Spectroscopic Properties of Chlorophyll f - The Journal of Physical

Article pubs.acs.org/JPCB

Spectroscopic Properties of Chlorophyll f Yaqiong Li,† Zheng-Li Cai,‡ and Min Chen*,† †

School of Biological Sciences, University of Sydney, NSW 2006, Australia School of Physics, University of New South Wales, NSW 2052, Australia

‡

S Supporting Information *

ABSTRACT: The absorption and fluorescence spectra of chlorophyll f (newly discovered in 2010) have been measured in acetone and methanol at different temperatures. The spectral analysis and assignment are compared with the spectra of chlorophyll a and d under the same experimental conditions. The spectroscopic properties of these chlorophylls have further been studied by the aid of density functional CAM-B3LYP and high-level symmetric adapted coupledcluster configuration interaction calculations. The main Q and Soret bands and possible sidebands of chlorophylls have been determined. The photophysical properties of chlorophyll f are discussed.

1. INTRODUCTION Chlorophylls are essential molecules for collecting and transduction of light energy in photosynthetic systems. There are five known types of chlorophylls with chlorophyll f (Chl f) as the newest member in this family, discovered in 2010.1 Chl f (Figure 1) has a similar chemical structure to Chl a with the notable exception of the C-2 position, where the methyl group in Chl a is replaced by a formyl group in Chl f .2 This difference causes the Qy absorption band maximum to shift to a longer wavelength, from 665 nm for Chl a to 707 nm for Chl f.1,3 A wavelength redshifted more than 40 nm from that of Chl a makes Chl f the most red-shifted chlorophyll found in oxygenic photosynthetic organisms. Chlorophylls possess two major absorption bands, B (or Soret) bands in the blue or near UV region and Q bands in the red or near-infrared region.4 The chemical structure of chlorophylls and their absorption spectra given in Figure 1 indicate the differences of the Q bands and the B (or Soret) bands in terms of the minor chemical structure modification. Such differences reflect the different molecular electronic energies of chlorophylls in terms of the four-orbital theoretical model.5 Chlorophyll absorption spectra result from the linear combination of one-electron promotions between the two highest occupied molecular orbitals (HOMOs) and the two lowest unoccupied molecular orbitals (LUMOs). The two higher-energy transitions are known as the B bands, and the two lowest-energy transitions are named as Q bands.6 The shifts in orbital energies caused by the chemical structural substitution in the different chlorophylls have consequences for the shifts in optical spectra, which can be predicted by quantum-chemical calculations.7 The absorption spectral properties of several chlorophylls have been investigated using semiempirical PM5 and ab initio MO/CI,8,9 conventional density functional theory (DFT) B3LYP,10−15 modern DFT CAM-B3LYP, and coupled cluster CI.16 Conventional B3LYP predicted numerous virtual © XXXX American Chemical Society

(or dark) excitations, while only CAM-B3LYP could predict the right excitations. The fluorescence measurement will reveal important features of the exited states of a chlorophyll molecule following photon absorption. However, detailed knowledge of the absorption and fluorescence properties of Chl f is lacking. The newly discovered cyanobacterium Halomicronema hongdechloris (H. hongdechloris) which contains both Chl a and Chl f was isolated from stromatolites found at Shark Bay, Western Australia, and cultured in the laboratory.3,17 However, the functions of Chl f and its role in photosynthetic reactions are unknown. The major question concerning Chl f in H. hongdechloris is, therefore, what role Chl f plays and whether uphill energy transfer occurs from Chl f to Chl a. The red-shifted Qy bands of Chl f at 707 nm (14144 cm−1) may require a net energy increase of 893 cm−1 compared with the absorption of Chl a at 665 nm (15037 cm−1).18,19 The characteristic absorption and fluorescence properties of this new chlorophyll are important parameters for understanding the roles in photosynthesis. A range of spectroscopic investigations to assess photochemical and photophysical properties of extracted Chl f were carried out. The modern density functional (CAM-B3LYP) and high-level symmetric adapted coupled cluster configuration interaction (SAC-CI) are further employed here to assist the assignment of absorption properties.20,21 The well-known properties of Chl a and Chl d were also used to compare with the defined spectroscopic properties of Chl f. The photophysical properties of these chlorophylls will briefly be discussed. Special Issue: Rienk van Grondelle Festschrift Received: March 9, 2013 Revised: April 23, 2013

