collected from a stromatolite colony from Shark Bay in Western Australia and is the most red-shifted Chl used in oxygenic photosynthesis (Chen et al. 2010). The cyanobacterium containing Chl f was isolated and named Halomicronemona hongdechloris (Chen et al. 2012). Chl f is not a major photosynthetic pigment in this organism and accounts only for maximum of 15 % of total Chls (with majority as Chl a) and is produced only upon growth in far-red light. It is hypothesized that Chl f is an accessory pigment in the photosynthetic process. However, that role will require a significant uphill energy transfer toward Chl a in the reaction center; thus, it is still under debate (Chen and Blankenship 2011). Even though computational studies of excited state properties of modified Chl f interacting with various axial ligands (Mg penta-coordinated) have been performed (Yamijala et al. 2011), experimentally the photophysical properties of Chl f are limited to detailed studies of properties of steady-state absorption, but more advanced studies have not been elucidated (Li et al. 2013). In addition, ICPMS spectroscopy was used to measure Mg content and obtain pigment extinction coefficients in various organic solvents (Li et al. 2012). Here, we present more detailed spectroscopic studies of Chl f in organic solvents with different Mg coordination characters (hexa- and penta-coordination) at room temperature and at 77 K. The studies utilize a combination of steady-state and time-resolved absorption and fluorescence techniques in order to obtain more insights to the spectral and temporal characteristics of the singlet- and tripletexcited states.
Materials and methods Culture growth Initially H. hongdechloris cells were kept on 1 % agar of K ? ES seawater solid plates (Chen et al. 2012; Miyashita et al. 1996) and cultured for 4 weeks under continuous
Photosynth Res
illumination of white-fluorescent light with an intensity of 5 lmol photons m-2 s-1 in order to accumulate more biomass. Subsequently, cultures were transferred to far-red light (740 nm, 27 nm spectral half width, same intensity), produced by lamps equipped with 17 W LumiBulb-FR (LumiBulb-Far RedTM bulb, LumiGrow, CA, USA). Isolation of Chl f Chl f was isolated from pelleted cells as follows. The pigment was extracted with 100 % technical grade methanol. The supernatant was removed and loaded directly into an Agilent Series 1100 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a reversephase Zorbax C-18 column (4.6 9 250 mm, Agilent Technologies Inc., Santa Clara, CA, USA) operated at 20 C. Pigment was eluted with 100 % methanol (Sigma, St. Louis, USA) pumped at a rate of 1.5 mL min-1. The major HPLC peak with Chl f spectral signature was collected (the sample is assumed to be free from Chl f0 forms), then dried under a stream of nitrogen gas, and stored under nitrogen in the dark at -20 C until needed. Samples preparation for spectroscopic measurements Purified Chl f was dissolved in Pyr, DE, or 2-MTHF (spectroscopic grade) prior to spectroscopic measurements. Measurements at 77 K were taken only in the 2-MTHF by cooling the sample with liquid nitrogen in a Janis SVP-100 Cryostat (Janis, Woburn, MA, USA). Femtosecond timeresolved absorption and picosecond time-resolved fluorescence measurements were performed on air-saturated samples. For triplet dynamics measurements samples were prepared by applying pump–freeze–thaw procedure as described previously (Niedzwiedzki and Blankenship 2010) under a vacuum quality of better than 1 mTorr compared to typical atmospheric pressure of 760 Torr. Samples were kept in vacuum conditions during spectroscopic measurements. Femtosecond time-resolved transient absorption spectroscopy Time-resolved pump-probe absorption experiments of Chl 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 described in detail previously (Niedzwiedzki et al. 2011). The samples were excited at Qy band (710 nm in Pyr, 695 nm in DE, 710 nm in 2-MTHF) with pump beam energy of 500 nJ in a spot size of 1-mm diameter corresponding to an intensity of
*2 9 1014 photons cm-2 per pulse with frequency of 1 kHz. The samples OD was adjusted to be 0.3–0.5 at Qy maximum (in 2-mm-path cuvette) corresponding to molecule concentration of 15–26 9 10-6 mol L-1 [9 9 1012 molecules lL-1) (e695 = 96.62 9 103 L mol-1 cm-1in DE was used (Li et al. 2012)]. Under the assumption that an individual laser pulse (2 9 1012 photons mm-2) will excite a (*0.8 mm2 9 2 mm) cylinder of the sample with volume of *1.6 mm3, which will contain *1.4 9 1013 molecules of Chl f, the photon-to-molecule ratio is *0.1. Even though this value is small, in penta-coordinating solvent like DE in which the dimeric form of Chl f can be formed, the chance of singlet–singlet annihilation effect is still possible and may slightly distort (decrease) the observed singlet-excited state lifetime. 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 an 250IS imaging spectrograph from Bruker. The emitted light was dispersed on 150 g mm-1 grading blazed at 800 nm. For focusing of the excitation beam on the sample and emitted light on the spectrograph standard optics setup, A8110-01 from Hamamatsu was used. The slit at spectrograph was set to 100 lm corresponding to 2 nm resolution. Excitation pulses were produced by Inspire 100, an ultrafast optical parametric oscillator (OPO; Radiantis-Spectra-Physics, 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. The excitation beam with power of *10 lW was focused on the sample in a circular spot of 0.5-mm diameter, corresponding to a photon intensity of *2 9 1010 photons cm-2 per pulse. The sample concentration was adjusted to *3 9 10-6 mol L-1 (1.8 9 1012 molecules lL-1) (based on OD *0.3 at the Qy band in a 1-cm cuvette; molar extinction coefficient in DE). These conditions correspond to a photon-to-molecule ratio of 0.005, which assures that in DE (Chl f may form dimers) fluorescence dynamics should be free of singlet–singlet annihilation. To assure an 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 excitation beam focus point was adjusted to be very close to the cuvette wall (1–3 mm from it) that was used to measure emission (at
123
Photosynth Res
right angle) so that self-absorption was practically negligible in all cases (maximum absorbance of 1–3 mm layer of sample is between 0.03 and 0.06 in the Qy band). Microsecond flash photolysis spectroscopy Experiments were performed on samples in a 1-cm-pathlength 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 10 Hz Nd:YAG laser (Quantel) producing 6-ns-duration laser pulses, second and third harmonic generators, and OPO (Opotek, Carlsbad, CA, USA). Energy of the excitation beam (1-cm dimension) was set to *10 mJ consistent to a photon density of *1016 photons cm-2 per pulse, and the sample was excited at 1 Hz frequency. The absorbance value at the maximum of the Qy band was adjusted to 0.3–0.4 in 1-cm path, which corresponds to a concentration of 3–4 9 10-6 mol L-1 (1.8 9 1015 molecules mL-1). Because the sample volume of 0.8 mL (*0.8 cm2 9 1 cm) will be excited, the photon-to-molecule ratio will be *7, and in this case, Chl f triplet dynamics that are taken in DE may be easily affected. 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. In order to obtain good quality results, the T-S spectra and triplet decay kinetics traces were averaged 5–20 times. Sample integrity was checked by taking steady-state absorption spectra before and after every experiment. Transient data processing and global analysis Group velocity dispersion of the TA spectra was corrected using Surface Xplorer 2.0 (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 datasets was performed using a custom modified version of ASUfit (http://www.public.asu.edu/ *laserweb/asufit/asufit.html). The FWHM 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 so that the reverse reaction rates are negligible. The spectral profiles obtained from this fitting of the TA datasets are termed Evolution-Associated Difference Spectra (EADS) (van
123
Fig. 2 Steady-state absorption and fluorescence spectra of Chl f in Pyr (black), DE (red) at RT, and 2-MTHF (blue) at 77 K
Stokkum et al. 2004). The sequential model quite realistically represents excitation decay path of Chl. Timeresolved fluorescence data of Chl f were first cleared of random noise by recomposing the data from the dominant principal components using singular value decomposition (van Stokkum et al. 2004) and then fitted globally with mono-exponentially decaying spectral-kinetic component convoluted by Gaussians with FWHM equal to that of the real IRF. The resulting spectrum is called Decay-Associated Fluorescence Spectrum (DAFS), which in shape is practically identical to steady-state fluorescence. The IRF of the streak camera tube was obtained by recording the profile of the scattered excitation laser beam in an appropriate streak camera time window.
