Absorption properties of chlorophylls in diethylether at room temperature
Chlorophylls are essential components in oxygenic photosynthesis, and chlorophyll (Chl)
In 1996, a unique cyanobacterium,
In 2010, a red-shifted chlorophyll was discovered in a methanolic extract of Shark Bay stromatolites, and was named Chl
In this chapter, we present systematic and essential physicochemical properties of chlorophylls
2. Chlorophylls in oxygenic photosynthesis
2.1. Popular chlorophylls: Chlorophylls
a and b
In 1818, the term chlorophyll (Chl), the green (Greek
2.2. Recently discovered chlorophylls in algae
d in a cyanobacterium, Acaryochloris marina
In 1943, Chl
f in a cyanobacterium, strain KC1, isolated from Lake Biwa
Note that in the strain KC1 Chl
2.3. Specially-tailored chlorophylls associated with reaction centers
2.3.1. Prime-type chlorophylls as the primary electron donors in PS I
a' and P700
The 132-epimer of Chl
d' and P740 in Acaryochloris marina
It is interesting to note that the primary electron acceptor, A0, in PS I of
18.104.22.168. Evolutionary relationship between chlorophylls and PS-I type reaction centers
Here we introduce our hypothesis about the evolution of the PS I-type RCs based on the structures of chlorophylls and quinones (Fig. 3). The prime-type chlorophylls, bacteriochlorophyll (BChl)
In Fig. 4, BChl
a as the primary electron acceptor in PS II
In 1974, pheophytin (Phe)
It is of interest to note that Phe
3. Physicochemical properties of chlorophylls
In the late 1970s, the high performance liquid chromatography (HPLC) technique was applied to the separation of plant pigments. In many cases the reversed-phase HPLC was preferred (Eskins et al. 1977; Shoaf et al. 1978; Schoch et al. 1978), and is still the main option to date. In that system, however, an eluent gradient is usually required for simultaneous separation of Chls and Phes and the gradient system is unfavorable for quantitative analysis, since the molar absorptivities of pigments strongly depend on solvents. In this context, an isocratic eluent system is favorable. In 1978, a simultaneous separation of Chls and Phes by normal-phase HPLC was attained by an isocratic procedure (Iriyama et al. 1978). In 1984, the isocratic normal-phase HPLC was established as a powerful tool for chlorophyll analysis (Watanabe et al. 1984).
3.1.1. Mixture of chlorophylls and pheophytins
A mixture of Chls and Phes was injected into a silica HPLC column (YMC-pak SIL, 250 x 4.6 mm i.d.) cooled to 277 K in an ice-water bath. The pigments were eluted isocratically with degassed hexane/2-propanol/methanol (100/0.7/0.2,
As illustrated in Fig. 6(F), eight Chls and four Phes are clearly separated. One can easily see that
3.1.2. Pigment extract from
Pigments were extracted from cell suspension (ca. 10 μL) by sonication in a ca. 300-fold volume of acetone/methanol (7/3,
As seen in Fig. 6(C),
3.1.3. Pigment extract from strain KC 1
Cells of the cyanobacterium strain KC1 were grown in BG-11 medium in a glass cell culture flask (1 L) at 297 K with continuous air-bubbling. Cells were incubated under continuous white fluorescent light (50 μmol photons/m2/s) or near-infrared LED light (see Fig. 5A, Tokyorika, Tokyo). Cells at the early stationary phase were harvested by centrifugation. See Akutsu et al. (2011) for more details.
Typical HPLC traces for acetone/methanol extracts from cells of the cyanobacterium strain KC1 cultivated under white fluorescent light or NIR LED light are shown in Figs. 6D and E, respectively. A large amount of Chl
3.2. Absorption spectra in four solvent varieties
The absorption spectrum is the simplest, most useful and extensively used analytical property to characterize chlorophylls. Absorption spectra of Chls show the electronic transitions along the x axis of the Chl running through the two nitrogen (N) atoms of rings II and IV, and along the y-axis through the N atoms of rings I and III (see Fig. 1). The two main absorption bands in the blue and red regions are called Soret and Q bands, respectively, and arise from π→π* transitions of four frontier orbitals (Weiss 1978; Petke et al. 1979; Hanson 1991).