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dx.doi.org/10.1021/jp402413d | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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Figure 1. Chemical structures and absorption spectra of chlorophyll a and chlorophyll f. The absorption spectra were recorded in 100% methanol at 193 K. Phytyl represents a phytol chain.

2. METHODS AND MATERIALS 2.1. Algal Culture and Chlorophyll Extraction. Synechocystis. PCC 6803 was cultured in fresh water BG11 medium for extracting Chl a, and Acaryochloris marina MBIC11017 was cultured in seawater KES medium for extracting Chl d. Both cultures were grown under continuous white light illumination at 27 °C as described previously.17 Chl f was extracted from Chl f containing cyanobacterium, H. hongdechloris, which was isolated from stromatolite, found at Shark Bay, Australia.3 Three chlorophylls were extracted from cyanobacteria in this experiment using prechilled 100% methanol (HPLC grade, Fisher scientific, USA), followed by preseparation from other pigments in the pigment extraction using a Strata C18-T reverse phase column (200 mg/3 mL, Phenomenex, USA) as described previously.17 The freshly collected pigment fraction containing chlorophylls from the Strata C18-T reverse phase column was concentrated under a stream of N2 gas to a final volume of approximately 100 μL and then purified further using highperformance liquid chromatography (HPLC). HPLC was performed on a Shimadzu VP series HPLC system with a reversed-phase C12 column (Synergi 4u MAX-RP 80A, 150 mm × 4.60 mm, 4 μm pore size, Phenomenex, USA). All experiments were carried out in the dark or under dim green light illumination to minimize any possible photodamage. For preparing the sample of chlorophylls in 100% methanol, the C12 column (Synerge Max-RP, Phenomenex) was initially equilibrated using 100% methanol and run with 100% methanol at 1 mL/min for 15 min. For preparing the sample of chlorophylls in 100% acetone, extracted methanolic chlorophylls were dried under a stream of N2 gas and were redissolved in 100% acetone and repurified by using the C12 column with a developed gradient program at a flow rate of 0.6 mL/min. The gradient program is as follows: Solvent A contained 90% acetone (water:100% acetone (v/v) = 1:9); solvent B was 100% acetone. The column was initially equilibrated using solvent A. The HPLC was run for 1.5 min with solvent A; then the moving phase was changed from 100% solvent A to 100% solvent B over 3.5 min following a linear gradient; then the HPLC was finished by running 10 min in 100% solvent B.