Results Steady-state absorption and fluorescence Steady-state absorption and fluorescence spectra of Chl f taken in three different environments are given in Fig. 2. In Pyr (hexa-coordinating solvent) the major absorption bands, Soret (By, Bx) and Qy, appear at, respectively, 413, 443, and 706 nm. The fluorescence spectrum is red shifted in respect to the Qy absorption band by 17 nm (723 nm, Stokes shift of *330 cm-1). Upon changing the environment to penta-coordinating DE, a substantial change in shapes and positions of the main bands (396, 439, and 694 nm) is observed. The fluorescence emission spectrum (706 nm) exhibits a slightly smaller Stokes shift (*280 cm-1). Upon lowering the temperature to 77 K (2-MTHF, hexa-coordinating solvent), some enhancement in spectral resolution is observed (bands narrow), but the positions of the individual transitions (absorption or emission) are very close to those recoded for Pyr at RT (Abs—410, 439 and 709 nm, Fluo—724 nm). In all cases, the (0–1) emissive vibronic band in the fluorescence spectrum is barely noticeable at *800 nm.
Photosynth Res
UF;Chlf ¼ UF;Chla
GradChlf ; GradChla
ð2Þ
where Grad is gradient of a linear function that simulates the dependency of fluorescence intensity (denoted as area under fluorescence spectrum) from concentration (denoted as absorbance at excitation wavelength) for a particular pigment. The results of determination of fluorescence quantum yield of Chl f are shown in Fig. 3. Quantum fluorescence yield of Chl f in Pyr calculated in the abovementioned way is 0.16 ± 0.01. Picosecond time-resolved fluorescence
Fig. 3 Determination of fluorescence quantum yield of Chl f using a relative method with Chl a as a fluorescence standard. a Fluorescence emission spectra of a concentration series of Chl a (blue) and Chl f (red) taken under the same experimental conditions. b Linear correlation of integrated fluorescence as a function of Chls absorbance at the excitation wavelength. The pigments were excited at the same wavelength (442 nm), and measurements were taken in degassed Pyr
Fluorescence quantum yield Fluorescence quantum yield determination for Chl f was performed in degassed Pyr using relative method with Chl a as the fluorescence quantum yield standard. According to this method, unknown fluorescence quantum yield of Chl f can be calculated based on Eq. 1 (Williams et al. 1983): EChlf =AChlf nChla 2 IChla UF;Chlf ¼ UF;Chla ; ð1Þ EChla =AChla nChlf IChlf where UF;Chla is the quantum fluorescence yield of standard (Chl a), A is absorbance of the solutions at excitation wavelengths, E is emission intensity, I is intensity of excitation light, and n is refractive index of the solutions. If both pigments are measured in the same solvent with the same excitation wavelength and intensity, and instead of one point, a series of different concentrations is done, the equation simplifies to
Time-resolved fluorescence profiles of Chl f recorded in Pyr, DE (RT), and in 2-MTHF (77 K) using the streak camera system are shown in Fig. 4. In order to obtain fluorescence decay lifetimes, the data were fitted using a global fitting procedure. The procedure employed a mono-exponentially decaying function convoluted with a Gaussian that mimics the IRF function. In the case of the TRF data taken in DE, a second short-lived decay component (not shown) was added to properly reconstruct scattering of the excitation beam visible as intense red spot in the 2D profile (Fig. 4b). The resulting decay components (DAFS) are shown in Fig. 4d, e along with their lifetimes. The representative kinetic traces of fluorescence decay probed at the maximum of the main fluorescence band accompanied with fits obtained from the global fitting and the real IRF function are given in Fig. 4g–i. Fitting results demonstrated that there is no significant difference in the fluorescence lifetime of Chl f between penta- and hexacoordinating solvents, at least in low pigment concentrations and very low laser intensity as used for the TRF experiment. On the other hand, the influence of low temperature is clearly noticeable, and the fluorescence lifetime lengthens *50 %. Femtosecond time-resolved absorption Results of TA spectroscopy and the data analysis are given in Fig. 5. Figure 5a–c shows individual TA spectra reordered at various delay times after excitation into Qy band. The spectra comprise multiple spectral features with the most evident negative bands coinciding with the Soret and Qy bands. The ratio between the bleaching of Soret and Qy bands is not preserved in the TA with respect to the steadystate absorption. This is due to the fact that the observed amplitude of the bleaching of the Qy band is greatly elevated by probe-driven stimulated emission that is indistinguishable by the detector. The emission spectrum is slightly shifted toward longer wavelengths overall, so that the ‘‘bleaching’’ of Qy band is also slightly (2–3 nm) ‘‘red-
123
Photosynth Res Fig. 4 Two-dimensional profiles of time-resolved fluorescence of Chl f in a Pyr, b DE at RT, and c 2-MTHF at 77 K taken by streak camera. The intense red dot in the DE data is associated with scattering of excitation laser beam. d–f DAFS spectra obtained from global fitting of the TRF datasets. Fluorescence lifetimes corresponding to the DAFS are also provided. g–i Representative kinetic traces of fluorescence emission decay taken at wavelengths corresponding to the maximum of the emission band. The red line represents monoexponential fit convoluted by Gaussian mimicking the IRF function (dashed line)
shifted’’ in respect to its position in the steady-state absorption. Results of global analysis performed using sequential decay path are shown in Fig. 5d, e. The fitting procedure resolved several kinetic components. Sub-nanosecond components may be associated with relaxation of the excited state and reorganization of the solvating medium upon promoting solvated molecules into the excited state. As can be expected, these effects are greatly reduced or become absent if the temperature is lowered to 77 K. The kinetic component with lifetime in the range of a few nanoseconds is associated with the decay of the first singlet excited state with which the Qy transition is associated. As can be seen, the values of lifetimes obtained from analysis of the TA datasets are, in the case of DE and 2-MTHF, significantly different from those obtained from TRF (DE: 5.6 ns vs. 4.7 ns and 2-MTHF: 8.5 ns vs. 5.1 ns) and are explained later in the text. Chl f triplet manifests in TA
123
fitting results as a non-decaying (in this time range) component. Figure 5g–i shows representative kinetic traces extracted from the TA datasets at Qy band and at wavelengths that exhibit gradual rise of DA signal as singlet excited state decays. This slowly appearing transient band is associated with the population of the chlorophyll triplet and represents the T1 ? Tn ESA band. Nanosecond time-resolved absorption This spectroscopic technique is particularly useful to monitor events that occur in micro/millisecond time regimes and fits perfectly to the time window of Chls triplet state lifetimes. In this case, the spectral profiles obtained from TA measurements are commonly called T-S (triplet-minus-singlet) spectra as they comprise the combination of the transient T1 ? Tn ESA band and bleaching of the ground state absorption. The T-S spectra obtained for Chl f in Pyr, DE
Photosynth Res Fig. 5 TA spectra of Chl f in a Pyr, b DE at RT, and c 2MTHF at 77 K taken at various delay times after excitation onto Qy band. d–f Results of global analysis (EADS) of the TA datasets shown in a–c. g–i Representative kinetic traces of recovery of the Qy band (709, 700, and 711 nm) and rise of the T1 ? Tn ESA band (575, 560, and 570 nm). The red and blue lines represent fits obtained from global analysis. The vertical scales have introduced gap breaks to better visualize the rise of the T1 ? Tn band