a, b, d and f
Absorption spectra of Chls
||447||688||Smith and Benitez (1955)|
|394.3b, 451.7b||691.4b||This work|
||421||692||Smith and Benitez (1955)|
|383.7b, 421.5b||691.0b||This work|
||395.6, 440.5||695.2||This work|
It is somewhat difficult to distinguish Chl
The Soret bands include several intense bands. In diethyl ether and benzene, the Soret band of Chl
In Fig. 5 are shown the absorption spectra of the strain KC1 grown under white fluorescent light and NIR LED light. The cells grown under NIR LED light show a clear shoulder over 700 nm, extending up to almost 800 nm (Fig. 5A). Absorption spectra in acetone solution of acetone/methanol extracts from the KC1 cells cultivated under NIR LED also exhibit a longer wavelength peak in the range of about 690 to 720 nm (Fig. 5B), due to the presence of Chl
We should note that inductive effects on the absorption wavelengths and intensities of QY-bands of chlorophylls strongly depend on the nature and position of substituent(s) on the macrocycle, due to the presence of two different electronic transitions polarized in the x and y directions (the axes of transition moments are depicted in Fig.1) (Gouterman 1961, Gouterman et al. 1963; Weiss 1978; Petke et al. 1979; Hanson 1991, Kobayashi et al. 2006b). Replacement of the electron-donating group, -CH3, on ring II of Chl
a, b, d and f
The free base related to Chl is called Phe. First of all, we emphasize that in natural photosynthesis only Phe
As seen in Fig. 7, Phe
3.3. Circular dichroism spectra
Circular dichroism (CD) spectra are very useful for distinction between the primed chlorophyll,
A spectropolarimeter Model FDCD-309 (JASCO) was used for CD measurements. Benzene was chosen as the solvent, in view of the sufficiently slow interconversion between epimeric species in this medium (Watanabe et al. 1984). The spectra were recorded from 800 nm to 300 nm at a scan rate of 200 nm/min with 20 scans at room temperature; time for measurement was ca. one hour.
The CD spectra of Chl
A series of QX transitions occur in the "valley" of the absorption spectrum as described in section 2. The positive CD activities derived from the QX(0,0) absorption (called bands III, see Fig. 2 in Petke et al. 1979) appear at 579, 535, 594, 557, 548, 604, 567 and 559 nm for Chl
The Chl and Phe Sorets contain many π-π* transitions characterized by a complex mixture of configurations. According to the results of molecular orbital calculations (Weiss 1978; Petke et al. 1979; Hanson 1991), band B in the Soret absorption consists of two nearly degenerate electronic transitions, BX(0,0) and BY(0,0). All the primed derivatives gave single and strongly positive CD spectra at this absorption peak, suggesting that the two transitions contribute to CD spectra in a similar manner (Watanabe et al. 1984). In contrast, the CD spectra of non-primed species, except Chl
3.4. Mass spectra
Chlorophylls in natural photosynthesis are sometimes present in very small amounts, and hence the use of mass spectrometry (MS) can be advantageous since only minute samples are required. MS can provide accurate and useful information not only on molecular weights and elemental compositions but also on the nature of functional groups attached to the macrocycle (
LC/MS experiments were performed on an LCQ mass spectrometer (Thermo Fisher Scientific Inc., MA, U.S.A.) equipped with an HPLC system (HP1100, Agilent, CA, U.S.A.) connected with a diode array detector. Each sample dissolved in dichloromethane before analysis was applied on a JASCO Finepak SIL C18S column (150 mm x 4.6 mm i.d.) cooled to 277 K in an ice-water bath, and separated using a mixture of ethanol/methanol/2-propanol/water (86/13/1/3,
As illustrated in Fig. 10 (left), Chl
It is interesting to note that a typical fast atom bombardment (FAB)-mass spectrum of Chl
As seen in Fig. 10 (right), the corresponding pheophytins prepared by acid treatment clearly showed the absence of magnesium (Fig. 1). For example, [M+H]+ of Phe
3.5. Nuclear magnetic resonance spectra
Nuclear magnetic resonance (NMR) spectroscopy can offer ample information about the molecular structure. Coupled use of NMR with HPLC, absorption-, CD- and mass-spectrometries has not only definitively identified the structures of several major naturally-occurring Chls but has also assisted recent studies of minor Chl pigments, present in minute quantities, such as electron donors and acceptors in the RC.