Eluted pigments were detected with a photodiode array detector (SPD-M10Avp, Shimadzu, Japan) at a wavelength range of 400−800 nm. Chlorophylls used in this study were collected from its elution peak, where the region was higher than the position at half-maximum monitoring peaks. The collected chlorophylls were used directly for spectral analysis. 2.2. Absorption Spectroscopy. Absorption spectra were recorded using a Shimadzu UV-2550 spectrophotometer (Japan) with the aid of a Cryostat attachment (Oxford Instruments, UK) for controlling the sample temperature. The absorption spectrophotometer was baselined using 100% methanol for the chlorophylls in methanol and using 100% acetone for the chlorophylls in acetone, respectively. Chlorophylls in these solvents for absorption spectroscopy analysis were freshly prepared as described in section 2.1, and diluted with the same solvent to the maximum absorbance at the Qy transition of range 0.1−1.0 at 295 K. Chlorophylls in different solvents were transferred into a 1 cm square quartz glass cuvette and covered by a lid immediately to avoid any evaporation during the measurement. Absorbance spectral analysis of these three chlorophylls in different solvents was performed over a temperature range from 295 to 183 K, and spectra were recorded in the wavelength range 350−800 nm at an interval reading of 0.5 nm. Each sample was read 10 times at each temperature point. The spectral profiles presented are the mean of 10 spectral readings. The spectra were deconvoluted by Gaussian fitting using Origin software (version 8.0). 2.3. Fluorescence Spectroscopy. Steady-state fluorescence spectral analysis was done using a Varian Cary Eclipse Fluorescence spectrophotometer with an aid of a Cryostat attachment (Oxford Instruments, UK) for controlling the sample temperature ranging from 295 to 183 K. Chlorophylls were freshly prepared and diluted with the same solvent to a maximum absorbance at the Qy transition of around 0.1 at 295 K as described in section 2.1. Spectra were recorded with a spectral bandwidth of 5 nm. To increase the signal-to-noise ratio, the fluorescence spectra were obtained from the mean of 15 repeats recorded and smoothed using the Savitzky−Golay method with a window of five points using the Origin software, version 8.0. B



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Figure 2. The absorption spectra of purified chlorophyll f in different solvents: (A) different temperature steady-state absorption spectra of Chl f in 100% acetone; (B) the assignment of absorption spectra of Chl f in 100% acetone; (C) different temperature steady-state absorption spectra of Chl f in 100% methanol; (D) the assignment of absorption spectra of Chl f in 100% methanol. Solid black line, absorption spectrum of purified Chl f recorded at 183 K; black dashed line, absorption spectrum of purified Chl f recorded at 243 K; solid gray line, absorption spectrum of purified Chl f recorded at 295 K. Gray dashed lines are assigned Gaussian-fitting peaks. The assigned wavelength maxima of fitting peaks are labeled.

2.4. Chlorophyll Derivatives (Impurities). The quality of chlorophylls after the spectral analysis was directly checked using the HPLC system with the same running program as described at section 2.1. Chlorophylls in 100% methanol were subjected to an HPLC analysis run with 100% methanol at a flow rate of 1.0 mL/ min for 15 min, while chlorophylls in 100% acetone were run with a gradient program from 90% acetone to 100% acetone with a flow rate of 0.6 mL/min for 15 min. 2.5. Computational Details. All calculations were performed using Gaussian 0922 with the 6-31G(d)23 basis set. All geometries of Chl a, d, and f on the ground state were optimized using density functional (DFT) CAM-B3LYP,20 while the excited-state properties in solvent (methanol) were calculated by the CAM-B3LYP and further the symmetric adapted coupledcluster configuration interaction (SAC-CI)21 using the SCRF (self-consistent reaction field) method.22 Both methods, as reported in previous work,16 predict reasonable results for chlorophylls and porphyrins.

region), which are also named as Soret bands. The origin bands are usually described as (0,0) bands, while the sidebands are labeled as (1,0).5,24 The spectral substructure (recorded at 183 K) shows a significant number of overlapping vertical electronic transitions (Figure 2B and D). The properties of those bands are resolved using Gaussian fitting deconvolution and assigned (Table 1). The absorption spectrum of Chl f in 100% acetone at Table 1. Observed (and Fitting) Q and Soret Bands of Chlorophyll f in Acetone and Methanol at 183 K in Terms of the Frequencies υmax, the Associated Wavelength λmax = 1/ υmax, and the Band Full Widths at Half-Maximum (fwhm) in 100% acetone

Qy(0,0) ??? Qy(1,0) Qx(0,0) Qx(1,0) Bx(0,0) Bx(1,0) By(0,0) By(1,0)

