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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Demetalation of chlorophyll (Chl) a and its analogs is an important reaction in oxygenic photosynthetic organisms, which produces the primary electron acceptors in photosystem II reaction centers and is crucial in the Chl degradation. From these viewpoints, demetalation reactions of four Chl a analogs, 3,8-divinyl-Chl a (DV-Chl a), 3-devinyl-3-ethyl-Chl a (mesoChl a), 132-demethoxycarbonyl-Chl a (pyroChl a) and protochlorophyll a (PChl a), were kinetically analyzed under weakly acidic conditions, and were compared with that of Chl a. DV-Chl a exhibited slower demetalation kinetics than did Chl a, whereas demetalation of mesoChl a was faster than that of Chl a. The difference in demetalation kinetics of the three chlorophyllous pigments originates from the electron-withdrawing ability of the vinyl group as the peripheral substituent compared with the ethyl group. Removal of the electron-withdrawing and homoconjugating 132-methoxycarbonyl group in Chl a (Chl a [RIGHTWARDS ARROW] pyroChl a) accelerated demetalation kinetics by two-fold. PChl a possessing the porphyrin-type skeleton exhibited slower demetalation kinetics than Chl a. The structure-dependent demetalation properties of Chl a analogs will be useful for understanding in vivo Chl demetalation reactions in oxygenic photosynthetic organisms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Chlorophyll (Chl) a is the most abundant photosynthetic pigment in nature, and plays important roles in photosynthetic activities, such as photoinduced charge separation, electron transfer and excitation energy transfer in oxygenic photosynthetic organisms [1]. Figure 1 shows the molecular structure of Chl a. Chl a has a central magnesium in the chlorin macrocycle, which is a 17,18-dihydroporphyrin, with an isocyclic five-membered ring. The saturation of the C17–C18 bond in the D-ring of Chl a is responsible for its spectral properties, which are appropriate for efficient light-harvesting and energy transduction during photosynthesis.

image

Figure 1. Molecular structures of Chl a, DV-Chl a, mesoChl a, pyroChl a and PChl a.

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Naturally occurring Chls in oxygenic photosynthetic organisms are commonly monovinyl pigments in terms of the peripheral substituents [1, 2]. The exceptions are divinyl (DV) pigments (3,8-divinyl-Chls (DV-Chls) a and b as well as Chls c2 and c3) and Chl d that has no vinyl group [1-7]. Marine cyanobacteria, Prochlorococcus species, possess DV-Chls a and b as major pigments [3-5]. The molecular structure of DV-Chl a is depicted in Fig. 1. DV-Chl a has a vinyl group at the 8-position, whereas this position is substituted by an ethyl group in Chl a. Owing to the 8-vinyl group, the Soret absorption bands of DV-Chls are redshifted by ca 10 nm relative to the corresponding 3-vinyl-8-ethyl-Chls. Such spectral properties would be advantageous for efficient absorption of blue light by Prochlorococcus species.

Oxygenic photosynthetic organisms have two types of reaction centers, namely photosystem (PS) I and PS II. A demetalation product of Chl a, namely pheophytin (Phe) a, functions as the primary electron acceptor in PS II of most oxygenic photosynthetic organisms [8-11]. In contrast, a mutant of Synechocystis sp. PCC6803, which is not able to hydrogenate the 8-vinyl group and accumulate DV-Chl a, was reported to possess DV-Phe a as the primary electron acceptor in PS II, instead of Phe a [12]. Biosynthesis of Phe a and DV-Phe a as the key cofactors of PS II remains completely unknown in spite of its biological importance. Chl a and DV-Chl a would be the precursors of Phe a and DV-Phe a, respectively, in PS II, as Chl synthases use chlorophyllide (Chlide) a and DV-Chlide a as substrates to esterify a long hydrocarbon chain at the 17-propionate and cannot use pheophorbide (Pheide) a lacking the central metal [13, 14]. Therefore, demetalation of Chl a and DV-Chl a is a key reaction in the biosynthesis of Phe molecules that function as the primary electron acceptors in PS II.

