Open access peer-reviewed chapter

Physicochemical Properties of Chlorophylls in Oxygenic Photosynthesis — Succession of Co-Factors from Anoxygenic to Oxygenic Photosynthesis

By Masami Kobayashi, Shinya Akutsu, Daiki Fujinuma, Hayato Furukawa, Hirohisa Komatsu, Yuichi Hotota, Yuki Kato, Yoshinori Kuroiwa, Tadashi Watanabe, Mayumi Ohnishi-Kameyama, Hiroshi Ono, Satoshi Ohkubo and Hideaki Miyashita

Submitted: May 7th 2012Reviewed: December 10th 2012Published: June 12th 2013

DOI: 10.5772/55460

Downloaded: 3000

1. Introduction

Chlorophylls are essential components in oxygenic photosynthesis, and chlorophyll (Chl) a(Fig. 1) is the major chlorophyll in cyanobacteria, algae and terrestrial plants. However, primary charge separation is initiated by a few specially-tailored chlorophylls in the reaction centers (RCs). Excitation of the primary donor reduces the primary and secondary electron acceptors, which again are often specially-tailored chlorophylls; e.g., the 132-epimer of Chl a,Chl a'(Fig. 1), constitutes the primary electron donor of P700 in PS I as a heterodimer of Chl a/a'(Kobayashi et al. 1988; Jordan et al. 2001), and a metal-free Chl a, pheophytin (Phe) a(Fig. 1), functions as the primary electron acceptor in PS II (Klimov et al. 1977a,b; Zouni et al. 2001) (Fig. 2).

In 1996, a unique cyanobacterium, Acaryochloris marina, with Chl das the dominant chlorophyll was discovered in colonial ascidians (Miyashita et al. 1996). Though P740 in PS I of A. marinais composed of Chl d',pheophytin in PS II is not d-type but a-type (Akiyama et al. 2001). One of the difficulties in finding Chl din nature was its overlap with Chl bon a reversed-phase HPLC trace. To make matters worse, Chl dhad been thought to have a lower oxidation potential than Chl aeven though no experimental evidence was available, mainly because a midpoint potential, Em, for P740 in A. marinawas shown to be +335 mV (Hu et al. 1998), marvelously more negative than that for P700 (ca. +470 mV) in the Chl a-type cyanobacteria. The fact that the QY-band of Chl dis at the longest wavelength compared with Chls aand bseems to have led to some misapprehensions concerning the oxidation potential of Chl d; one estimated that Chl dhad the lowest oxidation potential among Chls a, band d. In 2007, however, the Eox value of Chl din vitrowas first determined and found to be higher than that of Chl a(Kobayashi et al. 2007), and hence the Em of P740 was re-examined; the value was ca. + 435 mV (+430 mV: Benjamin et al. 2007, Telfer et al. 2007, and +439 mV: Tomo et al. 2008), being far positive of the initial report (+335 mV) and almost equal to the Chl a-type P700 values, around +470 mV (Brettel 1997, Krabben et al. 2000, Ke 2001, Itoh et al. 2001, Nakamura et al. 2004) (see Fig. 7 in Ohashi et al. 2008).

Figure 1.

Molecular structure and carbon numbering of chlorophylls, according to the IUPAC numbering system. Naturally occurring chlorophylls are designated by squares.

Figure 2.

Schematic comparison of photosynthetic electron transport in PS I-type RC and PS II-type RC. Components are placed according to their estimated or approximate midpoint potentials. The arrows indicate the direction of electron flow. In order to simplify the figure, some primary electron donors, P970, P850 and P865 are omitted here: P970 and P850 are the primary electron donors of BChlband Zn-BChlacontaining purple bacteria, respectively; P865 is the primary electron donor of green filamentous bacteria. Figure adapted fromAkutsu et al. (2011).

In 2010, a red-shifted chlorophyll was discovered in a methanolic extract of Shark Bay stromatolites, and was named Chl f(Chen et al. 2010); a Chl f-containing filamentous cyanobacterium was purified and named as Halomicronema hongdechloris(Chen et al. 2012). In 2011, a Chl f-like pigment was discovered in a unicellular cyanobacterium, strain KC1, isolated from Lake Biwa (Ohkubo et al. 2011), but there were also difficulties in its identification because there were not systematic physicochemical data as to Chls a, b, dand facquired under the same conditions. For example, the optical peak wavelengths of the red-shifted chlorophyll purified from the strain KC1 in methanol are almost the same as the reported values of Chl f(λ = 406 nm and 706 nm), while the reported ratio of Soret/QY-bands exhibited a large difference; the pigment purified from KC1 showed 0.9, but the reported ratio for Chl fwas surprisingly high, 1.9. What is worse, the 1H-NMR chemical shifts of formyl-H of Chl band fin acetone d6 at 263 K being presented in this chapter are 11.31 and 11.22, respectively, but the corresponding values reported were 11.22 for Chl bin CDCl3 at unknown temperature and 11.35 for Chl fin CD2Cl2/d5-pyridine(97/3, v/v) at 293 K, respectively, where our data are directly counter to the reported pair. These facts exhibit that chemical shift is highly sensitive to both solvents and temperature.

In this chapter, we present systematic and essential physicochemical properties of chlorophylls in vitroobtained under as common conditions as possible, introducing the detailed experimental procedures; HPLC, absorption, circular dichroism, mass, NMR and redox potential. We also introduce the succession of co-factors in PS I RCs from the viewpoints of minor but key chlorophylls (Fig. 3). In the future, our basic data will help researchers to identify the molecular structures of newly discovered chlorophylls and determine and/or predict their characteristics.

Figure 3.

Schematic arrangement of co-factors in the PS I-type RCs. Our hypothesis about the evolution of the PS I-type RCs from the viewpoint of the molecular modifications of chlorophylls and quinones are designated by solid arrows.

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2. Chlorophylls in oxygenic photosynthesis

2.1. Popular chlorophylls: Chlorophylls aand b

In 1818, the term chlorophyll (Chl), the green (Greek chloros) of leaf (Greek phyllon), was introduced for the pigments extracted from leaves with organic solvents (Pelletier and Caventou 1818). In 1903, a Russian botanist Tsvet(Tswett) (in Russian meaning "colour") separated leaf pigments by chromatography (from Greek chromaand grapheinmeaning "color" and "to write", respectively) into the blue Chl a, the green Chl band several yellow to orange carotenoids.

2.2. Recently discovered chlorophylls in algae

2.2.1. Chlorophyll din a cyanobacterium, Acaryochloris marina

In 1943, Chl d(Fig. 1) was first reported as a minor pigment in several red macroalgae (Manning and Strain 1943). In 1996, a unique cyanobacterium, Acaryochloris marina, was isolated from colonial ascidians containing Chl das the dominant chlorophyll (Miyashita et al. 1996; see Introduction in a review by Ohashi et al. 2008 for the short history of Chl ddiscovery).