3. RESULTS AND DISCUSSION 3.1. Absorbance Spectral Analysis and the Assignments. The observed electronic absorption spectra of Chl f recorded from 183 to 295 K in quartz glass are shown in Figure 2. These spectra are consistent with previously published spectra of Chl f in methanol (or acetone)17 but are at a higher resolution due to the lower temperature. It is noted that, in methanol, the Qy peak at 712 nm was red-shifted approximately by 5 nm at 183 K (Figure 2C and D) with respect to 707 nm at 295 K.17 A similar red-shifted Qy peak (from 699 to 704 nm) is observed at 183 K when Chl f is dissolved in 100% acetone (Figure 2A and B). Chlorophyll f shows the similar spectral properties as other chlorin molecules, in which up to eight readily discernible bands are found. These transitions correspond to four electronic origins known as Qx and Qy(in the red region) and Bx and By(in the blue

in 100% methanol

νmax (cm−1)

λmax (nm)

fwhm (cm−1)

νmax (cm−1)

λmax (nm)

fwhm (cm−1)

14209

704

438

15151 16496 ? 22505 23598 24826 26573

660 606 ? 444 424 403 376

1074 982 ? 1511 666 1744 1517

14043 14588 14871 15289 ? 22500 ? 24493 25807

712 685 672 654 ? 444 ? 408 387

448 357 677 1816 ? 2209 1171 1517

183 K (Figure 2B) indicates that Qy(0,0) lies at 14209 cm−1 (704 nm), Bx(0,0) at 22505 cm−1 (444 nm), and By(0,0) at 24826 cm−1 (403 nm). The side bands (1,0) of Qy, Bx, and By lie at 15151 cm−1 (660 nm), 23598 cm−1 (424 nm), and 26573 cm−1 (376 nm) (Table 1). Qx(0,0) is at 16496 cm−1 (606 nm); however, the sideband Qx(1,0) cannot be resolved due to low resolution in that region (Figure 2B and Table 1). C



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Table 2. Calculated CAM-B3LYP/6-31G(d) and SAC-CI/6-31g(d) Spectroscopic Properties (Wavenumber υ, Wavelength λ, and Oscillator Strength f) of Q and Soret Bands of Chlorophylls a, d, and f Qy

Δ(Qx−Qy)

Qx

Bx

Δ(By−Bx)

By

pigment

method

ν (cm−1)

λ (nm)

f

ν (cm−1)

λ (nm)

F

ν (cm−1)

λ (nm)

ν (cm−1)

λ (nm)

f

ν (cm−1)

λ (nm)

f

ν (cm−1)

λ (nm)

Chl a

CAM SACCI CAM SACCI CAM SACCI

17301 15083 16286 14286 16233 14025

578 663 614 700 616 713

0.31 0.30 0.31 0.29 0.39 0.34

20492 18832 19305 17668 19343 17953

488 531 518 566 517 557

0.07 0.03 0.06 0.03 0.08 0.04

3191 3749 3019 3382 3109 3928

90 132 96 134 99 156

27248 26954 26042 25706 26810 26247

367 371 384 389 373 381

1.05 0.92 0.95 0.86 0.89 0.73

29326 28902 28571 27701 28986 28571

341 346 350 361 345 350

0.88 0.86 0.90 0.72 1.15 0.86

2078 1948 2529 1995 2176 2324

26 25 34 28 28 31

Chl d Chl f

Figure 3. The absorption spectral comparison of purified chlorophyll d and chlorophyll a: (A) the absorption spectra of Chl d in 100% acetone at different temperatures; (B) the assignment of absorption spectra of Chl d in 100% acetone; (C) the absorption spectra of Chl d in 100% methanol at different temperatures; (D) the assignment of absorption spectra of Chl d in 100% methanol; (E) the absorption spectra of Chl a in 100% methanol at different temperatures; (F) the assignment of absorption spectra of Chl a in 100% methanol. Solid black line, spectrum recorded at 183 K; black dashed line, spectrum recorded at 243 K; solid gray line, spectrum recorded at 295 K; gray dashed lines, assigned peaks using Gaussian-fitting analysis. The assigned wavelength maxima of fitting peaks are labeled.