Demetalation of chlorophyllous pigments is also crucial in the Chl degradation pathway [15-18]. Chl degradation is one of the most large-scale bioprocesses on earth and a major origin of beautiful color changes of plant leaves in autumn. The first step in Chl a degradation would be removal of the central magnesium. The resulting Phe a is converted to Pheide a by hydrolysis of the phytyl ester by pheophytinase [19, 20]. From this point of view, demetalation properties of Chl a and its analogs have attracted considerable attention as well.

Physicochemical studies of demetalation properties of chlorophyllous pigments will provide useful information to unravel biologically important reactions in photosynthetic organisms described above, and in vitro demetalation of natural Chls and their model compounds have been examined [21-35]. Mackinney and Joslyn first reported difference in demetalation kinetics between Chls a and b in the early 1940s [21, 22]. Watanabe et al. examined detailed demetalation properties of Chls a, b and their epimers (Chls a′ and b′, respectively; [26, 27]). Kobayashi et al. analyzed demetalation of zinc bacteriochlorophyll (BChl) a [28], which was discovered in a purple photosynthetic bacterium, Acidiphilium rubrum [36, 37]. Recently, we examined demetalation properties of other naturally occurring (B)Chls [29-31] and synthetic zinc complexes [32-35], which were model compounds of natural Chls. These studies indicate that peripheral substituents on the chlorin macrocycle are responsible for physicochemical properties on demetalation of chlorophyllous pigments. However, there are few studies on demetalation of Chl a analogs concerning important metabolic intermediates. In addition, no information is available, to our best knowledge, on demetalation of DV-Chl a, which is a major photosynthetic pigment and would be a precursor of the primary electron acceptor in PS II in a Δslr1923 mutant of Synechocystis sp. PCC6803. Herein, we report demetalation properties of four Chl a analogs, DV-Chl a, 3-devinyl-3-ethyl-Chl a (mesoChl a), 132-demethoxycarbonyl-Chl a (pyroChl a) and protochlorophyll a (PChl a). Figure 1 shows molecular structures of the four Chl a analogs used in this study. DV-Chl a possesses two vinyl groups at the 3- and 8-positions, whereas ethyl groups occupy both the positions in mesoChl a. Comparison of demetalation properties among these two Chl a analogs as well as intact Chl a enables us to elucidate the substitution effects in the A- and B-rings of Chl a. pyroChl a lacks a methoxycarbonyl group at the 132-position, and is a good pigment to investigate the effect of the 132-methoxycarbonyl group in Chl a on demetalation. PChl a has the porphyrin-type macrocycle as the functional moiety and is an analog of the important intermediates, protochlorophyllide (PChlide) a, in the Chl a biosynthesis. Demetalation kinetics of PChl a, in which only the bond between C17 and C18 atoms differs from that in Chl a, allows us to understand how macrocyclic structures of magnesium cyclic tetrapyrroles affect their demetalation properties.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Apparatus

Visible absorption spectra were measured using a Shimadzu UV-2450 spectrophotometer (Shimadzu, Ltd., Kyoto, Japan), where the reaction temperature was regulated with a Shimadzu thermo-electric temperature-controlled cell holder TCC-240A. High-performance liquid chromatography (HPLC) was performed with a Shimadzu LC-20AT pump and an SPD-M20A or SPD-20AV detector.

Materials

DV-Chl a and Chl a were isolated from a Δslr1923 mutant of Synechocystis sp. PCC6803 and Spirulina geitleri, respectively [38, 39]. Three other chlorophyllous pigments, mesoChl a, pyroChl a and PChl a, were synthesized from Chl a according to previous reports [38-40]. The 3-vinyl group of Chl a was hydrogenated in the presence of palladium charcoal under hydrogen atmosphere to give mesoChl a [39]. Pyrolysis of Chl a in γ-collidine provided pyroChl a lacking the 132-methyoxycarbonyl group [40]. PChl a was obtained by oxidation of Chl a with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone [38, 39]. The four pigments were purified by reverse-phase HPLC just before measurements of demetalation kinetics.