(RT), and 2-MTHF (77 K), taken 1 ls after excitation are shown in Fig. 6a–c. The profiles clearly resemble bleaching of Soret and Qy bands (negative) and T1 ? Tn ESA (positive) in range of 560–570 nm. This ESA band is not wellresolved in DE but becomes very prominent in 2-MTHF if the temperature is lowered to 77 K. Representative kinetic traces of the T1 ? Tn ESA band decay recorded at 570 nm are given in Fig. 6d, e. The red lines represent mono-exponential fits done on the raw data. The fits were not convoluted by the IRF function upon the assumption that the rise of the signal is essentially instantaneous and does not affect the decay. Triplet state lifetimes obtained from fitting are also provided in the graphs. The numbers that were obtained from fitting do not represent true, intrinsic triplet lifetimes and are commonly called as observed lifetimes that are characteristic for a certain pigment concentration (Connolly et al. 1982; Fujimori and Livingston 1957). In order to obtain the intrinsic triplet lifetime of Chl f, a series of lifetime measurements of various concentrations of the pigment are necessary. That is demonstrated in Fig. 7. It is apparent that the observed rate of triplet decay (reciprocal of lifetime) is
linearly dependent on the concentration of Chl f, denoted as absorbance measured at Qy band. The relation can be described as a linear function kTobs ¼ kTint þ k½Chlf where kTobs is observed, and kTint is intrinsic triplet depopulation rate, and k is a rate of diffusion of Chl f molecules in Pyr. The intrinsic triplet depopulation rate obtained here is 6.54 9 103 s-1, which corresponds to an intrinsic lifetime of 153 ± 3 ls.
Discussion Excited state properties of Chl f An overview of the excited state properties of Chl f that were obtained here is shown in Table 1. For comparison purposes, the selective properties (in Pyr) of three other chlorin-type Chls utilized in oxygenic photosynthesis (Chl a, b, and d) are also added. Previous comparative studies of excited state properties of Chls and BChls demonstrated that there is no particular relation between fluorescence
123
Photosynth Res Fig. 6 Triplet-minus-singlet (T-S) spectra of Chl f in a Pyr, b DE at RT, and c 2-MTHF at 77 K taken 1 ls after excitation at 355 nm. The samples were adjusted to absorbance at the Qy band of 0.3–0.4. d–f Representative kinetic traces of decay of the T1 ? Tn ESA band taken at wavelength marked by arrow (570 nm). The red lines represent mono-exponential fits. Triplet state lifetime (observed) values are also provided
Fig. 7 Observed triplet decay rates as a function of Chl f concentration (measured as absorbance of the Qy band) in Pyr. The fit (red solid line) was done according to equation kTobs ¼ kTint þ k½Chlf . The intrinsic triplet lifetime (concentration independent) of Chl f obtained from this method is 153 ± 3 ls
lifetime and energy of the Qy transition within chlorin-type Chls and BChls (Niedzwiedzki and Blankenship 2010). Instead, there is strong correlation of fluorescence lifetime with the presence of the –CHO group in the –R7 position on the macrocycle. The presence of formyl group (–CHO) in the –R7 position reduces the lifetime by a factor of two,
123
as is the case for Chl b and BChl e compared to all other representatives of the chlorin-type (B)Chl family (Niedzwiedzki and Blankenship 2010). Chl f also fits into this pattern. With a –CH3 group in the –R7 position, its singlet excited state lifetime is *5.6 ns at RT, which is similar to the *6.2 ns lifetime observed for most of the representatives of the chlorin-like (B)Chls (Niedzwiedzki and Blankenship 2010). On the other hand, the presence of the formyl group at the –R2 position (Chl a vs. Chl f) has almost no influence. Disagreements in the value of the singlet excited state lifetime between TA and TRF methods observed in DE and 2-MTHF are very likely caused by two different effects. In the case of DE (penta-coordinating solvent), Chl f molecules are able to aggregate spontaneously into dimers, and upon laser intensities used in TA, there is small chance of singlet–singlet annihilation effects that will decrease the singlet excited state lifetime. On the other hand, Chl f frozen in 2-MTHF should not show any differences between both methods due to the hexa-coordinating nature of the solvent. However, a difference is still evident. This is most likely due to the fact that the same pool of molecules is excited over the course of the experiment, and the sample is photobleached (physically damaged). This artificially induced gradual reduction in signal amplitude will combine with natural decay and in overall dynamics will show a faster decay than expected. Even though photobleaching occurs in TRF, it will not influence the results (lifetimes).
411, 440
2-MTHF (H)
406, 463
446, 473
697
655
671
709
694
706
Qy
705
662
677
724
709
722
Fluor. kmax (nm)
c
b
a
Obtained in penta-coordinating solvent
Observed (measured) triplet lifetime
Intrinsic triplet lifetime
(H) Mg hexa-coordinated, (P) Mg penta-coordinated
Pyr
Chl d
Pyr
Chl b
Chl a Pyr
393, 421, 443
396, 439
DE (P)
Pyr
413,443
Soret
Absorption kmax (nm)
Pyr (H)
Chl f
Solvent
6.2
3.2
6.3–6.5
8.5 ± 0.1
5.1 ± 0.1
5.6 ± 0.1
4.7 ± 0.1
5.6 ± 0.1
5.6 ± 0.1
sS (ns)
a
312a
556a
413a
500b
98b
153 ± 3
sT (ls)
*0.16c
0.35
0.16 ± 0.01
UF
TRF
TA
TRF
TA
TRF
TA
Method
293
293
293
77
77
77
293
293
293
293
293
293
T (K)
Niedzwiedzki and Blankenship (2010)
Brody and Rabinowitch (1957), Latimer et al. (1956) and Niedzwiedzki and Blankenship (2010)
Connolly et al. (1982), Hindman et al. (1978), Natarajan et al. (1984) and Niedzwiedzki and Blankenship (2010)
This study
Source
Table 1 Spectroscopic properties of Chl f obtained in this study and three other Chls employed in oxygenic photosynthesis (Chl a, b, and d) obtained in Pyr
Photosynth Res
123
Photosynth Res
In this method, the emitted photons are also collected over some period of time (to obtain TRF profile). However, if intensity of emission drops during the time of experiment, fluorescence dynamics remain unchanged. Unlike singlet-excited state lifetimes, triplet-excited state lifetimes show a strong correlation with energy of Qy transition, as it was demonstrated previously, and the relation could be justified based on the energy gap law (Niedzwiedzki and Blankenship 2010). Based on this, the triplet lifetime of Chl f should fall in the 130–310 ls range with higher probability to be close to *250 ls. The 153 ls result that was obtained, even though it fits into the range, it is likely underscored and needs more evaluation in the future as other parameters not tested here may affect its lifetime (e.g., excitation beam intensity). As demonstrated in Fig. 6, temperature is an important factor in the dynamics of triplet state decay. Cryogenic temperatures greatly enhance the lifetime of the triplet state (500 ls at 77 K vs. *100 ls at RT), probably through weakening of triplet-singlet coupling. It was previously proposed that Chl f is accumulated in light harvesting antenna proteins and simply complements Chl a in the light-harvesting process (Chen and Blankenship 2011). However, taking under consideration its spectroscopic properties, Chl f does not seem to be really suitable for the role of accessory pigment. Resonant energy transfer to Chl a either within the same antenna complex or in the reaction center will require a significant uphill energy transfer. Taken together with a relatively lowfluorescence quantum yield, the overall performance of Chl f-to-Chl a energy transfer process becomes very questionable. In order to understand the actual role of Chl f in photosynthesis of the H. hongdechloris cyanobacterium, further spectroscopic studies on Chl f-containing complexes are necessary. Acknowledgments This research was 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 DESC 0001035 to R.E.B. M.C. is an Australian Future Fellow and thanks Australia Research Council for financial support.