Table 3. Observed (and Fitting) Q and Soret Bands of Chlorophyll d in Acetone and Methanol and Chlorophyll a in Methanol at 183 K in Terms of the Frequencies υmax, the Associated Wavelength λmax = 1/υmax, and the Band Full Widths at Half-Maximum (fwhm) chlorophyll d

chlorophyll a

in 100% acetone −1


in 100% methanol −1

−1

in 100% methanol −1

νmax (cm )

λmax (nm)

fwhm (cm )

νmax (cm )

λmax (nm)

fwhm (cm )

14452

692

368

15483 16771 17795 22036 23311 25244 26711

646 596 562 454 429 396 374

1096 562 1025 952 1368 1292 1073

14284 14970 15367 15900 16918 21299 22492 24675 25942

700 668 651 628 591 470 445 405 385

452 377 626 923 1494 950 1684 1022 1608

−1


νmax (cm )

λmax (nm)

fwhm (cm−1)

14912 15354 16172 16476 17668 22503 23560 25493 26490

671 651 618 607 566 444 424 392 378

299 669 581 1328 2248 663 1281 1816 2295

14043 cm−1 (712 nm), 22500 cm−1 (444 nm), and 24493 cm−1 (408 nm), respectively. The Qy side bands (1,0) lie at 14871 cm−1 (672 nm), and 25807 cm−1 (387 nm) may be suggested as By(1,0) (Figure 2D and Table 1). The side bands of Qx and Bx cannot be determined due to the limited resolution of the spectra in the range.

The absorption spectrum of Chl f in 100% methanol shows a similar profile as in 100% acetone (Figure 2C). The origin bands and a few possible sidebands of Qx, Qy, Bx, and By can be determined on the basis of the Gaussian-fitting analysis (Figure 2D and Table 1). The four origin bands Qx(0,0), Qy(0,0), Bx(0,0), and By(0,0) in methanol are at 15289 cm−1 (654 nm), D



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Figure 4. The purities of chlorophyll d and chlorophyll a in 100% methanol were monitored by HPLC after spectral measurement. (A) HPLC chromatography of Chl d samples after experiments. Inset I: The detailed chromatography of Chl d (100×). Inset II: The online absorption spectrum of the peaks labeled in inset I. The black dashed line represents the absorption spectrum for Chl d in 100% methanol, whereas the impurity peak 4 which is drawn with a solid black line shows an obvious spectral blue shift (Qy = ∼660 nm) compared with the maximum absorbance at the Qy transition of Chl d (Qy = 696 nm). Other peaks show the same spectral properties as Chl d. (B) HPLC chromatography of Chl a samples after experiments. Inset I: Detailed chromatography highlighted area (100×). Inset II: The absorption spectrum of the peaks labeled in inset I. Black dashed lines represent the absorption spectrum for Chl a in 100% methanol, whereas the impurity peak 2 which is drawn with a solid black line shows an obvious spectral blue shift (Qy = 658 nm) compared with the maximum absorbance at the Qy transition of Chl a (Qy = 665 nm). Other peaks show the same spectral properties as Chl a.

The DFT (CAM-B3LYP) and SAC-CI results of Chl a, d, and f are shown in Table 2. For Chl f, both CAM and SACI-CI predict the spacing of Bx(0,0) − By(0,0), being 2176 and 2324 cm−1, respectively, in agreement with the current experimental value of 2321 cm−1 (in acetone) or 1993 cm−1 (in methanol). The predicted spacing of Qx(0,0) − Qy(0,0), 3928 cm−1 (SAC-CI) and 3109 cm−1 (CAM) for Chl f, respectively, is greater than the corresponding values obtained from the experiments. The spectral assignments give a spacing of 2279 cm−1 (in acetone) and 1246 cm−1 (in methanol). The difference may be ascribed to the solvent interaction and vibronic coupling. It is known that both CAM-B3LYP and SAC-CI overestimate the energy spacing for chlorins and porphyrins in general.16 The spectroscopic properties of Chl a and Chl d have been studied widely by various experimental measurements and lowlevel theoretical calculations.8−14,25−30 In 2011, Yamijala et al. performed computational studies of modified chlorophyll f by the aid of convention B3LYP and provided some excitation energies, but as described in our previous work on Chl a,16 conventional B3LYP is a poor method for studying chlorophylls and porphyrins. To verify the assignment of Chl f, the spectroscopic properties of Chl a and Chl d are analyzed under the same experimental conditions as the analysis for Chl f. The four main bands Qy, Qx, Bx, and By of Chls a and d are reassigned