Measurements of demetalation kinetics

A 3.0 mL acetone solution of purified chlorophyllous pigments (Soret absorbance = 1.0 at the 1 cm pathlength) was mixed with 1.0 mL of distilled water. A 10 μL of aqueous hydrochloric acid for volumetric analysis (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to the solutions, and absorbance at the Soret peak positions of chlorophyllous pigments was followed in time under the control of reaction temperatures at 25°C. The proton concentrations were estimated by assumption of complete dissociation of hydrochloric acid in a mixture of acetone and water (3/1, vol/vol), as was ensured in the cases of proton concentrations below 10−1 m in previous reports [26, 28].

Pigment analysis after demetalation

After demetalation reactions of DV-Chl a, mesoChl a and pyroChl a, the solutions were neutralized with aqueous 4% NaHCO3, and the pigments were extracted with CH2Cl2. The CH2Cl2 solutions were washed with aqueous saturated NaCl, and dried over anhydrous Na2SO4. The solutions were filtered and dried with nitrogen gas. In the case of demetalation of PChl a, the solutions after reactions were directly analyzed to avoid degradation of products through the extraction processes. The residues were analyzed on a reverse-phase HPLC column 5C18-AR-II (6 mmφ × 250 mm) with a guard column 5C18-AR-II (4.6 mmφ × 10 mm) with methanol/acetone (85/15, vol/vol) or a normal-phase column 5SL-II (6 mmφ × 250 mm) with hexane/2-propanol (99/1, vol/vol) at a flow rate of 1.0 mL min−1.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Spectral changes

Figure 2 depicts spectral changes in DV-Chl a, mesoChl a, pyroChl a and PChl a in aqueous acetone (acetone/water = 3/1, vol/vol) at 25°C. The 440-nm Soret absorption band of DV-Chl a gradually decreased and a new absorption band appeared at 419 nm under the present conditions, as shown in Fig. 2A. The 419-nm band was ascribed to the Soret band of the free-base of DV-Chl a, namely DV-Phe a. The Qy absorbance at 665 nm of DV-Chl a also decreased by incubation in the acidic aqueous acetone. The isosbestic points were present at 429, 466 and 561 nm in this spectral change.

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Figure 2. Spectral changes of DV-Chl a (A), mesoChl a (B), pyroChl a (C) and PChl a (D) in acetone/water (3/1, vol/vol). DV-Chl a: spectra from 0 to 150 min at 10-min intervals at the proton concentration of 2.5 × 10−4 m. mesoChl a: spectra from 0 to 150 min at 10-min intervals at the proton concentration of 1.2 × 10−4 m. pyroChl a: spectra from 0 to 150 min at 10-min intervals at the proton concentration of 1.2 × 10−4 m. PChl a: spectra from 0 to 450 min at 30-min intervals at the proton concentration of 2.5 × 10−4 m. The arrows show the direction of the absorbance changes.

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The other three Chl a analogs exhibited essentially the same spectral changes under the acidic conditions. The Soret and Qy absorption bands at 427 and 652 nm, respectively, of mesoChl a decreased, accompanying increase in the 406-nm Soret band of mesoPhe a (Fig. 2B). The isosbestic points could be observed at 415, 450 and 554 nm. In the case of pyroChl a, 433-nm Soret and 664-nm Qy absorbance became small with appearance of the 411-nm Soret band, which was derived from the production of pyroPhe a (Fig. 2C). This spectral change had the isosbestic points at 421, 457 and 557 nm. PChl a showed disappearance of the 435-nm Soret band and appearance of the 418-nm band, which was the Soret band of the pheophytinized PChl a, namely PPhe a (Fig. 2D). The small bands in the wavelength range between 500 and 700 nm gradually changed with several isosbestic points through demetalation of PChl a under the acidic conditions.