References Brody SS, Rabinowitch E (1957) Excitation lifetime of photosynthetic pigments in vitro and in vivo. Science 125:555 Chen M, Blankenship RE (2011) Expanding the solar spectrum used by photosynthesis. Trends Plant Sci 16:427–431 Chen M, Schliep M, Willows RD, Cai ZL, Neilan BA, Scheer H (2010) A red-shifted chlorophyll. Science 329:1318–1319
123
Chen M, Li YQ, Birch D, Willows RD (2012) A cyanobacterium that contains chlorophyll f—a red-absorbing photopigment. FEBS Lett 586:3249–3254 Connolly JS, Janzen AF, Samuel EB (1982) Fluorescence lifetimes of chlorophyll a—solvent, concentration and oxygen dependence. Photochem Photobiol 36:559–563 Fujimori E, Livingston R (1957) Interactions of chlorophyll in its triplet state with oxygen, carotene, etc. Nature 180:1036–1038 Gouterman M (1959) Study of the effects of substitution on the absorption spectra of porphyrin. J Chem Phys 30:1139–1161 Gouterman M (1961) Spectra of porphyrins. J Mol Spectrosc 6:138–163 Gouterman M (1978) Optical spectra and electronic structure of porphyrins and related rings. In: Dolphin D (ed) Physical chemistry: Part A. Academic Press, New York, pp 1–166 Hindman JC, Kugel R, Svirmickas A, Katz JJ (1978) Stimulated fluorescence and fluorescence quenching in chlorophyll a and bacteriochlorophyll a. Chem Phys Lett 53:197–200 Latimer P, Bannister TT, Rabinowitch E (1956) Quantum yields of fluorescence of plant pigments. Science 124:585–586 Li YQ, Scales N, Blankenship RE, Willows RD, Chen M (2012) Extinction coefficient for red-shifted chlorophylls: chlorophyll d and chlorophyll f. Biochim Biophys Acta 1817:1292–1298 Li Y, Cai ZL, Chen M (2013) Spectroscopic properties of chlorophyll f. J Phys Chem B 117:11309–11317 Manning WM, Strain HH (1943) Chlorophyll d, a green pigment of red algae. J Biol Chem 151:1–19 Miyashita H, Ikemoto H, Kurano N, Adachi K, Chihara M, Miyachi S (1996) Chlorophyll d as a major pigment. Nature 383:402 Natarajan LV, Ricker JE, Blankenship RE, Chang R (1984) Solvent influences on the singlet quenching of chlorophyll a by 2,5dimethyl-para-benzoquinone. Photochem Photobiol 39:301–306 Niedzwiedzki DM, Blankenship RE (2010) Singlet and triplet excited state properties of natural chlorophylls and bacteriochlorophylls. Photosynth Res 106:227–238 Niedzwiedzki DM, Fuciman M, Frank HA, Blankenship RE (2011) Energy transfer in an LH4-like light harvesting complex from the aerobic purple photosynthetic bacterium Roseobacter denitrificans. Biochim Biophys Acta 1807:518–528 Pelletier PJ, Caventou JB (1818) Sur la matie`re verte des feuilles. Ann Chim Phys 9:194–196 Strain HH (1958) Chloroplast pigments and chromatographic analysis. Pennsylvania State University, University Park van Stokkum IH, Larsen DS, van Grondelle R (2004) Global and target analysis of time-resolved spectra. Biochim Biophys Acta 1657:82–104 Weiss C (1972) p-electron structure and absorption spectra of chlorophylls in solution. J Mol Spectrosc 44:37–80 Williams ATR, Winfield SA, Miller JN (1983) Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer. Analyst 108:1067–1071 Yamijala SRKCS, Periyasamy G, Pati SK (2011) Computational studies on structural and excited-state properties of modified chlorophyll f with various axial ligands. J Phys Chem A 115:12298–12306 Zapata M, Garrido J, Jeffrey SW (2006) Chlorophyll c pigments: current status. In: Grimm B, Porra RJ, Rudiger W, Scheer H (eds) Chlorophylls and bacteriochlorophylls: biochemistry, biophysics, function and applications (advances in photosynthesis and respiration). Springer, New York, pp 39–53
REGULAR PAPER
Excited state properties of chlorophyll f in organic solvents at ambient and cryogenic temperatures Dariusz M. Niedzwiedzki • Haijun Liu Min Chen • Robert E. Blankenship
•
Received: 13 November 2013 / Accepted: 5 February 2014 Springer Science+Business Media Dordrecht 2014
Abstract Chlorophyll f is a photosynthetic pigment that was discovered in 2010. In this study, we present investigations on spectral and dynamic characteristics of singletexcited and triplet states of Chl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The pigment was studied at room temperature in two organic solvents: pyridine and diethyl ether that have different characters of coordination of the chlorophyll magnesium (Mg) atom (hexa- and penta-coordination, respectively). Cryogenic measurements (77 K) were performed in 2-methyltetrahydrofuran (hexa-coordination). The singlet-excited state lifetime was measured to be 5.6 ns at room temperature regardless of Mg coordination and 8.1 ns at 77 K. The fluorescence quantum yield of Chl f was also determined in pyridine to be 0.16. The triplet state lifetime was studied in detail in pyridine at room temperature, and the inherent lifetime was estimated to *150 ls. Selective measurements at 77 K demonstrated that the
D. M. Niedzwiedzki (&) H. Liu R. E. Blankenship Photosynthetic Antenna Research Center, Washington University in St. Louis, Campus Box 1138, St. Louis, MO 63130, USA e-mail: [email protected] H. Liu R. E. Blankenship Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA M. Chen School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia R. E. Blankenship Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA
metastability of the triplet state greatly enhances, and its lifetime increases by a factor of more than three. Keywords Chlorophyll f Transient absorption Singlet-excited state Triplet state Time-resolved fluorescence Halomicronemona hongdechloris Abbreviations 2-MTHF 2-Methyl tetrahydrofuran CCD Charge couple device Chl Chlorophyll DE Diethyl ether EADS Evolution-associated difference spectra ESA Excited state absorption FWHM Full width at half maximum HOMO Highest occupied molecular orbital ICP-MS Inductively coupled plasma-mass spectrometry LUMO Lowest unoccupied molecular orbital OPO Optical parametric oscillator Pyr Pyridine RT Room temperature TA Transient absorption TRF Time-resolved fluorescence T-S Triplet-minus-singlet
Introduction Chlorophylls (Chls) serve several important roles in the process of photosynthesis including light absorption, excitation transfer, and primary charge separation in reaction centers. Basic spectroscopic properties of Chls can be deduced from Gouterman’s ‘‘four-orbital’’ model that assumes that the major bands of electronic absorption are
123
Photosynth Res
Fig. 1 Chemical structures of Chl a, b, d, and f and their room temperature steady-state absorption spectra taken in Pyr. Absorption spectra of Chl a, b, and d were taken from Niedzwiedzki and Blankenship (2010)
associated with p ? p* electronic transitions that derive from electron promotions involving the two HOMOs and two LUMOs. The resulting electronic transitions are polarized along either the x or y-axis of the macrocycle (Fig. 1) and are referred to Qy, Qx, By, and Bx bands (Gouterman 1959, 1961, 1978; Weiss 1972). In Chls, B transitions appear between 400 and 500 nm and are collectively called the Soret band. The Qy band can span the spectral region from 620 to 700 nm, depending on the degree of macrocycle saturation and the presence of specific side chain modifications. The Qx band is greatly reduced in amplitude but is expected to appear between 500 and 600 nm. Chls can be divided into two subgroups characterized by the degree of unsaturation of the macrocycle: porphyrin-type (fully unsaturated macrocycle) containing group of Chl c pigments (c1, c2, c3, and numerous clike forms), and chlorin-type (single bond between C-17 and C-18 carbons in the macrocycle) that comprises Chl a, b, d, and f. Chlorophylls: a and b (Fig. 1) were discovered by two French chemists, Caventou and Pelletier in 1818 (Pelletier and Caventou 1818) who also introduced the name ‘‘chlorophyll’’ from a combination of the Greek words, chloros (green) and phyllos (leaves). Chl c was discovered in the early 1900s in aquatic algae, and currently, the name