2.2.2. Chlorophyll fin a cyanobacterium, strain KC1, isolated from Lake Biwa

A Chl f-producing cyanobacterium, strain KC1, was discovered by Miyashita, one of the authors of this chapter. The discovery story was a kind of fortunate accident, similar to that of Chl d-producing cyanobacterium, A. marina(Miyashita et al. 1996, Ohashi et al. 2008). Strain KC1 was a by-product during the hunting of chlorophyll d-producing cyanobacteria in freshwater environment. Chl dhad been only produced by cyanobacteria in the genus Acaryochloris, and the strains in Acaryochlorishad been isolated only from saline environments such as the marine or salty lake but not from freshwater environments at all (Murakami et al. 2004, Miller et al. 2005, Mohr et al. 2010, Behrendt et al. 2011). Moreover, the strain A. marinaMBIC11017 dose not grow in freshwater media and requires sodium chloride for its growth at more than 1.5% (w/v) in the medium (Miyashita et al. 1997). Chlorophyll dwas, however, detected in the sediment at the bottom of Lake Biwa, the largest freshwater lake in Japan (Kashiyama et al. 2008), bringing us the idea that Chl d-containing algae exist in this lake. We collected algal mats and lake water from a shore zone of Lake Biwa at 24th, Apr. 2008. The samples were suspended in several media for freshwater algae, diluted and dispensed into cell culture plate or on agar plates. Those culture/agar plates were kept in an incubator with near infrared (NIR) light as the sole light source, because we expected that a Chl d-containing alga should grow faster than other algae under such light condition. Colony of Acaryochloriscells grown under NIR light looked yellow-green rather than blue-green of common cyanobacteria, and we also saw several yellow-green colonies (Oct. 2008). Our attempt to isolate a Chl d-containing freshwater Acaryochlorissp. from Lake Biwa turned out to be a success (Feb. 2010) (details will be reported elsewhere). Miyashita checked the culture/agar plates, which were left for a long time in the incubator under NIR light, and some unusual cyanobacterial colonies were found (Oct. 2009). The colonies were different from that of Acaryochlorissp. in color; being dark-blue-green rather than yellow-green. The cells grew under NIR light as the sole light source when we put them in a freshwater medium. Morphological features of the cells were similar to those of Acaryochlorissp. in that the cell was unicellular, spherical to subspherical and aggregated. We expected that the organism was a new Chl d-containing cyanobacterium which was closely related to the genus Acaryochloris, phylogenetically. The results of HPLC analysis disappointed us, since the cyanobacterium possessed no Chl dat all, and Chl aas the major chlorophyll like common cyanobacteria. Immediately thereafter, however, came an excitement when an unusual chlorophyll was detected (22nd. Jan. 2010). The pigment showed typical two absorption peaks in the Soret (406 nm) and QY (707 nm) regions in MeOH; they were clearly different from those of known chlorophylls. We concluded that the pigment was a new chlorophyll that should be named “Chl f”. We started mass culture of the cells for chemical characterization such as detailed spectral properties, molecular mass and chemical structure. At around the same time, Chen et al. (2010) reported the discovery of Chl fin a methanolic extract of Shark Bay stromatolites incubated under NIR light for the initial purpose of the isolation of new Chl d-containing phototrophs. Discoveries are mostly accidental.

Note that in the strain KC1 Chl fwas not induced under white fluorescent light even if NIR LED was also used as additional light. Further, neither Chl f'nor Phe fwas detected, suggesting that Chl a'and Phe afunction as P700 and the primary electron acceptor in PS II, respectively, as in common cyanobacteria (Akutsu et al. 2011). The results indicate that Chl fmay function as not an electron transfer component but an antenna part. Chl fis not the major photopigment, and may function as an accessory chlorophyll, although the function of Chl fin energy storage is under debate, because uphill energy transfer is needed to deliver the excitation energy to Chl amolecules in the RC (Chen and Blankenship 2011).

2.3. Specially-tailored chlorophylls associated with reaction centers

2.3.1. Prime-type chlorophylls as the primary electron donors in PS I

2.3.1.1. Chlorophyll a'and P700

The 132-epimer of Chl a, Chl a'("a-prime") (Fig. 1), was first reported in 1942 (Strain and Manning 1942). In 1988, it was proposed that Chl a'constitutes P700 as a heterodimer of Chl a/a'(Fig. 2) (Kobayashi et al. 1988), and the idea has been confirmed in 2001 (Jordan et al. 2001). As seen in Fig. 2, it has also been shown that P798 consists of BChl g'in the RC of heliobacteria in 1991 (Kobayashi et al. 1991), and that P840 consists of BChl a'in green sulfur bacteria in 1992 (Kobayashi et al. 1992, 2000), suggesting that prime-type chlorophylls are essential as the primary electron donors in the PS I-type RCs (see Figs. 2 and 3). For more details, see Chapter 4 in Kobayashi et al. (2006).

2.3.1.2. Chlorophyll d'and P740 in Acaryochloris marina

Chl d', the 132-epimer of Chl d(Fig. 1), was always detected in A. marinaas a minor component, while Chl a'was absent (see Fig. 6(C)) (Akiyama et al. 2001). P740, the primary electron donor of PS I in A. marina, was initially proposed to be a homodimer of Chl d(Hu et al. 1998), then a homodimer of Chl d'(Akiyama et al. 2001), and finally a Chl d/d'heterodimer (Fig. 2) (Akiyama et al. 2002, 2004; Kobayashi et al. 2005, 2007: Ohashi et al. 2008), just like the Chl a/a'for P700 in other cyanobacteria and higher plants (Figs. 2 and 3): a dimer model for P740 was supported by FTIR spectroscopy (Sivakumar et al. 2003). The finding of Chl d’in A. marinaappears to ensure our hypothesis that prime-type chlorophyll is a general feature of the primary electron donor in the PS I-type RCs (see Figs. 2 and 3). The homology of PsaA and PsaB between A. marinaand other cyanobacteria is low (Swingley et al. 2008), which may reflect the replacement of Chl aby Chl d, also Chl a'by Chl d', in the PS I RC of A. marina(see Fig. 3).

It is interesting to note that the primary electron acceptor, A0, in PS I of A. marinais not Chl dbut Chl a(Figs. 2 and 3), which was first shown by laser photolysis experiment (Kumazaki et al. 2002), and then supported by flash-induced spectral analysis (Itoh et al. 2007). The results support our hypothesis that Chl a-derivative is a general feature of A0 in the PS I-type RCs (see Figs. 2 and 3).

2.3.1.3. 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) a'in green sulfur bacteria, BChl g'in heliobacteria, Chl a'in Chl a-type PS I, and Chl d'in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a')2 and (BChl g')2 in anoxygenic organisms, or heterodimers, Chl a/a'and Chl d/d'in oxygenic photosynthesis. BChl g/g'may be a convincing ancestor of Chl a/a', because the BChl g/g'→ Chl a/a'conversion takes place spontaneously under mild conditions in vitro(Kobayashi et al. 1998). Further, a Chl a/a'→ Chl d/d'conversion also occurs with ease in vitro(Kobayashi et al. 2005: Koizumi et al. 2005), supporting the succession from the Chl a-type cyanobacteria to A. marina(Fig. 3). Chl fis produced in very small amounts in a Chl a-type special cyanobacterium, only when cultivated under NIR light, suggesting that Chl fappeared after the birth of Chl a. As mentioned above, the primary electron acceptors, A0, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, and the side chains appear to have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in PS I of oxygenic photosynthetic species (Ohashi et al. 2010).

In Fig. 4, BChl g/g'and their derivatives functioning in natural photosynthesis are illustrated. Chlorophyll a/a'are produced from BChl g/g'by isomerization, and Chl d/d'are then produced from Chl a/a'by oxidation. These three primed chlorophylls function as the primary electron donors of PS I-type RCs. Note that Chls b'and f 'are not found in natural photosynthesis. Chlorophylls band fare produced from Chl aby oxidation, but function as antenna pigments.

Figure 4.

Bacterlochlorophyllg/g'and their derivatives functioning in natural photosynthesis.