Demetalation kinetics

Demetalation reactions of four Chl a analogs were quantitatively analyzed by monitoring absorbance changes at their Soret peak positions. The apparent demetalation rate constant of demetalation is denoted as k′ in this article, and the k′ is followed by an equation, k′ = k[H+]n. Figure 3 depicts kinetic plots of the Soret absorbance of four Chl a analogs as well as Chl a in acetone/water (3/1, vol/vol) at the proton concentration of 1.2 × 10−3 m. Their demetalation reactions were regarded as pseudo-first-order reactions because of linear time-dependency of the logarithm of Soret absorbance in their reactions. This is consistent with the reaction conditions in which the proton concentration was much higher than the concentration of the Chl a analogs. Such pseudo-first-order demetalation kinetics was also observed at the lower proton concentrations of 2.5 × 10−4 and 1.2 × 10−4 m. Therefore, apparent reaction rate constants of the demetalation, k′s, could be estimated by fitting the time courses of the Soret absorbance of the Chl a analogs to the following kinetic equation:

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where A0, A and A are their Soret absorbance at the onset of the measurement, at time t, and at the complete demetalation, respectively. The k′-values of the four Chl a analogs are summarized in Fig. 4, and are compared with those of Chl a. The k′s in Fig. 4 are the averages of more than three independent measurements; their standard deviations were less than 6, 11, 12, 5 and 4% of the averages of the k′s for DV-Chl a, mesoChl a, pyroChl a, PChl a and Chl a, respectively. The logarithm of apparent demetalation rate constants, log k′, of all the Chl a analogs linearly increased with that of proton concentrations, log [H+]. The slopes of log k′ plots against log [H+] in Fig. 4 were determined to be 1.9, 2.1, 2.0, 2.2 and 1.9 for DV-Chl a, mesoChl a, pyroChl a, PChl a and Chl a, respectively. The relationships between log k′ and log [H+] allow us to estimate the number of protons that participate in the rate-limiting step of the demetalation reactions, as the k′s are followed by the equation, k′ = k[H+]n, where n denotes the proton number involved in the rate-limiting step [26-28]. The present results indicate the participation of two protons in the rate-limiting step of removal of the central magnesium from Chl a analogs under the acidic conditions. This is consistent with previous works, which mentioned that the proton number in the rate-limiting steps of demetalation of Chl a/a′, BChl a, Zn-Chl a and Zn-BChl a was ca 2 [26-28]. These suggest that the rate-limiting step of (B)Chl demetalation is the attack of two protons on two nitrogen atoms in the cyclic tetrapyrroles under acidic conditions.

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Figure 3. Kinetic plots for demetalation of DV-Chl a (open circle), mesoChl a (open square), pyroChl a (open triangle), PChl a (open diamond) and Chl a (closed square) in acetone/water (3/1, vol/vol) at the proton concentration of 1.2 × 10−3 m. Absorbance changes were monitored at 440, 427, 433, 435 and 431 nm for DV-Chl a, mesoChl a, pyroChl a, PChl a and Chl a respectively. A0, A and A are Soret absorbance of Chl a analogs at the onset of measurements, at time t, and at the complete demetalation, respectively.

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Figure 4. Demetalation rate constants, k′s, of DV-Chl a (open circle), mesoChl a (open square), pyroChl a (open triangle), PChl a (open diamond) and Chl a (closed square) in acetone/water (3/1, vol/vol) dependent on the examined proton concentrations [H+].

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Effects of 3- and 8-vinyl groups on demetalation

Comparison of apparent demetalation rate constants, k′s, among DV-Chl a, mesoChl a and Chl a enables us to unravel the effects of the 3- and 8-vinyl groups on demetalation properties of Chl a analogs. The k′-values of DV-Chl a were smaller than those of Chl a in the proton concentration range between 1.2 × 10−3 and 1.2 × 10−4 m. In contrast, the k′s of mesoChl a were larger than those of Chl a under the acidic conditions. These reveal that the substitution of the ethyl with vinyl group(s) in the A- and/or B-ring(s) of Chl a analogs provides the resistance to demetalation under the acidic conditions. The relative ratios of k′s of DV-Chl a to Chl a, k′(DV-Chl a)/k′(Chl a), were 0.39, 0.42 and 0.45 at the proton concentrations of 1.2 × 10−3, 2.5 × 10−4 and 1.2 × 10−4 m, respectively, and k′(Chl a)/k′(mesoChl a) were estimated to be 0.45, 0.53 and 0.63. The former values were slightly smaller than those of the latter. Such a slight difference might be ascribable to inherent differences of the vinyl groups conjugated to the A- and B-rings.