123
embraces a group of many sub-types (Chl c1, c2, etc.) (Zapata et al. 2006). The next representative, Chl d (Fig. 1), was first reported in the 1940s in red algae (Manning and Strain 1943) but for a long time was considered to be an extraction artifact. In 1996, a marine cyanobacterium named Acaryochloris marina, containing almost exclusively Chl, d was discovered (Miyashita et al. 1996). Chl e was reported in 1943 as a minor pigment in algae Tribonema bombycinum (Strain 1958) with absorption bands at 415 and 654 nm in methanol; however, its structure and properties have never been elucidated, and until now its status remains uncertain. The latest member of the Chls family, Chl f (Fig. 1), was discovered in 2010 in cyanobacteria collected from a stromatolite colony from Shark Bay in Western Australia and is the most red-shifted Chl used in oxygenic photosynthesis (Chen et al. 2010). The cyanobacterium containing Chl f was isolated and named Halomicronemona hongdechloris (Chen et al. 2012). Chl f is not a major photosynthetic pigment in this organism and accounts only for maximum of 15 % of total Chls (with majority as Chl a) and is produced only upon growth in far-red light. It is hypothesized that Chl f is an accessory pigment in the photosynthetic process. However, that role will require a significant uphill energy transfer toward Chl a in the reaction center; thus, it is still under debate (Chen and Blankenship 2011). Even though computational studies of excited state properties of modified Chl f interacting with various axial ligands (Mg penta-coordinated) have been performed (Yamijala et al. 2011), experimentally the photophysical properties of Chl f are limited to detailed studies of properties of steady-state absorption, but more advanced studies have not been elucidated (Li et al. 2013). In addition, ICPMS spectroscopy was used to measure Mg content and obtain pigment extinction coefficients in various organic solvents (Li et al. 2012). Here, we present more detailed spectroscopic studies of Chl f in organic solvents with different Mg coordination characters (hexa- and penta-coordination) at room temperature and at 77 K. The studies utilize a combination of steady-state and time-resolved absorption and fluorescence techniques in order to obtain more insights to the spectral and temporal characteristics of the singlet- and tripletexcited states.
Materials and methods Culture growth Initially H. hongdechloris cells were kept on 1 % agar of K ? ES seawater solid plates (Chen et al. 2012; Miyashita et al. 1996) and cultured for 4 weeks under continuous
Photosynth Res
illumination of white-fluorescent light with an intensity of 5 lmol photons m-2 s-1 in order to accumulate more biomass. Subsequently, cultures were transferred to far-red light (740 nm, 27 nm spectral half width, same intensity), produced by lamps equipped with 17 W LumiBulb-FR (LumiBulb-Far RedTM bulb, LumiGrow, CA, USA). Isolation of Chl f Chl f was isolated from pelleted cells as follows. The pigment was extracted with 100 % technical grade methanol. The supernatant was removed and loaded directly into an Agilent Series 1100 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a reversephase Zorbax C-18 column (4.6 9 250 mm, Agilent Technologies Inc., Santa Clara, CA, USA) operated at 20 C. Pigment was eluted with 100 % methanol (Sigma, St. Louis, USA) pumped at a rate of 1.5 mL min-1. The major HPLC peak with Chl f spectral signature was collected (the sample is assumed to be free from Chl f0 forms), then dried under a stream of nitrogen gas, and stored under nitrogen in the dark at -20 C until needed. Samples preparation for spectroscopic measurements Purified Chl f was dissolved in Pyr, DE, or 2-MTHF (spectroscopic grade) prior to spectroscopic measurements. Measurements at 77 K were taken only in the 2-MTHF by cooling the sample with liquid nitrogen in a Janis SVP-100 Cryostat (Janis, Woburn, MA, USA). Femtosecond timeresolved absorption and picosecond time-resolved fluorescence measurements were performed on air-saturated samples. For triplet dynamics measurements samples were prepared by applying pump–freeze–thaw procedure as described previously (Niedzwiedzki and Blankenship 2010) under a vacuum quality of better than 1 mTorr compared to typical atmospheric pressure of 760 Torr. Samples were kept in vacuum conditions during spectroscopic measurements. Femtosecond time-resolved transient absorption spectroscopy Time-resolved pump-probe absorption experiments of Chl 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 described in detail previously (Niedzwiedzki et al. 2011). The samples were excited at Qy band (710 nm in Pyr, 695 nm in DE, 710 nm in 2-MTHF) with pump beam energy of 500 nJ in a spot size of 1-mm diameter corresponding to an intensity of
*2 9 1014 photons cm-2 per pulse with frequency of 1 kHz. The samples OD was adjusted to be 0.3–0.5 at Qy maximum (in 2-mm-path cuvette) corresponding to molecule concentration of 15–26 9 10-6 mol L-1 [9 9 1012 molecules lL-1) (e695 = 96.62 9 103 L mol-1 cm-1in DE was used (Li et al. 2012)]. Under the assumption that an individual laser pulse (2 9 1012 photons mm-2) will excite a (*0.8 mm2 9 2 mm) cylinder of the sample with volume of *1.6 mm3, which will contain *1.4 9 1013 molecules of Chl f, the photon-to-molecule ratio is *0.1. Even though this value is small, in penta-coordinating solvent like DE in which the dimeric form of Chl f can be formed, the chance of singlet–singlet annihilation effect is still possible and may slightly distort (decrease) the observed singlet-excited state lifetime. 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 an 250IS imaging spectrograph from Bruker. The emitted light was dispersed on 150 g mm-1 grading blazed at 800 nm. For focusing of the excitation beam on the sample and emitted light on the spectrograph standard optics setup, A8110-01 from Hamamatsu was used. The slit at spectrograph was set to 100 lm corresponding to 2 nm resolution. Excitation pulses were produced by Inspire 100, an ultrafast optical parametric oscillator (OPO; Radiantis-Spectra-Physics, 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. The excitation beam with power of *10 lW was focused on the sample in a circular spot of 0.5-mm diameter, corresponding to a photon intensity of *2 9 1010 photons cm-2 per pulse. The sample concentration was adjusted to *3 9 10-6 mol L-1 (1.8 9 1012 molecules lL-1) (based on OD *0.3 at the Qy band in a 1-cm cuvette; molar extinction coefficient in DE). These conditions correspond to a photon-to-molecule ratio of 0.005, which assures that in DE (Chl f may form dimers) fluorescence dynamics should be free of singlet–singlet annihilation. To assure an 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 excitation beam focus point was adjusted to be very close to the cuvette wall (1–3 mm from it) that was used to measure emission (at
123
Photosynth Res