The NMR spectra were recorded on a Bruker Avance 800 spectrometer (Bruker Biospin, Karlsruhe, Germany), with a frequency of 1H at 800 MHz and 13C at 201 MHz, equipped with TCI CryoProbe using a microtube (Shigemi Inc., Tokyo) and about 0.5 mg of sample in 0.3 mL of acetone-
As observed in one-dimensional 1H-NMR spectra (Fig. 11, Table 2), marked differences are seen in the signals arising from the formyl group. Each low-field singlet signal characteristic of the formyl moiety observed around 11 ppm in the spectra of Chls
Here we note that the 31-H vinylic proton shows a large downfield shift in Chl
The pair signals of 32- and 32'-H vinylic protons are well resolved in the spectra of Chls
In the 13C-NMR spectra (Fig. 12, Table 3), marked differences are noted in the range of 0 ppm to 20 ppm, 120 ppm to 140 ppm, and 180 ppm to 200 ppm, relating to the -CH3, -CH=CH2 and -CHO moieties. Compared to Chl
|21||3.343 (3.36)1 (s)||3.316 (3.40)2*(s)||3.724 (3.68)3(s)||11.215 (11.35)4**(s)|
|31||8.162 (8.18)1(dd)||8.043(7.95)2*(dd)||11.460 (11.40)3(s)||8.534(dd)|
|32||6.242 (6.24)1(dd), 6.028 (6.03)1(dd)||6.302(6.25)2*(dd), 6.055 (6.04)2*(dd)||-||6.324(dd),6.365(dd)|
|5||9.410 (9.40)1(s)||10.192 (10.04)2*(s)||10.294(10.20)3(s)||9.770 (9.79)4**(s)|
|71||3.300 (3.30)1(s)||11.305 (11.22)2*(s)||3.365 (3.33)3(s)||3.351(s)|
|81||3.817 (3.82)1(q)||4.243||3.876 (3.86)3(q)||3.754(q)|
|82||1.696 (1.69)1(t)||1.815||1.723 (1.73)3(t)||1.705(t)|
|10||9.749 (9.75)1(s)||9.934 (9.64)2*(s)||9.873 (9.8)3(s)||9.838 (9.86)4**(s)|
|121||3.619 (3.61)1(s)||3.606 (3.65)2*(s)||3.668 (3.65)3(s)||3.637(s)|
|132||6.234 (6.24)1(s)||6.189 (6.19)2*(s)||6.335 (6.28)3(s)||6.318(s)|
|134||3.829 (3.83)1(s)||3.842 (4.02)2*(s)||3.851 (3.83)3(s)||3.887(s)|
|17||4.175 (4.16)1||4.128||4.242 (4.25)3||4.230|
|171||2.589 (2.60)1, 2.461 (2.45)1||2.43,2.593||2.484,2.622||2.467,2.632|
|172||2.431 (2.35)1, 2.159 (2.05)1||2.08,2.44||1.98,2.418||2.08,2.47|
|18||4.572 (4.57)1(q)||4.524(q)||4.660 (4.63)3(q)||4.634(q)|
|181||1.772 (1.77)1, 1.762(1.76)1(d)||1.768,1.759(1.78)2*(d)||1.812, 1.802(1.82)3(d)||1.800, 1.791(d)|
|20||8.582 (8.58)1(s)||8.480(8.20)2*(s)||8.867 (8.81)3(s)||9.533 (9.77)4**(s)|
|P1||4.342 (4.33)1,4.224 (4.21)1||4.364, 4.247||4.343 (4.26)3, 4.227 (4.36)3||4.361, 4.263|
|P2||4.955 (4.95)1||4.980||4.944 (5.04)3||4.987|
|P31||1.509 (1.51)1||1.519||1.505 (1.54)3||1.525|
|P4||1.822 (1.82)1||1.845||1.832 (1.85)3||1.845|
|P71||0.811,(0.81)1, 0.803(0.80)1||0.785, 0.777||0.778, 0.770 (0.79)3||0.785, 0.777|
|P111||0.783(0.79)1,0.774 (0.78)1||0.809,0.801||0.806, 0.797 (0.81)3||0.807, 0.798|
|P15||1.500 (1.50)1||1.489||1.497 (1.51)3||1.495|
|P151||0.854(0.86)1,0.845 (0.84)1||0.851,0.842||0.850 (0.85)3, 0.842 (0.85)3||0.849, 0.841|
|P16||0.854(0.86)1,0.845 (0.84)1||0.851,0.842||0.850 (0.85)3, 0.842 (0.85)3||0.849, 0.841|
|P151||23.013 (28.66)1||23.014||23.015 (23.15)2||23.006|
|P16||22.903 (22.91)1||22.914||22.905 (23.07)2||22.906|
The signals of 71-CH3 of Chls
Two-dimensional NMR spectra provide further information about a molecule than one-dimesional NMR spectra. NOESY is one of several types of two-dimensional NMR, where the nuclear Overhauser effect (NOE) between nuclear spins is used to establish the correlations. The cross-peaks in the two-dimensional spectrum connect resonances from spins that are spatially close to each other.