(Figure 3 and Table 3) and confirmed using the more efficient DFT CAM-B3LYP and high level SAC-CI methods (Table 2). The properties of four main vibration bands of Chl d in 100% acetone are well resolved using Gaussian fitting deconvolution (Figure 3A and B): Qy(0,0) lies at 14452 cm−1 (692 nm), Qx(0,0) is at 16771 cm−1 (596 nm), Bx(0,0) at 22036 cm−1 (454 nm), and By(0,0) at 25244 cm−1 (396 nm). The side bands (1,0) of Qy, Qx, Bx, and By lie at 15483 cm−1 (646 nm), 17795 cm−1 (562 nm), 23311 cm−1 (429 nm), and 26711 cm−1 (374 nm) (Table 3), respectively. The absorption spectrum of Chl d in 100% methanol shows similar profiles as in 100% acetone (Figure 3C and D) and the four main vibration bands are resolved well, although an impurity peak is observed in the spectrum recorded in 100% methanol, which agrees well with the previous report.25 The spectral assignment generated from purified Chl a in 100% methanol at 183 K (Figure 3E and F and Table 3) is consistent with a previous report.31 The spectroscopic experimental spacings of Qx(0,0) − Qy(0,0)1564 cm−1 for Chl a in methanol, 2319 cm−1 for Chl d in acetone, and 1613 cm−1 for Chl d in methanolare consistent with the spectral assigned results of Chl f, which are smaller than the corresponding values obtained from computer predicted spacing. The predicted spacings of Qx(0,0) − Qy(0,0) are 3749 cm−1 (SAC-CI) and 3191 cm−1 (CAM) for Chl a and 3382 cm−1 (SAC-CI) and 3019 cm−1 (CAM) for Chl d. The E



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Figure 5. HPLC chromatography of Chl f samples after experiments: (A) HPLC chromatography of Chl f samples in 100% methanol running by isocratic 100% methanol; (B) HPLC chromatography of Chl f samples in 100% acetone running via a gradient program (90−100% acetone). Inset I: The rectangle highlighted area is magnified to 100 times (100×) to identify the minor products formed during the experimental procedure. Inset II: The online recorded absorption spectra of the peaks shown in inset I. Gray solid lines represent the absorption spectrum for Chl f; the solid black lines are the impurity products identified by the spectral comparison. The impurity formed in 100% methanol indicates a Qy peak at ∼660 nm, which is less than 1.0% of total chlorophyll according to the ratio of areas. The impurity formed in 100% acetone is less than 0.02% of the total chlorophyll, according to a sideband (∼657 nm) observed in trace 4.

for the well-resolved peak in the absorption spectrum according to the spectral properties. Unfortunately, there is no report related to the structure information for this “impurity”. There is an uncharacterized peak at 685 nm in the absorption spectrum of methanolic Chl f (Figure 2D). To characterize the property of the “impurity”, the Chl f was rechecked using HPLC immediately after its spectral measurement (Figure 5). The main fraction of Chl f is resolved at a retention time of 5.7 min using an isocratic program of 100% methanol. A tiny fraction at 5.1 min is resolved, and the online absorption spectra confirmed that the “impurity” possesses a maximal absorption peak at ∼660 nm with a ratio of Soret band to Qy band bigger than 2.0 (Figure 5A, trace 1). It is uncertain whether it is responsible for the uncharacterized peak of 685 nm assigned in the absorption spectrum of methanolic Chl f according to current spectroscopic data. However, it cannot be further analyzed because of the small percentage (less than 1% of the main Chl f fraction) and unseparated retention time with the main Chl f fraction. To avoid the impurity generated by methanol interaction, we used 100% acetone for the spectral analysis. The HPLC analysis on Chl f after spectral measurements indicated that Chl f in 100% acetone is more stable during the experimental procedure (Figure 5B). No resolved byproduct is observed in HPLC chromatography, although there is a tiny side peak that can be observed in trace 4, which is less than 0.1% of total chlorophyll