It has been reported that electron-withdrawing groups such as formyl and acetyl groups directly conjugated with the π-system of cyclic tetrapyrroles slow down removal of the central metal [31, 33, 34]. Such peripheral electron-withdrawing groups would decrease the electron densities of the core nitrogen atoms, preventing electrophilic attack of protons to pyrrole nitrogen atoms. As vinyl group is more electronegative than an ethyl group [41], the vinyl group(s) at the 3- and/or 8-position(s) of Chl a analogs also resulted in slower demetalation kinetics. These effects are in line with the previous report, in which zinc 3-vinyl-Chl derivative exhibited slower removal of the central zinc than the corresponding zinc 3-ethyl-chlorin [34].

The substitution effect of weakly electron-withdrawing 8-vinyl group on demetalation properties of Chls is much smaller than the strongly electron-withdrawing 7-formyl effects based on the comparison of the present k′-value of DV-Chl a with the reported one of Chl b under the identical acidic conditions [31]. Such a difference between the 8-vinyl and 7-formyl groups would allow DV-Phe a to function as the primary electron acceptor in PS II by in vivo demetalation of DV-Chl a in oxygenic photosynthetic organisms. Further studies will be required to unravel the formation and selection mechanisms of the primary electron accepting Phe molecules in PS II.

Effects of 132-methoxycarbonyl group on demetalation

The apparent demetalation rate constants, k′s, of pyroChl a were compared with those of Chl a to examine the effect of the 132-methoxycarbonyl group on demetalation of Chl a. The k′-values of pyroChl a were about twice larger than those of Chl a in the proton concentration range between 1.2 × 10−3 and 1.2 × 10−4 m, indicating that the 132-methoxycarbonyl group in Chl a was one of the structural factors to slow down demetalation kinetics. This study first revealed the effect of the 132-methoxycarbonyl group in Chl a on demetalation properties without other structural effects, although the effect of the stereochemistry at the 132-position in Chl a was already reported by comparing Chl a with its epimer Chl a[26, 27]. Mazaki and Watanabe reported 1.4-times faster demetalation kinetics of Chl a′ relative to Chl a in acidic aqueous acetone, and explained the difference by invoking larger steric repulsion between the 132-methoxycarbonyl group and the 17-propionate residue possessing the phytyl ester in Chl a[26, 27]. In contrast, faster demetalation kinetics of pyroChl a would mainly originate from electronic effects in the isocyclic E-ring rather than steric effects in the case of Chl a′. The E-ring of Chl a possesses a β-ketoester system, whereas that of pyroChl a is a cyclopentanone. This study suggests that the difference in the E-ring would influence electronic states in the chlorin π-macrocycle, especially pyrrole nitrogen atoms, resulting in the difference of demetalation properties between Chl a and pyroChl a. Homoconjugation of the electron-withdrawing 132-methoxycarbonyl group with the 13-keto group (C131–C132–COOCH3) and chlorin π-system (C15–C132–COOCH3) in Chl a would decrease the k′-values as expected (vide supra). It is noted that the cross-talk effects between the 132-methoxycarbonyl group and the C17–C18 double bond might be taken into consideration to understand structure-dependent demetalation properties more precisely.

Effects of macrocycles on demetalation

The apparent demetalation rate constants, k′s, of PChl a, whose macrocyclic structure was the porphyrin skeleton, were four to six times slower than those of Chl a under the acidic conditions. The slow demetalation kinetics of PChl a relative to Chl a can be ascribed to the porphyrin-type tetrapyrroles, as PChl a possessed the same structure as Chl a except the bond between C17 and C18 atoms. The effect of the porphyrin skeleton on demetalation kinetics was in line with previous reports [32, 35], in which zinc complexes of methyl protopyropheophorbides (porphyrin-type complexes) also exhibited slower demetalation kinetics than the corresponding zinc methyl pyropheophorbides (chlorin-type complexes). These indicate that the porphyrin macrocycle is responsible for resistance to demetalation of PChl-type cyclic tetrapyrroles due to the electronic states and/or flexibility of π-macrocycles.