To obtain further evidence for the structural identity of Chl
To obtain further evidence for Chl
HSQC is a two-dimensional inverse correlation technique that allows for the determination of connectivity between two different nuclear species, and HSQC is selective for direct coupling. As illustrated in the 1H-13C HSQC spectra of Chls
HMBC is also a two-dimensional inverse correlation method that allows for the determination of connectivity between two different nuclear species like HSQC, but HMBC gives longer range coupling (2-4 bond coupling) than HSQC.
Three meso-Hs in Chl
3.6. Redox potentials
To understand the charge separation in the RC, electrochemical characterization of chlorophylls is of crucial importance. In this section, the redox potentials of Chls and Phes
Acetonitrile (Aldrich, anhydrous grade: water < 50 ppm) was deoxygenated and dried before use. The solvent was subjected to freeze-pump-thaw cycles at least three times under about 10-5 torr. Under a nitrogen atmosphere, the deoxidized solvent was then dried for 24 h with activated molecular sieves (4A 1/16, Wako), pretreated
The redox potentials of chlorophylls were measured by square wave voltammetry (SWV). The signal-to-noise ratio of SWV is generally better than that of CV, especially for measuring redox couples at such low concentration (ca. 0.5 mM) as in the present case (Cotton et al. 1979, Wasielewski et al. 1980). Measurements were done with an ALS model 620A electrochemical analyzer. Parameters for SWV were Vstep = 5.0 mV, AC signal (Vpulse) = 25 mV, and p-p at 8 Hz. The measurements were carried out in an air-tight electrochemical cell containing a small compartment for a sample solution (ca. 0.5 mM) equipped with a glass filter that can be degassed and filled with dry N2. A platinum disk electrode with 1.6 mm in diameter (outer diameter: 3 mm) was used as the working electrode, and a platinum black wire fabricated in the small compartment (internal diameter: 8.9 mm) as the counter electrode. An Ag/AgCl electrode, chosen for good reproducibility despite possibility of junction potential, was connected through a salt bridge to the outer electrolytic solution of the small components. After measurement, the redox potentials of the ferrocene-ferrocinium were measured as +0.45 V vs. Ag/AgCl in acetonitrile.
Typical square wave voltammograms (SWVs) for Chls
In Table 4 are summarized the redox potentials for Chls
The -CHO substituent on Chls
The redox behavior of a compound is related to the energy levels of its molecular orbitals:
As clearly seen in Fig. 17, the order of absolute values of the first reduction potentials,
The primary redox potential difference, Δ
In 1959, the domination of inductive effects of the central metal over a conjugative macrocycle has first been formulated (Gouterman 1959). The redox potential of chlorophyll shows a systematic shifts with the electronegativity of the central metal, and such a trend is rationalized in terms of an electron density decrease in the chlorin π-system by the presence of an electron negative metal in the center of chlorophylls (Watanabe and Kobayashi 1991; Hanson 1991; Noy et al. 1998). Inspection of Table 4 demonstrates that such a trend is essential for the pair of Chls and Phes; the electronegativity of 2.2 for H is significantly higher than that of 1.2 for Mg, which renders Phes more difficult to oxidize than the corresponding Chls.
4. Evolutionary and ecological aspects of chlorophylls
4.1. Diversification of chlorophylls during the evolution of photosynthetic organisms
Chlorophylls are distributed among oxygenic photosynthetic organisms, including cyanobacteria, algae and terrestrial plants (Falkowski et al. 2004). It is generally accepted that plastids first arose by endosymbiosis between photosynthetic prokaryotes (ancestral to present cyanobacteria) and non-photosynthetic eukaryotic hosts (Fig. 18). There are two different types of hypothesis for the evolution of Chl
4.2. Ecology of the red-shifted chlorophylls
Acquisition of new or additional chlorophylls by photosynthetic organisms is thought to be an adaptation to the light quality of their niches. The two red-shifted chlorophylls, Chls
We thank Dr. Nobuaki Ishida (Ishikawa Prefectural Univ.), Dr. Yoshihiro Shiraiwa and Dr. Koji Iwamoto (Univ. Tsukuba) for their invaluable help. This work was supported in part by Special Project of Organization for the Support and Development of Strategic Initiatives (Green Innovation) (Univ. Tsukuba) to M.K.
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