spectroscopic assignment suggests that there is a similar spectral structure for isolated Chl a, Chl d, and Chl f. 3.2. Presence of “Impurities”. According to the absorption spectrum, there are unwanted “impurity” peaks of 685 nm in the methanolic Chl d absorbance spectrum but not in Chl d-acetone solution (Figure 3D). Further HPLC analysis confirms that the impurity is a byproduct of interaction between Chl d and methanol, designated as “iso-Chl d” by Manning and Strain (Figure 4A).32 It is noted that, in methanol, solvent interaction also gives rise to an “impurity” in Chl a, having a peak at ∼658 nm (Figure 4B), which agrees well with the observation of the allomerization reaction of Chl a in methanol.25,33,34 According to HPLC analysis, the unwanted “impurity” has been reported that, in methanol solution, the purified Chl d gradually forms some methanolic isomerization products with an absorption band at ∼661 nm, which was named as isochlorophyll d.25,32 To avoid such a byproduct, we used chlorophylls directly collected from HPLC. The HPLC rechecking was performed immediately after the spectral measurement. The HPLC chromatography showed that Chl d with a retention time at 5.5 min remains stable during the experiment; over 99% of the chlorophylls are Chl d. Only a very tiny fraction (less than 1.0% of total chlorophyll) with a retention time at 6.2 min showed a different spectral property, with about 35 nm blue-shifted Qy band at 661 nm (Figure 4A, trace of 4), which agrees with the spectral property of “iso-Chl d”. This fraction may be responsible F



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Figure 6. The fluorescence emission spectra of Chl f in 100% acetone recorded using different excitation wavelengths. The scattering peaks arising from incident light (660−705 nm) were determined and removed by Gaussian fitting assignments. (A and B) The fluoresence spectra were obtained using excitation wavelengths in the Q region: (A) recorded at 193 K; (B) recorded at 295 K. (C) The spectra were recorded at room temperature using excitation wavelengths in the Soret region.

according to the ratio of fraction areas resolved in HPLC chromatography (Figure 5B). 3.3. Fluorescence after Excitation at Soret Bands. The fluorescence spectra of Chl f in 100% acetone at room temperature (295 K) and low temperature (193 K) after a series of changed excitation wavelength at Q bands (660−700 nm) are shown in Figure 6. While temperature changes from 295 to 193 K, the emission maximum of 13996 cm−1 (714 nm) moves evenly to 13927 cm−1 (718 nm), but the intensity of fluorescence increases (Figure 6). The maximal fluorescence intensity is observed when the excitation wavelength is 700 nm, which is the same at room temperature (295 K) and a low temperature of 193 K. The emission maximum central wavelength remains unchanged following a series of changed excitation wavelength in the range 400−450 nm (Soret band region); however, the fluorescence intensity increases approximately 2 times, compared with the fluorescence intensity using an excitation wavelength of 700 nm (Figure 6). The fluorescence spectra of Chl f in 100% methanol using excitation at 407 nm (By(0,0)) at various temperatures (193, 240, 270, and 295 K) is shown in Figure 7 (inset). The fluorescence intensities increased proportionally when the temperature was decreased from 295 to 193 K, which agrees with Maxwell− Boltzaman statistics. The emission maximum lying at 13680 cm−1 (731 nm) at 193 K moves evenly to 13792 cm−1 (725 nm) when the temperature rises to 295 K. The intensity of fluorescence falls from 193 to 295 K, with the bandwidth increasing slightly with respect to temperature (Figure 7). The same fluorescence emission profiles were observed after excitation at 440 nm at various temperatures (data not shown). The emission peak at 14749 cm−1 (678 nm) observed at 193 K is assigned as the fluorescence of “impurity”, which is a product of interaction between chlorophyll and methanol (Figure 4). This emission peak is absent in the analysis when 100% acetone is used (Figure 6), which confirms that no such solvent interaction is generated in 100% acetone. The comparison of the