2.3.2. Pheophytin aas the primary electron acceptor in PS II

In 1974, pheophytin (Phe) a(Fig. 1), a demetallated Chl a, was first postulated to be the primary electron acceptor in PS II (Fig. 2) (van Gorkom 1974), and the idea was experimentally confirmed in 1977 (Klimov et al. 1977b). In 1975, bacteriopheophytin (BPhe) awas found to function as a primary electron acceptor in the RC of purple bacteria (Fig. 2) (Parson et al. 1975; Rockley et al. 1975; Fajer et al. 1975; Kaufmann et al. 1975), and shortly thereafter BPhe bwas also found to perform the same function (Fig. 2) (Klimov et al. 1977c). In 1986, BPhe awas also found to function in green filamentous bacteria (Fig. 2) (Kirmaier et al. 1986; Shuvalov et al. 1986). In 2001, the primary electron acceptor of PS II in A. marinawas first found to be Phe a(Fig. 2) (Akiyama et al. 2001) and later supported by Tomo et al. (2007). For more details, see Chapter 4 in Kobayashi et al. (2006) and a review by Ohashi et al. (2008).

It is of interest to note that Phe aas well as Chl a', d'and Chl dare artifacts easily produced invitro(see Fig. 4): Phe acan be readily produced from Chl aunder acidic conditions, primed chlorophylls from non-primed ones by epimerization under basic conditions, and Chl dfrom Chl aunder oxidative conditions. These artifacts, however, function as key components in natural photosynthesis, while Phes b, d, fand Chls b', f 'are not found in nature.

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3. Physicochemical properties of chlorophylls in vitro

3.1. HPLC

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

Chls aand bwere extracted with acetone/methanol (7/3, v/v) mixure at 277 K from parsley (Petroselinum crispumNym.), Chl dfrom A. marina, and Chl ffrom a cyanobacterium strain KC1 grown under near-infrared LED light. The extract was applied to a preparative-scale HPLC (Senshupak 5251-N, 250 mm x 20 mm i.d.) and eluted with hexane/2-propanol/methanol (100/2/0.4, v/v) at a flow rate of 7 mL min-1 at 277 K, as described elsewhere (Kobayashi et al. 1991). Other authentic pigments, Chl a', Chl f', Phe aand Phe f, were prepared by epimerization and pheophytinization of Chl aand Chl fas described elsewhere (Watanabe et al. 1984).

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, v/v) at a flow rate of 0.9 mL min-1, and were monitored with a JASCO UV-970 detector (λ = 670 nm) and a JASCO Multiwavelength MD-915 detector (λ = 300 - 800 nm) in series.

As illustrated in Fig. 6(F), eight Chls and four Phes are clearly separated. One can easily see that Synechocystissp. PCC6803 possesses Phe aand Chl a'as well as Chl a, and that Chlorellavulgarishas also Chl b.

3.1.2. Pigment extract from A. marina

Pigments were extracted from cell suspension (ca. 10 μL) by sonication in a ca. 300-fold volume of acetone/methanol (7/3, v/v) mixture for 2 min in the dark at room temperature. The extract was filtered and dried in vacuo. The whole procedure was completed within 5 min. The solid material thus obtained was immediately dissolved in 10 μL of chloroform, and injected into a silica HPLC column.

As seen in Fig. 6(C), A. marinahas three minor chlorophylls, Phe a, Chl aand Chl d', in addition to the major Chl d(Akiyama et al. 2001). Pheophytin afunctions as the primary electron acceptor in PS II, Chl aas the primary electron acceptor in PS I, and Chl d'as the primary electron donor P740 as a heterodimer of Chl d/d', like the Chl a/a'in P700 (Fig. 2).

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 a, as well as small amounts of Chl a'and Phe a, were detected in both cells. We should note that only the strain KC1 grown under NIR LED light showed the presence of Chl fas a minor pigment and that Chl f'and Phe fwere not detected at all (Fig. 6E).

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).

Figure 5.

Absorption spectra of (A) the cells of strain KC1 grown under white fluorescent light (- - -), near infrared (NIR) LED light (―) and (B) acetone solution of acetone/methanol extracts from the corresponding KC1 cells. Emission spectrum of NIR LED (-•-•-) is inserted in (A).

Figure 6.

Normal-phase HPLC profiles for acetone/methanol extracts of (A)Synechocystissp. PCC6803, (B)Chlorella vulgaris, (C)A. marina, (D) the cyanobacterium strain KC1 grown under white fluorescent light, (E) the cyanobacterium strain KC1 grown under near-infrared LED light, and (F) a mixture of Chls and Phes. Detection wavelength is 670 nm.

3.2.1. Chlorophylls a, b, dand f

Absorption spectra of Chls a, b, dand fin four kinds of solvent measured at room temperature are shown in Fig. 7 (top). As compared to Chl a, Chl bshows red-shifted Soret bands and blue-shifted weak QY bands, while the QY bands of Chls dand fare intensified and shifted to longer wavelengths. The Qx exhibits practically no intensity. The ratios of Soret/QY band intensities show remarkable differences, e.g., in diethyl ether ca. 1.3 in Chl a, ca. 2.8 in Chl b, ca. 0.85 in Chl d, and ca. 0.65 in Chl f(Table 1). Note that the Soret/QY-band ratios in Chl bis more than 2 and those in Chls dand fare below 1 in all solvents in Fig. 7, while the ratios of Chl ain three solvents excluding methanol are around 1.3, but slightly below 1 in methanol. So one can easily distinguish Chl bfrom Chls a, dand f; also Chl afrom Chls dand f, by their absorption spectra in any solvents used here.

Figure 7.

Comparison of the absorption spectra of Chlsa,b,d,f(top), and Phesa,b,d,f(bottom) in diethyl ether, acetone, methanol and benzene at room temperature. Spectra were scaled to the Soret- or QY-band maximum.