right angle) so that self-absorption was practically negligible in all cases (maximum absorbance of 1–3 mm layer of sample is between 0.03 and 0.06 in the Qy band). Microsecond flash photolysis spectroscopy Experiments were performed on samples in a 1-cm-pathlength 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 10 Hz Nd:YAG laser (Quantel) producing 6-ns-duration laser pulses, second and third harmonic generators, and OPO (Opotek, Carlsbad, CA, USA). Energy of the excitation beam (1-cm dimension) was set to *10 mJ consistent to a photon density of *1016 photons cm-2 per pulse, and the sample was excited at 1 Hz frequency. The absorbance value at the maximum of the Qy band was adjusted to 0.3–0.4 in 1-cm path, which corresponds to a concentration of 3–4 9 10-6 mol L-1 (1.8 9 1015 molecules mL-1). Because the sample volume of 0.8 mL (*0.8 cm2 9 1 cm) will be excited, the photon-to-molecule ratio will be *7, and in this case, Chl f triplet dynamics that are taken in DE may be easily affected. 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. In order to obtain good quality results, the T-S spectra and triplet decay kinetics traces were averaged 5–20 times. Sample integrity was checked by taking steady-state absorption spectra before and after every experiment. Transient data processing and global analysis Group velocity dispersion of the TA spectra was corrected using Surface Xplorer 2.0 (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 datasets was performed using a custom modified version of ASUfit (http://www.public.asu.edu/ *laserweb/asufit/asufit.html). The FWHM 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 so that the reverse reaction rates are negligible. The spectral profiles obtained from this fitting of the TA datasets are termed Evolution-Associated Difference Spectra (EADS) (van
123
Fig. 2 Steady-state absorption and fluorescence spectra of Chl f in Pyr (black), DE (red) at RT, and 2-MTHF (blue) at 77 K
Stokkum et al. 2004). The sequential model quite realistically represents excitation decay path of Chl. Timeresolved fluorescence data of Chl f were first cleared of random noise by recomposing the data from the dominant principal components using singular value decomposition (van Stokkum et al. 2004) and then fitted globally with mono-exponentially decaying spectral-kinetic component convoluted by Gaussians with FWHM equal to that of the real IRF. The resulting spectrum is called Decay-Associated Fluorescence Spectrum (DAFS), which in shape is practically identical to steady-state fluorescence. The IRF of the streak camera tube was obtained by recording the profile of the scattered excitation laser beam in an appropriate streak camera time window.
Results Steady-state absorption and fluorescence Steady-state absorption and fluorescence spectra of Chl f taken in three different environments are given in Fig. 2. In Pyr (hexa-coordinating solvent) the major absorption bands, Soret (By, Bx) and Qy, appear at, respectively, 413, 443, and 706 nm. The fluorescence spectrum is red shifted in respect to the Qy absorption band by 17 nm (723 nm, Stokes shift of *330 cm-1). Upon changing the environment to penta-coordinating DE, a substantial change in shapes and positions of the main bands (396, 439, and 694 nm) is observed. The fluorescence emission spectrum (706 nm) exhibits a slightly smaller Stokes shift (*280 cm-1). Upon lowering the temperature to 77 K (2-MTHF, hexa-coordinating solvent), some enhancement in spectral resolution is observed (bands narrow), but the positions of the individual transitions (absorption or emission) are very close to those recoded for Pyr at RT (Abs—410, 439 and 709 nm, Fluo—724 nm). In all cases, the (0–1) emissive vibronic band in the fluorescence spectrum is barely noticeable at *800 nm.
Photosynth Res
UF;Chlf ¼ UF;Chla
GradChlf ; GradChla
ð2Þ
where Grad is gradient of a linear function that simulates the dependency of fluorescence intensity (denoted as area under fluorescence spectrum) from concentration (denoted as absorbance at excitation wavelength) for a particular pigment. The results of determination of fluorescence quantum yield of Chl f are shown in Fig. 3. Quantum fluorescence yield of Chl f in Pyr calculated in the abovementioned way is 0.16 ± 0.01. Picosecond time-resolved fluorescence
Fig. 3 Determination of fluorescence quantum yield of Chl f using a relative method with Chl a as a fluorescence standard. a Fluorescence emission spectra of a concentration series of Chl a (blue) and Chl f (red) taken under the same experimental conditions. b Linear correlation of integrated fluorescence as a function of Chls absorbance at the excitation wavelength. The pigments were excited at the same wavelength (442 nm), and measurements were taken in degassed Pyr
Fluorescence quantum yield Fluorescence quantum yield determination for Chl f was performed in degassed Pyr using relative method with Chl a as the fluorescence quantum yield standard. According to this method, unknown fluorescence quantum yield of Chl f can be calculated based on Eq. 1 (Williams et al. 1983): EChlf =AChlf nChla 2 IChla UF;Chlf ¼ UF;Chla ; ð1Þ EChla =AChla nChlf IChlf where UF;Chla is the quantum fluorescence yield of standard (Chl a), A is absorbance of the solutions at excitation wavelengths, E is emission intensity, I is intensity of excitation light, and n is refractive index of the solutions. If both pigments are measured in the same solvent with the same excitation wavelength and intensity, and instead of one point, a series of different concentrations is done, the equation simplifies to
Time-resolved fluorescence profiles of Chl f recorded in Pyr, DE (RT), and in 2-MTHF (77 K) using the streak camera system are shown in Fig. 4. In order to obtain fluorescence decay lifetimes, the data were fitted using a global fitting procedure. The procedure employed a mono-exponentially decaying function convoluted with a Gaussian that mimics the IRF function. In the case of the TRF data taken in DE, a second short-lived decay component (not shown) was added to properly reconstruct scattering of the excitation beam visible as intense red spot in the 2D profile (Fig. 4b). The resulting decay components (DAFS) are shown in Fig. 4d, e along with their lifetimes. The representative kinetic traces of fluorescence decay probed at the maximum of the main fluorescence band accompanied with fits obtained from the global fitting and the real IRF function are given in Fig. 4g–i. Fitting results demonstrated that there is no significant difference in the fluorescence lifetime of Chl f between penta- and hexacoordinating solvents, at least in low pigment concentrations and very low laser intensity as used for the TRF experiment. On the other hand, the influence of low temperature is clearly noticeable, and the fluorescence lifetime lengthens *50 %. Femtosecond time-resolved absorption Results of TA spectroscopy and the data analysis are given in Fig. 5. Figure 5a–c shows individual TA spectra reordered at various delay times after excitation into Qy band. The spectra comprise multiple spectral features with the most evident negative bands coinciding with the Soret and Qy bands. The ratio between the bleaching of Soret and Qy bands is not preserved in the TA with respect to the steadystate absorption. This is due to the fact that the observed amplitude of the bleaching of the Qy band is greatly elevated by probe-driven stimulated emission that is indistinguishable by the detector. The emission spectrum is slightly shifted toward longer wavelengths overall, so that the ‘‘bleaching’’ of Qy band is also slightly (2–3 nm) ‘‘red-
123