Figure 7. The comparison of the chlorophyll f absorption spectrum with its fluorescence spectra. All spectra were recorded in 100% methanol at 193 K. The spectra are normalized at the Qy bands. Solid black line, absorption spectrum of Chl f in 100% methanol at 193 K; black dashed line, fluorescence excitation spectrum of Chl f in 100% methanol by using an emission wavelength of 750 nm at 193 K; gray solid line, fluorescence emission spectrum by using an excitation wavelength of 407 nm. The inset is the fluorescence emission spectra of Chl f in 100% methanol recorded using an excitation wavelength of 407 nm at various temperatures (193−295 K). The arrow indicates the emission peak generated from an “impurity”, the product of interaction of methanol and Chl f.

fluorescence excitation spectrum of Chl f using an emission wavelength of 750 nm with the absorption spectrum of Chl f in methanol at room temperature indicates the existence of an “impurity” in methanolic Chl f (Figure 7). Excitation at 700 nm (in acetone) drives the Qy(0,0) transition, producing a Chl f molecule in its ground vibration state, the main vibration modes. Some excitation of Soret bands and other low-frequency excitation showed the same emission profiles (Figure 7) because the low frequency excitation is relaxed in its Qy state (Figure 8). It then emits through the Qy(0,0) transition. The most significant difference between G



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Figure 8. The photophysical processes model according to the assignments of absorptional spectra and fluorescence spectra of purified Chl f. Assigned lines in the absorption and emission spectra are shown as vibronic transitions among the ground state (GS) and excited states Qy, Bx, and By(Qx is omitted for clarity). Between absorption and emission, relaxation processes (e.g., R1 and R2) and thermal equilibration occurs. Shown are the absorption spectrum and emission spectrum of purified chlorophyll f in 100% methanol recorded at 183 K. The characteristic vibrational spacing Δν of 2000−3100 cm−1 is indicated with bold double arrows (↔).

4. CONCLUSION

excitation at those different wavelengths is the nonradiative processes of the higher states may also result in ground-state transfer and, therefore, decrease in the fluorescence intensity (Figure 8). The maximal fluorescence intensity is observed by excitation at Qy(0,0), instead of the high-energy Soret bands, which agrees with in vivo low temperature fluorescence emission spectra being centered in the far-red region.3

Detailed study on the spectroscopic properties of isolated Chl f in different solvents and at different temperatures reveals that Chl f has a large gap between Soret bands and Q bands. The Qy(0,0) of Chl f shifted from 707 to 712 nm when the temperature decreased from 295 to 183 K. The same red-shifted Qy band, H



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from 699 to 704 nm, is observed when 100% acetone is used as the solvent. The fluorescence emission is emitted primarily through the Qy(0,0) transition at 718 nm in 100% acetone and 731 nm in 100% methanol at a low temperature of 183 K (Supporting Information).

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Absorption and fluorescence spectra of isolated chlorophyll f in 100% methanol or in 100% acetone. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author

*Phone: +61-2-9036 5006. Fax: +61-2-9351 4119. E-mail: min. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.C. holds an Australian Research Council Future Fellowship, and M.C. thanks the Australian Research Council for support. Y.L. is the recipient of a scholarship supported by the China Scholarship Council.

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REFERENCES

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Spectroscopic Properties of Chlorophyll f - The Journal of Physical

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