Compoundλmax,blue [nm]λmax,red [nm]
(ε[103 M-1cm-1])(ε[103 M-1cm-1])Ref
Chl a428.4660.3Watanabe et al.(1984)
(115)(89.8)ibid.
429.1b661.6bibid.
(100)b(81.3)bibid.
432.5d665.4dibid.
(101)d(79.7)dibid.
429.0660.9Kobayashi et al.(2006)
(1.00)a(0.775)aibid.
431.3b662.2bThis work
(1.00)a,b(0.828)a,bibid.
432.5c665.8cibid.
(0.944)a,c(1.00)a,cibid.
432.5d665.3dibid.
(1.00)a,d(0.785)a,dibid.
Phe a408.4667.9Watanabe et al.(1984)
(107)(52.6)ibid.
409.2b665.9bibid.
(104)b(46.0)bibid.
414.8d671.6dibid.
(108)d(53.1)dibid.
408.4667.3Kobayashi et al.(2006)
(1.00)a(0.497)aibid.
408.9b665.4bThis work
(1.00)a,b(0.440)a,bibid.
409.2c665.7cibid.
(1.00)a,c(0.464)a,cibid.
414.5d670.8dibid.
(1.00)a,d(0.798)a,dibid.
Chl b451.9641.9Watanabeet al.(1984)
(159)(56.7)ibid.
455.8b644.6bibid.
(136)b(47.6)bibid.
457.9d646.2dibid.
(152)d(56.2)dibid.
452.4642.5Kobayashi et al.(2006)
(1.00)a(0.355)aibid.
458.7b646.0bThis work
(1.00)a,b(0.355)a,bibid.
469.4c652.2cibid.
(1.00)a,c(0.355)a,cibid.
458.3d646.5dibid.
(1.00)a,d(0.364)a,dibid.
Phe b432.7654.6Watanabe et al.(1984)
(172)(34.8)ibid.
433.8b653.3bibid.
(153)b(29.3)bibid.
439.5d656.7dibid.
(152)d(4.718)dibid.
433.2654.5Kobayashi et al.(2006)
(1.00)a(0.202)aibid.
434.4b653.3bThis work
(1.00)a,b(0.195)a,bibid.
436.0c653.9cibid.
(1.00)a,c(0.245)a,cibid.
440.0d656.9dibid.
(1.00)a,d(0.216)a,dibid.
Chl d447688Smith and Benitez (1955)
(87.6)(98.9)ibid.
447688French (1960)
(87.6)(98.5)ibid.
390, 445686Miyashita et al.(1997)
392b, 447b688bibid.
400c, 455c697cibid.
445.6686.2Kobayashi et al.(2006)
(0.853)a(1.00)aibid.
394.3b, 451.7b691.4bThis work
(0.559)a,b(0.826)a,b(1.00)a,bibid.
400.8c, 455.5c698.1cibid.
(0.735)a,c(0.706)a,c(1.00)a,cibid.
394.2d, 450.2d693.7dibid.
(0.532)a,d, (0.885)a,d(1.00)a,dibid.
Phe d421692Smith and Benitez (1955)
(84.9)(72.2)ibid.
421692French (1960)
(84.9)(72.2)ibid.
382.7, 421.3692.0Kobayashi et al.(2006)
(0.881)a,(1.00)a(0.911)aibid.
383.7b, 421.5b691.0bThis work
(0.888)a,b, (1.00)a,b(0.761)a,bibid.
384.0c, 410.7c693.1cibid.
(1.00)a,c, (0.964)a,c(0.637)a,cibid.
387.8d, 428.8d697.3dibid.
(0.802)a,d, (1.00)a,d(0.915)a,dibid.
Chl f395.6, 440.5695.2This work
(0.657)a (0.648)a(1.00)aibid.
398.2b, 442.0b701.0bibid.
(0.780)a,c, (0.576)a,b(1.00)a,bibid.
400.9d, 444.0d700.9dibid.
(0.668)a,d, (0.658)a,d(1.00)a,dibid.
406.7c708.3cAkutsu et al.(2011)
(0.904)a,c(1.00)a,cibid.
406706Chen et al.(2010)
(1.00)a,d(0.527)a,dibid.
Phef409.3696.9This work
(1.00)a(0.727)aibid.
409.3b697.9bibid.
(1.00)a,b(0.610)a,bibid.
410.0c699.8cibid.
(1.00)a,c(0.561)a,cibid.
415.0d701.9dibid.
(1.00)a,d(0.776)a,dibid.

Table 1.

Absorption properties of chlorophylls in diethylether at room temperature

a: relative values


b: in acetone


c: in methanol


d: in benzene


It is somewhat difficult to distinguish Chl ffrom Chl d, when one roughly compares the absorption spectrum of Chl fin diethyl ether (Fig. 7A"') with that of Chl din methanol (Fig. 7C"), because their spectral shapes are very similar. In contrast, in diethyl ether one can easily distinguish Chl ffrom Chl dwithout spectrophotometer, because Chl flooks blue-green as Chl a, while Chl dlight-green as Chl b, indicating that the naked eye is often powerful for colour judgement.

The Soret bands include several intense bands. In diethyl ether and benzene, the Soret band of Chl fis clearly split into two bands, most probably the so-called B-bands (longer wavelength) and η-bands (shorter wavelength), while Chl dshows such a split not in those solvents but in methanol, and hence one can easily distinguish them by comparing their optical spectra in the same solvents, e.g., diethyl ether (Figs. 7A" and A"').

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 f. We had better pay attention that the NIR LED emission spectrum seen in Fig. 5A overlaps the absorption spectrum of the strain KC1 cells grown under white fluorescent light, indicating that the cells without Chl fcan absorb NIR LED light, where some Chl amolecules possessing longer wavelength absorption may act as a trigger for Chl fbiosynthesis under NIR LED light. If this hypothesis holds, a much longer wavelength LED could not induce Chl fbiosynthesis. In such a study, one should give a lot of care to the shorter wavelength foot of emission spectrum of NIR LED not to overlap the absorption of cells at all.

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 aby the electron-withdrawing group, -CHO, yielding Chl b, causes the blue-shift and significant intensity reduction of the QY-band (Fig. 7). In contrast, replacement of -CH3 on ring I of Chl aby -CHO, yielding Chl f, causes the red-shift and intensity increase of the QY-band (Fig. 7). A similar phenomenon is clearly seen in Chl d, where -CH=CH2 on ring I of Chl ais replaced with -CHO. These results indicate it is a general feature that substitution by the electron-withdrawing group on ring II causes the blue-shift and intensity reduction of the QY-band and that the same substitution on ring I leads the opposite, namely, the red-shift and intensity increase of the QY-band. Moreover, it looks that substitution on ring I by the electron-withdrawing group generates the well-split Soret band, while showing heavy dependence on solvent as described above.

3.2.2. Pheophytins a, b, dand f

The free base related to Chl is called Phe. First of all, we emphasize that in natural photosynthesis only Phe afunctions, and Phes b, dand fare not functional. In general, the more structured shape and red shifted Soret band of Chls distinguishes them from the corresponding Phes. In contrast to Chls, the η bands in the Soret band was poorly resolved in any Phes except Phe d(Fig. 7). Removal of the central Mg increases deviation from planarity and reduces the molecular symmetry, thus increasing Soret and QX transition. The Soret/QY-band ratios noticeably increases by pheophytinization; in diethyl ether Phe bshows the highest value of around 5, Phe athe secondary highest about 2, and Phe dthe lowest near 1 (compare bottom with top in Fig. 7, see also Table 1). Therefore, contamination of pheophytins in a Chl sample is often noticed from the optical spectra.

As seen in Fig. 7, Phe bcan be easily distinguished from Phe aby its blue shifted QY-band, red shifted Soret band, and by the marginally higher Soret/QY band ratio. Phes dand fcan be distinguished from Phes aand bby their red shifted QY band and intense QY bands, i.e., the Soret/QY-band ratios in Phes dand fare not high and almost the same as those seen in Chl a. We should also pay attention to Phe f, because in methanol its optical shape is somewhat similar to Phe a(compare Figs. 7G with 7G"'), although they can be distinguished by the QY wavelength difference. We must emphasize again that Phes possess relatively strong and characteristic QX-bands in the region of 490-570 nm; the QX bands in Phes aand dare better resolved to the QX(0,0) and QX(1,0) transitions. Phe dalso shows significant splitting of the Soret and QX bands in all solvents as illustrated in Fig. 7. In contrast, the Qx band corresponding to the QX(1,0) transition (shorter wavelength) of Phe flooks unclear. It is of interest to note that in diethyl ether Phe dassumes a pale pink color, while both Phes band fshow a dull color. These characteristics will help us to discern Phes from Chls, and among Phes.

3.3. Circular dichroism spectra

Circular dichroism (CD) spectra are very useful for distinction between the primed chlorophyll, e.g., Chl a', and the corresponding non-primed one, Chl a, although the absorption characteristics of the primed derivatives (Chls a', b', d', f', Phes a', b', d'and f') are identical with those of the non-primed ones (Wolf and Scheer 1973; Weiss 1978; Watanabe et al. 1984; Kobayashi et al. 2006b).