Photosynth Res Fig. 4 Two-dimensional profiles of time-resolved fluorescence of Chl f in a Pyr, b DE at RT, and c 2-MTHF at 77 K taken by streak camera. The intense red dot in the DE data is associated with scattering of excitation laser beam. d–f DAFS spectra obtained from global fitting of the TRF datasets. Fluorescence lifetimes corresponding to the DAFS are also provided. g–i Representative kinetic traces of fluorescence emission decay taken at wavelengths corresponding to the maximum of the emission band. The red line represents monoexponential fit convoluted by Gaussian mimicking the IRF function (dashed line)
shifted’’ in respect to its position in the steady-state absorption. Results of global analysis performed using sequential decay path are shown in Fig. 5d, e. The fitting procedure resolved several kinetic components. Sub-nanosecond components may be associated with relaxation of the excited state and reorganization of the solvating medium upon promoting solvated molecules into the excited state. As can be expected, these effects are greatly reduced or become absent if the temperature is lowered to 77 K. The kinetic component with lifetime in the range of a few nanoseconds is associated with the decay of the first singlet excited state with which the Qy transition is associated. As can be seen, the values of lifetimes obtained from analysis of the TA datasets are, in the case of DE and 2-MTHF, significantly different from those obtained from TRF (DE: 5.6 ns vs. 4.7 ns and 2-MTHF: 8.5 ns vs. 5.1 ns) and are explained later in the text. Chl f triplet manifests in TA
123
fitting results as a non-decaying (in this time range) component. Figure 5g–i shows representative kinetic traces extracted from the TA datasets at Qy band and at wavelengths that exhibit gradual rise of DA signal as singlet excited state decays. This slowly appearing transient band is associated with the population of the chlorophyll triplet and represents the T1 ? Tn ESA band. Nanosecond time-resolved absorption This spectroscopic technique is particularly useful to monitor events that occur in micro/millisecond time regimes and fits perfectly to the time window of Chls triplet state lifetimes. In this case, the spectral profiles obtained from TA measurements are commonly called T-S (triplet-minus-singlet) spectra as they comprise the combination of the transient T1 ? Tn ESA band and bleaching of the ground state absorption. The T-S spectra obtained for Chl f in Pyr, DE
Photosynth Res Fig. 5 TA spectra of Chl f in a Pyr, b DE at RT, and c 2MTHF at 77 K taken at various delay times after excitation onto Qy band. d–f Results of global analysis (EADS) of the TA datasets shown in a–c. g–i Representative kinetic traces of recovery of the Qy band (709, 700, and 711 nm) and rise of the T1 ? Tn ESA band (575, 560, and 570 nm). The red and blue lines represent fits obtained from global analysis. The vertical scales have introduced gap breaks to better visualize the rise of the T1 ? Tn band
(RT), and 2-MTHF (77 K), taken 1 ls after excitation are shown in Fig. 6a–c. The profiles clearly resemble bleaching of Soret and Qy bands (negative) and T1 ? Tn ESA (positive) in range of 560–570 nm. This ESA band is not wellresolved in DE but becomes very prominent in 2-MTHF if the temperature is lowered to 77 K. Representative kinetic traces of the T1 ? Tn ESA band decay recorded at 570 nm are given in Fig. 6d, e. The red lines represent mono-exponential fits done on the raw data. The fits were not convoluted by the IRF function upon the assumption that the rise of the signal is essentially instantaneous and does not affect the decay. Triplet state lifetimes obtained from fitting are also provided in the graphs. The numbers that were obtained from fitting do not represent true, intrinsic triplet lifetimes and are commonly called as observed lifetimes that are characteristic for a certain pigment concentration (Connolly et al. 1982; Fujimori and Livingston 1957). In order to obtain the intrinsic triplet lifetime of Chl f, a series of lifetime measurements of various concentrations of the pigment are necessary. That is demonstrated in Fig. 7. It is apparent that the observed rate of triplet decay (reciprocal of lifetime) is
linearly dependent on the concentration of Chl f, denoted as absorbance measured at Qy band. The relation can be described as a linear function kTobs ¼ kTint þ k½Chlf where kTobs is observed, and kTint is intrinsic triplet depopulation rate, and k is a rate of diffusion of Chl f molecules in Pyr. The intrinsic triplet depopulation rate obtained here is 6.54 9 103 s-1, which corresponds to an intrinsic lifetime of 153 ± 3 ls.
Discussion Excited state properties of Chl f An overview of the excited state properties of Chl f that were obtained here is shown in Table 1. For comparison purposes, the selective properties (in Pyr) of three other chlorin-type Chls utilized in oxygenic photosynthesis (Chl a, b, and d) are also added. Previous comparative studies of excited state properties of Chls and BChls demonstrated that there is no particular relation between fluorescence
123
Photosynth Res Fig. 6 Triplet-minus-singlet (T-S) spectra of Chl f in a Pyr, b DE at RT, and c 2-MTHF at 77 K taken 1 ls after excitation at 355 nm. The samples were adjusted to absorbance at the Qy band of 0.3–0.4. d–f Representative kinetic traces of decay of the T1 ? Tn ESA band taken at wavelength marked by arrow (570 nm). The red lines represent mono-exponential fits. Triplet state lifetime (observed) values are also provided
Fig. 7 Observed triplet decay rates as a function of Chl f concentration (measured as absorbance of the Qy band) in Pyr. The fit (red solid line) was done according to equation kTobs ¼ kTint þ k½Chlf . The intrinsic triplet lifetime (concentration independent) of Chl f obtained from this method is 153 ± 3 ls
lifetime and energy of the Qy transition within chlorin-type Chls and BChls (Niedzwiedzki and Blankenship 2010). Instead, there is strong correlation of fluorescence lifetime with the presence of the –CHO group in the –R7 position on the macrocycle. The presence of formyl group (–CHO) in the –R7 position reduces the lifetime by a factor of two,
123
as is the case for Chl b and BChl e compared to all other representatives of the chlorin-type (B)Chl family (Niedzwiedzki and Blankenship 2010). Chl f also fits into this pattern. With a –CH3 group in the –R7 position, its singlet excited state lifetime is *5.6 ns at RT, which is similar to the *6.2 ns lifetime observed for most of the representatives of the chlorin-like (B)Chls (Niedzwiedzki and Blankenship 2010). On the other hand, the presence of the formyl group at the –R2 position (Chl a vs. Chl f) has almost no influence. Disagreements in the value of the singlet excited state lifetime between TA and TRF methods observed in DE and 2-MTHF are very likely caused by two different effects. In the case of DE (penta-coordinating solvent), Chl f molecules are able to aggregate spontaneously into dimers, and upon laser intensities used in TA, there is small chance of singlet–singlet annihilation effects that will decrease the singlet excited state lifetime. On the other hand, Chl f frozen in 2-MTHF should not show any differences between both methods due to the hexa-coordinating nature of the solvent. However, a difference is still evident. This is most likely due to the fact that the same pool of molecules is excited over the course of the experiment, and the sample is photobleached (physically damaged). This artificially induced gradual reduction in signal amplitude will combine with natural decay and in overall dynamics will show a faster decay than expected. Even though photobleaching occurs in TRF, it will not influence the results (lifetimes).