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/a', b/b', d/d'and f/f'in benzene are illustrated in Fig. 8. It is immediately seen that, for a given pair of epimers, the CD spectra are considerably different, although the absorption spectra are practically identical with each other. For each of Chls a', b', d'and f', an intense negative CD is associated with QY(0,0) and a well-defined weakly negative satellite with QY(1,0). On the other hand, the non-primed species, Chls a, b, dand f, show complicated, very weak negative and/or positive activities at these transitions. In Fig. 9, all pheophytins show negative activities, and primed ones reveal stronger and red-shifted signals compared to the non-primed ones, although the absorption spectra of the primed derivatives are also identical with those of the non-primed ones. The findings suggest that the QY maximum transition consists of at least two bands, and shorter wavelength band shows stronger activity in primed Phe and longer wavelength band stronger in non-primed Phe.

Figure 8.

CD spectra for (A) Chlsa/a', (B) Chlsb/b', (C) Chlsd/d', (D) Chlsf/f', and (A')-(D') the corresponding absorption spectra in benzene at room temperature. [θ] denotes the molar ellipticity.

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 a', Phe a', Chl d', Phes d, d', Chl f', Phes fand f', respectively, while Chl fand Phe aexhibit weakly negative activities at 609 nm and 535 nm, respectively. Such definite activities are not observed in Chls a, b/b', dand Phe b/b'; optical activities of b-type pigments, Chl b/b'and Phe b/b', are extremely weak, which may be related to the very diffuse feature of their absorption spectra. The CD activity associated with the QX(0,1) absorption satellites (band IV) at the shorter wavelength also is very weak and vague in all the pigments examined.

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 fand Phes, apparently reflect the existence of the two transitions: they show a maximum and a minimum with the center wavelength roughly coinciding with the Soret absorption maximum. Different feature of CD spectrum for Chl famong Chls may come from its characteristically splitted Soret absorption arising from B-bands and so-called η-bands in benzene. Primed Chls exhibit relatively intense negative CD spectra in the near ultraviolet at η-bands of the Soret region (Welss 1978; Petke et al. 1979), whereas non-primed ones exhibit positive activities. Phes aand falso show positive CD spectra at η-bands in the near ultraviolet region, but such a tendency is not clear in Phe b, and Phe dexhibits negative activity (Fig. 9), although all the primed Phes show negative activity and Phe d'shows the most intense activity.

Figure 9.

CD spectra for (A) Phesa/a', (B) Phesb/b', (C) Phesd/d', (D) Phesf/f', and (A')-(D') the corresponding absorption spectra in benzene at room temperature. [θ] denotes the molar ellipticity.

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 (e.g.phytol) and of the central metal (see reviews by Smith 1975; Hunt and Michalski 1991; Porra and Scheer 2000; Kobayashi et al. 2006).

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, v/v) at a flow rate of 300 μL min-1. The eluate was monitored by the UV-Vis absorption in a range of 220-800 nm, and was introduced into the mass spectrometer from 5 to 55 min after sample injection. Atmospheric pressure chemical ionization (APCI) mass and MS/MS spectra were recorded in the positive-ion mode in the mass range of m/z150-2,000. Helium was used as collision gas for MS/MS experiments, followed by the isolation of ions over a selected mass window of 2 Da. The mass spectrometer was initially tuned using a standard Chl asolution as follows: APCI vaporizer temp., 723 K; spray voltage, 4 kV; capillary temperature, 423 K; capillary voltage, 8 V; sheath gas (nitrogen); flow rate, 56 (arbitrary unit); auxiliary gas flow rate, 9 (arbitrary unit).

As illustrated in Fig. 10 (left), Chl a(C55H72MgN4O5, monoisotopic mass; 892.535. Hereafter the value in the bracket shows the monoisotopic mass of the molecule or the ion) gives the protonated molecule ([M+H]+) at m/z893.2 producing the dominant fragment ion at m/z615.1. The mass difference 278 between [M+H]+ and the product ion corresponds to C20H38. This shows the presence of a phytyl chain in Chl a. The other product ions at m/z583.0 and m/z555.2 corresponding to [M+H-278-32]+ and [M+H-278-60]+, respectively, are supposed to be the results of the loss of carboxymethyl group followed by the cleavage of phytol. The losses of 278, 310 and 338 from the precursor ion in MS/MS spectra are seen in all the pigments examined here reveals the presence of a phytyl chain.

Figure 10.

MS/MS spectra of Chlsa,b,d,f(left column) and Phesa,b,dandf(right column). Each mass spectrum of the chlorophyll fraction is shown in the shaded square. MS/MS spectra of the protonated molecules ([M+H]+) of Chlsa,b,dandfgive product ions of [M+H-278]+, [M+H-278-32]+ and [M+H-278-60]+. Pheophytinsa,b,dandfgive product ions of [M+H-278]+ and [M+H-278-60]+.

It is interesting to note that a typical fast atom bombardment (FAB)-mass spectrum of Chl ashows two intense molecular ion peaks, [M]+ at m/z892.6 and [M + H]+ at 893.6, as well as the dominant fragment ion [M-C20H38+H]+ at m/z614.3 (see Fig. 4 in Kobayashi et al. 2000). Both spectra, however, reveal that Chl ahas a phytyl chain (C20H39). We should make sure that the mass spectra of chlorophyll molecular ion peak(s) and the fragment ion peak(s) vary by m/z1.0 according to the ionization methods.

Chlorophyll d(C54H70MgN4O6, 894.515) gives the protonated molecule ([M+H]+) at m/z895.2 and the prominent fragment ion at m/z617.1. Though chlorophylls band f(both C55H70MgN4O6, 906.515) are eluted at different LC retention times, they show the same mass and MS/MS spectral patterns, [M+H]+ at m/z907 and the dominant product ion at m/z629, which correspond to [C55H71MgN4O6]+ (907.522) and [M-C20H38+H]+ (629.225). These results suugest that Chl falso possesses a phythyl long chain in such molecules as Chls a, band d, and that most probably one -CH3 moiety of Chl ais substituted for -CHO group in Chl flike Chl b, yielding [2-formyl]-Chl a, [12-formyl]-Chl aor [18-formyl]-Chl a.

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 ais observed at m/z871.3 which is 22 Da smaller than that of Chl a, showing the substitution of Mg with two H atoms by pheophytinization (see Fig. 1). Pheophytins band f(C55H74N4O6, 884.545) and Phe d(C54H72N4O6 872.545) show the similar pattern, supporting that all of them do not possess Mg as central metal.

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-d6 with tetramethylsilane (TMS) as an internal standard. The chemical shifts are given in δ-scale [ppm] downfield from TMS. The measurements were performed at 273 K. The typical experimental conditions for the 1H NMR spectra were 256 scans, a spectral width of 17 ppm, 128k data points. The 13C spectra were acquired using a power gated decoupling with 48k scans. The spectral width of 220 ppm was acquired in 64k data points. The 2D-homonuclear (Nuclear Overhauser and Exchange Spectroscopy (NOESY)) and 2D-heteronuclear (H,C-Heteronuclear Single Quantum Coherence (H,C-HSQC) and H,C-Heteronuclear Multiple Bond Correlation (H,C-HMBC)) experiments were performed for the structural assignments of the 1H and 13C signals using standard 2D-NMR pulse sequences of Bruker software.

3.5.1. 1H-NMR

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 b(71), d(31) and f(21) is absent from the spectrum of Chl a. Similarly, double doublet signal of 31-H vinylic proton at 8-8.5 ppm in the spectra of Chls a, band fis not seen in the spectrum of Chl d. These results reconfirm that Chl dis 3-desvinyl-3-formyl Chl a([3-formyl]-Chl a).

Here we note that the 31-H vinylic proton shows a large downfield shift in Chl f(8.534 ppm) and that is slightly upfield shifted in Chl b(8.043 ppm), as compared to Chl a(8.162 ppm), suggesting that Chl fshould be formylated along the y-axis and that interaction between -CH=CH2 and -CHO in Chl fis rather strong than that in Chl b. The -CHO substitution position in Chl fis hence most probably at C2, next to the 3-vinyl group.

The pair signals of 32- and 32'-H vinylic protons are well resolved in the spectra of Chls a(6.242 ppm and 6.028 ppm) and b(6.302 ppm and 6.055 ppm), while the corresponding pair signals in Chl fshow low resolution (6.365 ppm and 6.324 ppm), suggesting that the environment of 3-vinyl moiety in Chls aand dis very similar to each other, but is profoundly different from that in Chl f, most probably due to the presence of formyl moiety at the neighboring C2 in Chl f. The 1H singlet signals for 71-CH3 in Chls a, dand fare at 3.3 ppm and absent from the spectrum of Chl b, while the 1H signal for 71-CHO in Chl bappears on a much lower field at 11.3 ppm. Another marked difference seen in Chl fspectrum is the disappearance of the singlet signal of 21-CH3 proton; the corresponding signals are observed at 3.3-3.72 ppm in the spectra of Chls a, band d, implying that 21-CH3 of Chl ais substituted in Chl ffor some other moiety, most probably -CHO. No other change is clear in the one-dimensional 1H-NMR spectra.

3.5.2. 13C-NMR

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 a, in the spectrum of Chl f, the 13C signal of 21-CH3 is absent, the 71-CH3 and 31,2-CH=CH3 carbon signals appear at 11 ppm, 130 ppm and 126 ppm, respectively, similar to Chl a(11 ppm, 131 ppm and 120 ppm). Further, a new carbon signal appears at 189 ppm, close to the signals of -CHO in Chls b(188 ppm) and d(190 ppm), supporting the presence of -CHO at C2 in Chl f.

Figure 11.

1H-NMR spectra of Chlsa,b,dandfmeasured in acetone-d6 at 273 K. Signals corresponding to 1H atoms of the macrocycle are labeled with the numbers of the corresponding carbons. The peak at 2.06-2.11 ppm is acetone and 3.07-3.10 ppm is H2O, respectively.

IUPAC no.ofcarbon atomChl aChl bChl dChl f
213.343 (3.36)1 (s)3.316 (3.40)2*(s)3.724 (3.68)3(s)11.215 (11.35)4**(s)
3----
318.162 (8.18)1(dd)8.043(7.95)2*(dd)11.460 (11.40)3(s)8.534(dd)
326.242 (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)
4----
59.410 (9.40)1(s)10.192 (10.04)2*(s)10.294(10.20)3(s)9.770 (9.79)4**(s)
713.300 (3.30)1(s)11.305 (11.22)2*(s)3.365 (3.33)3(s)3.351(s)
8----
813.817 (3.82)1(q)4.2433.876 (3.86)3(q)3.754(q)
821.696 (1.69)1(t)1.8151.723 (1.73)3(t)1.705(t)
109.749 (9.75)1(s)9.934 (9.64)2*(s)9.873 (9.8)3(s)9.838 (9.86)4**(s)
11----
12----
1213.619 (3.61)1(s)3.606 (3.65)2*(s)3.668 (3.65)3(s)3.637(s)
13----
1326.234 (6.24)1(s)6.189 (6.19)2*(s)6.335 (6.28)3(s)6.318(s)
133----
1343.829 (3.83)1(s)3.842 (4.02)2*(s)3.851 (3.83)3(s)3.887(s)
174.175 (4.16)14.1284.242 (4.25)34.230
1712.589 (2.60)1, 2.461 (2.45)12.43,2.5932.484,2.6222.467,2.632
1722.431 (2.35)1, 2.159 (2.05)12.08,2.441.98,2.4182.08,2.47
184.572 (4.57)1(q)4.524(q)4.660 (4.63)3(q)4.634(q)
1811.772 (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)
208.582 (8.58)1(s)8.480(8.20)2*(s)8.867 (8.81)3(s)9.533 (9.77)4**(s)
P14.342 (4.33)1,4.224 (4.21)14.364, 4.2474.343 (4.26)3, 4.227 (4.36)34.361, 4.263
P24.955 (4.95)14.9804.944 (5.04)34.987
P3----
P311.509 (1.51)11.5191.505 (1.54)31.525
P41.822 (1.82)11.8451.832 (1.85)31.845
P51.311.311.301.195
P60.97,1.170.98,1.181.97,1.160.97,1.17
P71.311.331.311.324
P710.811,(0.81)1, 0.803(0.80)10.785, 0.7770.778, 0.770 (0.79)30.785, 0.777
P81.01,1.231.02,1.221.01,1.221.01,1.22
P91.15,1.281.15,1.281.14,1.281.15,1.27
P101.01,1.231.02,1.221.01,1.221.01,1.22
P111.311.321.321.32
P1110.783(0.79)1,0.774 (0.78)10.809,0.8010.806, 0.797 (0.81)30.807, 0.798
P121.01,1.231.02,1.221.01,1.221.01,1.22
P131.23,1.281.23,1.281.23,1.281.23,1.28
P141.121.121.121.12
P151.500 (1.50)11.4891.497 (1.51)31.495
P1510.854(0.86)1,0.845 (0.84)10.851,0.8420.850 (0.85)3, 0.842 (0.85)30.849, 0.841
P160.854(0.86)1,0.845 (0.84)10.851,0.8420.850 (0.85)3, 0.842 (0.85)30.849, 0.841

Table 2.

1H-chemcal shifts of Chls a, b, dand fin acetone-d6 at 273K

1Kobayashi et al. (2000), 2Wu et al. (1985), 3Miyashita et al. (1997), 4Chen et al. (2010)


* in CDCl3, **in CD2Cl2/d5-pyridine(97/3, v/v)


Figure 12.

NMR spectra of Chlsa,b,dandfmeasured in acetone-d6 at 273 K. Signals corresponding to 13C atoms of the molecules are labeled. The peak at 30-32 ppm is acetone.

IUPAC no. ofcarbon atomChlaChlbChldChlf
1155.47(155.46)1159.43151.47(151.81)2151.02
2136.28(136.24)1136.98147.66(147.33)2136.49
2112.65(12.70)112.3312.58(11.75)2189.27
3139.76(139.68)1142.53134.77(135.12)2138.17
31131.30(131.29)1130.83189.59(189.54)2130.22
32120.33(120.33)1120.80-126.36
4148.96(148.99)1150.15146.00(146.36)2150.20
5100.58(100.51)1103.63106.24(104.41)2105.39
6152.78(152.80)1157.25152.27(152.55)2149.24
7134.68(134.65)1140.76136.19(136.31)2133.35
7111.15(11.16)1188.6211.25(11.39)211.17
8145.08(145.02)1148.32145.02(145.13)2143.83
8119.91(19.88)119.5119.94(20.12)219.87
8218.12(18.18)119.9118.12(18.12)218.07
9146.74(146.74)1149.29148.28(148.64)2144.59
10108.50(108.53)1111.53107.68(107.8)2107.82
11148.34(148.34)1149.94149.97(150.35)2159.99
12134.57(134.46)1138.66136.50(136.63)2132.12
12112.65(12.66)112.4712.79(12.89)212.82
13131.53(131.41)1132.13132.76(133.04)2131.19
131190.30(190.37)1190.66190.49(190.50)2190.68
13266.00(65.95)165.7766.19(66.47)266.13
133171.32(171.36)1171.04171.20(171.33)2173.30
13452.67(52.71)152.7952.79(52.84)252.83
14162.55(162.58)1164.11162.30(162.68)2168.67
15106.35(106.27)1105.94106.82(107.04)2106.03
16156.50(156.54)1160.38158.12(158.60)2163.04
1750.98(50.92)151.1451.54(52.05)251.74
17130.28(30.03)130.2830.66(30.64)230.90
17230.66(30.09)130.6631.27(31.37)230.35
173173.29(173.39)1173.35173.26(173.45)2189.25
1849.73(49.69)149.6549.44(49.80)249.26
18123.01(23.88)123.6824.13(24.25)224.20
19169.69(167.74)1168.03168.09(168.34)2171.15
2093.78(93.79)193.8695.26(95.36)297.60
P161.31(61.32)161.4261.33(61.67)261.35
P2120.17(119.12)1119.19119.13(119.53)2119.18
P3142.46(142.48)1143.42142.49(142.70)2142.49
P3116.11(16.10)116.1616.11(16.40)216.13
P440.19(40.19)140.2340.19(40.46)240.20
P525.54(25.52)125.5725.53(25.86)225.55
P637.1137.1438.11(37.40)237.11
P733.43(33.44)133.4433.22(33.47)233.50
P7120.03(20.02)120.0319.98(20.19)220.02
P837.9437.9537.94(38.21)237.94
P925.04(25.06)125.0625.04(25.25)225.04
P1038.0138.0238.01(38.26)238.01
P1133.22(33.23)133.2533.42(33.65)233.42
P11119.98(19.98)119.9920.02(20.23)219.98
P1237.8737.8937.88(38.14)237.88
P1325.49(25.52)125.4925.49(25.64)225.48
P1440.00(39.98)140.0040.00(40.27)240.00
P1528.65(28.66)128.6528.65(28.85)228.65
P15123.013 (28.66)123.01423.015 (23.15)223.006
P1622.903 (22.91)122.91422.905 (23.07)222.906