411, 440
2-MTHF (H)
406, 463
446, 473
697
655
671
709
694
706
Qy
705
662
677
724
709
722
Fluor. kmax (nm)
c
b
a
Obtained in penta-coordinating solvent
Observed (measured) triplet lifetime
Intrinsic triplet lifetime
(H) Mg hexa-coordinated, (P) Mg penta-coordinated
Pyr
Chl d
Pyr
Chl b
Chl a Pyr
393, 421, 443
396, 439
DE (P)
Pyr
413,443
Soret
Absorption kmax (nm)
Pyr (H)
Chl f
Solvent
6.2
3.2
6.3–6.5
8.5 ± 0.1
5.1 ± 0.1
5.6 ± 0.1
4.7 ± 0.1
5.6 ± 0.1
5.6 ± 0.1
sS (ns)
a
312a
556a
413a
500b
98b
153 ± 3
sT (ls)
*0.16c
0.35
0.16 ± 0.01
UF
TRF
TA
TRF
TA
TRF
TA
Method
293
293
293
77
77
77
293
293
293
293
293
293
T (K)
Niedzwiedzki and Blankenship (2010)
Brody and Rabinowitch (1957), Latimer et al. (1956) and Niedzwiedzki and Blankenship (2010)
Connolly et al. (1982), Hindman et al. (1978), Natarajan et al. (1984) and Niedzwiedzki and Blankenship (2010)
This study
Source
Table 1 Spectroscopic properties of Chl f obtained in this study and three other Chls employed in oxygenic photosynthesis (Chl a, b, and d) obtained in Pyr
Photosynth Res
123
Photosynth Res
In this method, the emitted photons are also collected over some period of time (to obtain TRF profile). However, if intensity of emission drops during the time of experiment, fluorescence dynamics remain unchanged. Unlike singlet-excited state lifetimes, triplet-excited state lifetimes show a strong correlation with energy of Qy transition, as it was demonstrated previously, and the relation could be justified based on the energy gap law (Niedzwiedzki and Blankenship 2010). Based on this, the triplet lifetime of Chl f should fall in the 130–310 ls range with higher probability to be close to *250 ls. The 153 ls result that was obtained, even though it fits into the range, it is likely underscored and needs more evaluation in the future as other parameters not tested here may affect its lifetime (e.g., excitation beam intensity). As demonstrated in Fig. 6, temperature is an important factor in the dynamics of triplet state decay. Cryogenic temperatures greatly enhance the lifetime of the triplet state (500 ls at 77 K vs. *100 ls at RT), probably through weakening of triplet-singlet coupling. It was previously proposed that Chl f is accumulated in light harvesting antenna proteins and simply complements Chl a in the light-harvesting process (Chen and Blankenship 2011). However, taking under consideration its spectroscopic properties, Chl f does not seem to be really suitable for the role of accessory pigment. Resonant energy transfer to Chl a either within the same antenna complex or in the reaction center will require a significant uphill energy transfer. Taken together with a relatively lowfluorescence quantum yield, the overall performance of Chl f-to-Chl a energy transfer process becomes very questionable. In order to understand the actual role of Chl f in photosynthesis of the H. hongdechloris cyanobacterium, further spectroscopic studies on Chl f-containing complexes are necessary. Acknowledgments This research was 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 DESC 0001035 to R.E.B. M.C. is an Australian Future Fellow and thanks Australia Research Council for financial support.
References Brody SS, Rabinowitch E (1957) Excitation lifetime of photosynthetic pigments in vitro and in vivo. Science 125:555 Chen M, Blankenship RE (2011) Expanding the solar spectrum used by photosynthesis. Trends Plant Sci 16:427–431 Chen M, Schliep M, Willows RD, Cai ZL, Neilan BA, Scheer H (2010) A red-shifted chlorophyll. Science 329:1318–1319
123
Chen M, Li YQ, Birch D, Willows RD (2012) A cyanobacterium that contains chlorophyll f—a red-absorbing photopigment. FEBS Lett 586:3249–3254 Connolly JS, Janzen AF, Samuel EB (1982) Fluorescence lifetimes of chlorophyll a—solvent, concentration and oxygen dependence. Photochem Photobiol 36:559–563 Fujimori E, Livingston R (1957) Interactions of chlorophyll in its triplet state with oxygen, carotene, etc. Nature 180:1036–1038 Gouterman M (1959) Study of the effects of substitution on the absorption spectra of porphyrin. J Chem Phys 30:1139–1161 Gouterman M (1961) Spectra of porphyrins. J Mol Spectrosc 6:138–163 Gouterman M (1978) Optical spectra and electronic structure of porphyrins and related rings. In: Dolphin D (ed) Physical chemistry: Part A. Academic Press, New York, pp 1–166 Hindman JC, Kugel R, Svirmickas A, Katz JJ (1978) Stimulated fluorescence and fluorescence quenching in chlorophyll a and bacteriochlorophyll a. Chem Phys Lett 53:197–200 Latimer P, Bannister TT, Rabinowitch E (1956) Quantum yields of fluorescence of plant pigments. Science 124:585–586 Li YQ, Scales N, Blankenship RE, Willows RD, Chen M (2012) Extinction coefficient for red-shifted chlorophylls: chlorophyll d and chlorophyll f. Biochim Biophys Acta 1817:1292–1298 Li Y, Cai ZL, Chen M (2013) Spectroscopic properties of chlorophyll f. J Phys Chem B 117:11309–11317 Manning WM, Strain HH (1943) Chlorophyll d, a green pigment of red algae. J Biol Chem 151:1–19 Miyashita H, Ikemoto H, Kurano N, Adachi K, Chihara M, Miyachi S (1996) Chlorophyll d as a major pigment. Nature 383:402 Natarajan LV, Ricker JE, Blankenship RE, Chang R (1984) Solvent influences on the singlet quenching of chlorophyll a by 2,5dimethyl-para-benzoquinone. Photochem Photobiol 39:301–306 Niedzwiedzki DM, Blankenship RE (2010) Singlet and triplet excited state properties of natural chlorophylls and bacteriochlorophylls. Photosynth Res 106:227–238 Niedzwiedzki DM, Fuciman M, Frank HA, Blankenship RE (2011) Energy transfer in an LH4-like light harvesting complex from the aerobic purple photosynthetic bacterium Roseobacter denitrificans. Biochim Biophys Acta 1807:518–528 Pelletier PJ, Caventou JB (1818) Sur la matie`re verte des feuilles. Ann Chim Phys 9:194–196 Strain HH (1958) Chloroplast pigments and chromatographic analysis. Pennsylvania State University, University Park van Stokkum IH, Larsen DS, van Grondelle R (2004) Global and target analysis of time-resolved spectra. Biochim Biophys Acta 1657:82–104 Weiss C (1972) p-electron structure and absorption spectra of chlorophylls in solution. J Mol Spectrosc 44:37–80 Williams ATR, Winfield SA, Miller JN (1983) Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer. Analyst 108:1067–1071 Yamijala SRKCS, Periyasamy G, Pati SK (2011) Computational studies on structural and excited-state properties of modified chlorophyll f with various axial ligands. J Phys Chem A 115:12298–12306 Zapata M, Garrido J, Jeffrey SW (2006) Chlorophyll c pigments: current status. In: Grimm B, Porra RJ, Rudiger W, Scheer H (eds) Chlorophylls and bacteriochlorophylls: biochemistry, biophysics, function and applications (advances in photosynthesis and respiration). Springer, New York, pp 39–53