Table 3.

13C-chemcal shifts of Chls a, b, dand fin acetone-d6 at 273K

1Kobayashi et al. (2000), 2Miyashita et al. (1997)


3-6 assignment interchangeable


The signals of 71-CH3 of Chls a, dand fshow almost the same chemical shifts, 11.15 ppm, 11.24 ppm and 11.17 ppm, respectively, indicating the interaction between the -CHO and 71-CH3 moieties in Chls dand fare negligibly small, and hence the -CHO substituent is not so close to the 71-CH3 moiety in Chls dand f. The chemical shifts of 32-vinyl carbons in Chls aand bare almost identical (120.3-120.8 ppm), but Chl fshows a slight but significant downfield shift (126.3 ppm), suggesting that the formyl substituent in Chl fis positioned very close to the 3-vinyl group, most probably on C2. The signals of -CHO moiety of Chls dand fexhibits almost the same chemical shift (189.59 ppm and 189.27 ppm), but a slight upfield C-formyl signal is observed at 188.62 ppm in Chl b, indicating that the environment of -CHO in Chls dand fis very similar, supporting that the -CHO moieties of Chls dand fare positioned at the same ring I, while that of Chl bis at ring II.

3.5.3. NOESY

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 f, the signals were investigated using NOESY spectra. First of all, we had better see well-defined coherent correlations on the NOESY spectrum of Chl a. Here, we will trace the coherent correlations from meso-20-H, because Chl fposseses -CHO most probably at C2 near to C20. Coherent correlations can be easlily traced from 20-H on the NOESY spectrum of Chl a(Fig. 13), where the signal of 20-H at 8.582 ppm shows three cross peaks with the signals of 181-H at 1.771 ppm, 21-H at 3.343 ppm, and 18-H at 4.572 ppm. Good coherent correlations can also be traced from meso-5-H and meso-10-H; (1) the signal of 5-H at 9.410 ppm shows three cross peaks with the signal of 71-H at 3.300 ppm, 32-H at 6.242 ppm, and 31-H at 8.162 ppm, and (2) the signal of 10-H at 9.749 ppm shows two cross peaks with the signal of 82-H at 1.696 ppm and 121-H at 3.619 ppm. In Chl band Chl d, similar nice correlations are seen (Fig. 13).

Figure 13.

1H-1H-NOESY spectra of Chlsa,b,dandfmeasured in acetone-d6 at 273 K.

To obtain further evidence for Chl f,a NOESY spectrum is illustrated in Fig. 13. As expected, nice coherent correlations can be traced from meso-20-H, the signal of 20-H at 9.553 ppm shows three cross peaks with the signal of 18-H at 4.634 ppm, 181-H at 1.816 ppm, and 21-H, most probably assigned to -CHO moiety, at 11.215 ppm. The signal of meso-5-H at 9.770 ppm shows three cross peaks with the signal of 71-H at 3.351 ppm, 32-H at 6.347 ppm, and 31-H at 8.534 ppm. Similarly, coherent correlations of meso-10-H with 81-H (3.754 ppm) and 121-H (3.637 ppm) are clearly seen. These results indicate that the C-20 methine, among the three methines, is nearest to the -CHO moiety, supporting the substitution position by -CHO is most likely at C2 in Chl f.

3.5.4. HSQC

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 a, b, dand f(Fig. 14), all substituents on the macrocycle show the corresponding cross peaks. The results support that one methyl group of Chl ais replaced with a formyl one in Chl f.

Figure 14.

1H-13C-HSQC spectra of Chlsa,b,dandfmeasured in acetone-d6 at 273 K. □ in Chld: δC189.59 (folded from outside the spectral window)

3.5.5. HMBC

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 aexhibit one to three cross peaks as seen in Fig. 15A. The formyl-H in Chls b, dand fshows two to four cross peaks. For example, in the 1H-13C HMBC spectrum of Chl f, the 1H-signal for 21-CHO has four cross peaks, one is with the signal for 31-vinylic carbons, one is with the signal for 1C carbon, and the other two are with the signal for 21-CHO carbon, where the cross peak split is due to the direct coupling. Conclusively, the Chl f-like pigment isolated from the strain KC1 has been identified as Chl f, namely, 2-desmethyl-2-formyl-Chl a([2-CHO]-Chl a).

Figure 15.

1H-13C-HMBC spectra of Chlsa,b,dandfmeasured in acetone-d6 at 273 K.

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 in vitroare presented.

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 in vacuoat 473 K over 24 h. Tetra-n-butylammonium perchlorate (Bu4NClO4, TBAP) (Aldrich, Electrochemical grade: > 99.0 %), was used as the supporting electrolyte, which had been recrystallized from methanol solution and then dried in vacuo at 333 K over 24 h.

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 a, b, dand fin acetonitrile are illustrated in Fig. 16. Four peaks are observed in each voltammogram and the potentials in anodic sweep and cathodic sweep (data not shown) are identical to each other, indicating that the four redox reactions are reversible. Similar trends are observed for Phes a, band d(data not shown). The measurement for Phe fis now underway.

In Table 4 are summarized the redox potentials for Chls a, b, d, f, Phes a, band d. Chl dshows higher oxidation potentials than Chl a, lower than Chl b, and much lower than Phe a, Phe band Phe d. Chl fexhibits higher oxidation potentials than Chls a, d, and lower than Chl b. The results can be explained by invoking the inductive effect of substituent groups on the macrocycle, because the redox potentials of chlorophylls are sensibly affected by the nature of substituent groups on the π-electron system (Fuhrhop 1975, Watanabe and Kobayashi 1991, Kobayashi et al. 2007).

The -CHO substituent on Chls b, dand fis an electron-withdrawing group (→CHO), and hence reduces the electronic density in the π-system of chlorophyll. The replacements of -CH3 at C7 or C2 of Chl aby →CHO to yield Chl bor Chl fcause the macrocycle to be electron poor, thus rendering the molecule less oxidizable (E1ox: Chl b, f> Chl a). Similarly, replacement of -CH=CH2 at C3 of Chl aby →CHO to yield Chl dmakes the first oxidation potential, E1ox, more positive than that of Chl a(E1ox: Chl d> Chl a). Therefore, the E1ox order becomes Chls b, d, f> Chl a(see Figs 16 and 17). When one pays attention to the group of -CH3 at C7 of Chl dand the group of -CH=CH2 at C3 of Chl bor C7 of Chl f, the -CH3 group is more electron-donating (←CH3), thus making the macrocycle of Chl dmore electron rich, and hence its oxidation potential less positive (Chls b, f> d); the E1ox order results in Chls b, f> Chl d> Chl a. As expected from the inductive effect of substituent groups, Chls band fshow the almost the same E1ox values. Consequently, as seen in Fig. 17, the E1ox order results in Chl b> Chl f> Chl d> Chl a; a little higher oxidation potential of Chl bthan that of Chl f, 20 mV, cannot be explained from the primitive way used here.

Figure 16.

Square wave voltammograms of Chlsa,b,dandfin acetonitrile.

E2redE1redE1oxE2oxE1ox - E1red
V vs. SHE
Chl a-1.46-1.120.811.041.93
Chl b-1.41-1.020.941.151.96
Chl d-1.27-0.910.881.091.79
Chl f-1.12-0.750.921.131.67
Phe a-1.00-0.751.141.491.89
Phe b-1.05-0.641.251.581.89
Phe d-0.87-0.631.211.501.84

Table 4.

Redox potentials of Chls a, b, d, f, Phes a, band din acetonitrile

Figure 17.

Schematic comparison of redox potentials of Chlsa,b,dandfin acetonitrile.

The redox behavior of a compound is related to the energy levels of its molecular orbitals: E1ox is intimately related to the highest occupied molecular orbital (HOMO) and E1red to the lowest unoccupied molecular orbital (Watanabe and Kobayashi 1991; Hanson 1991). The order of HOMO energy levels well parallels the E1ox values, while the correlation between the LUMO levels and the E1red values is less conspicuous (see Fig. 7 in Watanabe and Kobayashi 1991).

As clearly seen in Fig. 17, the order of absolute values of the first reduction potentials, E1red, of Chls a, dand fis Chl a> Chl d> Chl f, which can be well explained by substituent inductive effect, like E1ox as mentioned above. This simple rule, however, does not hold for Chls dand f; their values of E1red are remarkably more positive than those of Chls aand b; compared to Chl a, Chl bis harder to oxidize by 130 mV, and easier to reduce by 100 mV, namely, similar in degree, while Chls dand fpeculiarly easier to reduce by 210 mV and 370 mV. The findings indicate that the inductive effect of substituent groups at ring II conspicuously appears for E1red. Anyway, this irregularity may come from the fact that the LUMO energy levels are to a lesser extent correlated with the E1red values.

The primary redox potential difference, ΔE= E1ox - E1red, seen in Fig. 17 can be taken as an index for the QY excitation energy (Watanabe and Kobayashi 1991; Hanson 1991). For example, ΔEfor Chl ais 1.93 eV in Fig. 17, which corresponds to the QY excitation wavelengths to a certain extent, 661-666 nm for Chl ain Fig.17. Similarly. ΔE= 1.96 eV, 1.79 eV and 1.67 eV for Chls b, dand falso correlate to the QY wavelengths, 644-662 nm, 686-698 nm. and 695-708 nm. Pheophytins also behave in a similar fashion.

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.

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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 b. One is based on vertical transfer of the gene for Chl bbiosynthesis, which includes the lost of the gene. The other is based on the lateral gene transfer for Chl bbiosynthesis. In the hypothesis of the former one, the ancestral cyanobacteria contained both Chls aand b, and the gene for Chl bbiosynthesis had been lost. In many cyanobacterial linage as well as the linages of the red algae and glaucophytes, the gene had been lost, whereas a few linage of cyanobacteria, (Prochloronand Prochlorothrix) and the green algae (and terrestrial plants) retained it. This hypothesis is partly supported by the high homology of the enzyme for Chl bbiosynthesis (CAO) (Tomitani et al. 1999). In the latter hypothesis, the gene for Chl bbiosynthesis was firstly evolved in the ancestor of green algae or cyanobacteria, and the gene was transferred into different linages. This is supported by the fact that one gene transfer was enough for the acquisition of Chl bbiosynthesis in cyanobacerium. The secondary endosymbioses of green algae gave rise to euglenophytes, chlorarachniophytes and "green" dinoflagellates, which were containing Chls aand b. The secondary or tertiary plastids of cryptophytes, haptophytes, heterokonts and dinoflagellates contain Chl cin addition to Chl a. Most of present-day cyanobacteria contain only Chl a, and only a few genera are known to keep Chl b(Prochloronand Prochlorothrix). This suggests that Chl bhad been lost in polyphyletic lineage during the evolution of cyanobacteria. Some cyanobacteria contain divinyl chlorophylls (DVChls) aand b(Prochlorococcus), Chl d(Acaryochloris) and Chl f(strain KC1 and Halomicronema hongdechloris). These chlorophylls are only found in cyanobacteria, and it has yet to be revealed when and how they acquired these pigments.

Figure 18.

The distribution of various chlorophylls among oxygenic photosynthetic organisms.

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 dand f, are also thought be acquired and selected by a part of cyanobacteria for their growth and survival by sustaining oxygenic photosynthesis using the light available at their niches.

Chl d-containing cyanobacterium, Acaryochloris marina, was firstly found as a minor symbionts in colonial ascidians which had large numbers of cyanobacterial symbionts, Prochloron, which contain Chls aand bas their photosynthetic chlorophylls (Miyashita et al. 1996). Subsequently, Acaryochlorisspp. has been found on the surface of coastal macroalgae (Murakami et al. 2004), under those colonial ascidians (Kühl et al. 2005), and in coastal microbial mats at a saline lake (Miller et al. 2005). In all of those niches, Acaryochlorisspp. competes for light for their oxygenic photosynthesis. However, due to the possession of Chl das primary chlorophyll, Acaryochlorisspp. can absorb and utilized far-red light from 700 nm to 740 nm for oxygenic photosynthesis (Miyashita et al. 1997). The ability has advantage for their growth and survival, since another oxygenic phototrophs which contain Chl aas their primary chlorophyll cannot absorb and utilize the light.

Chl f-containing cyanobacterium was firstly reported in microbial mat, where the competition for the light also occurs among the phototrophs (Chen et al. 2010). While the distribution and detailed niche of Chl f-containing cyanobacteria have not been elucidated yet, Chl fmust contribute to the survival of those cyanobacteria. Since Chl fin cyanobacteria also absorbed far-red light around 720 nm and Chl f-possessing cyanobacteria can grow under far-red LED light as a sole light source (Chen and Blankenship 2011).

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Acknowledgments

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.

© 2013 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Masami Kobayashi, Shinya Akutsu, Daiki Fujinuma, Hayato Furukawa, Hirohisa Komatsu, Yuichi Hotota, Yuki Kato, Yoshinori Kuroiwa, Tadashi Watanabe, Mayumi Ohnishi-Kameyama, Hiroshi Ono, Satoshi Ohkubo and Hideaki Miyashita (June 12th 2013). Physicochemical Properties of Chlorophylls in Oxygenic Photosynthesis — Succession of Co-Factors from Anoxygenic to Oxygenic Photosynthesis, Photosynthesis, Zvy Dubinsky, IntechOpen, DOI: 10.5772/55460. Available from:

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