PChl a is an esterified pigment of PChlide a, which is an important biosynthetic intermediate and sometimes a photosynthetically active pigment in photosynthetic organisms. Demetalation properties of PChl a, therefore, are useful for understanding the in vivo states of PChlide a by invoking the effect of an esterifying phytyl chain. Comparison of demetalation kinetics among Chl a, Chlide a, Chlide a methyl ester and Chlide a ethyl ester suggested that the effects of esterifying chains on demetalation properties of chlorophyllous pigments were slight [23]. This is in line with no conjugation of an esterifying chain with the chlorin π-macrocycle. Thus, the present results suggest that PChlide a has more tolerance to demetalation than Chl a and Chlide a.

HPLC analysis of reaction products

Reaction products of four Chl a analogs under the same acidic conditions as shown in Fig. 3 were analyzed by reverse-phase or normal-phase HPLC as shown in Fig. 5. The products of DV-Chl a after reaction under this condition for 100 min exhibited the main fraction at 79 min (Fig. 5A). This fraction had the Soret and Qy bands at 417 and 666 nm, respectively, in the HPLC eluent, and was ascribed to DV-Phe a. In addition, slight fractions due to by-products during the demetalation reaction were detected at 5 and 13 min in this chromatogram. These by-products could not be assigned because of their small amounts. By incubating mesoChl a in the acidic aqueous acetone for 20 min, the main fraction was eluted at 16 min, which exhibited the Soret and Qy bands at 405 and 658 nm, and few fractions except a slight fraction at 13 min were observed (Fig. 5B). The main product at 16 min was ascribable to mesoPhe a, and the slight fraction at 13 min would be ascribable to the 132-stereoisomer of mesoPhe a, mesoPhe a′. As the product of pyroChl a after 30-min demetalation reaction, sole pyroPhe a was detected at 12 min, and no by-product was formed during the reaction (Fig. 5C). In the case of demetalation reaction of PChl a for 90 min, the demetalation product of PChl a, namely PPhe a, which was eluted at 61 min, was predominantly formed (Fig. 5D). A slight fraction at 5 min was produced by side reactions during the reaction process. These HPLC analyses revealed predominant conversion of Chl a analogs to the corresponding demetalated pigments (Phes) with few side reactions under the present conditions.

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Figure 5. HPLC elution patterns of demetalation products of DV-Chl a (A), mesoChl a (B), pyroChl a (C) and PChl a (D) under the conditions in Fig. 3 for 100, 20, 30 and 90 min, respectively. Reaction products of DV-Chl a (A) and PChl a (D) were eluted on a reverse-phase column 5C18-AR-II (6 mmφ × 250 mm) with a guard column 5C18-AR-II (4.6 mmφ × 10 mm) with methanol/acetone (85/15, vol/vol) at a flow rate of 1.0 mL min−1. Reaction products of mesoChl a (B) and pyroChl a (C) were eluted on a normal-phase column 5SL-II (6 mmφ × 250 mm) with hexane/2-propanol (99/1, vol/vol) at a flow rate of 1.0 mL min−1. Chromatograms (A–D) were recorded at 417, 405, 411 and 418 nm, respectively. The signal denoted by × in the chromatogram (D) was due to direct injection of the reaction mixture.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

This study demonstrated structure-dependence on demetalation properties of Chl a analogs under weakly acidic conditions. The vinyl group directly conjugated with the chlorin π-system in the A- and/or B-ring(s) of Chl a analogs was responsible for slower demetalation kinetics than the ethyl group. The substitution effect was derived from the larger electron-withdrawing ability of a vinyl group than that of an ethyl group. Removal of the methoxycarbonyl group at the 132-position in Chl a resulted in faster demetalation kinetics, which was also ascribed to its electron-withdrawing moiety through homconjugation. The porphyrin-type macrocycle of PChl a provided the resistance to demetalation. These physicochemical properties on demetalation of Chl a analogs will be a clue to solving an enigma concerning in vivo Chl demetalation such as production of the primary electron acceptors in PS II reaction centers and the early processes in the Chl a degradation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References

We thank Dr. Yuichiro Kashiyama, Dr. Taichi Yoshitomi, Ms. Yuki Kimura, Mr. Daisuke Takekoshi, and Ms. Haruna Yokota of Ritsumeikan University for their assistance in the preparation of chlorophyllous pigments. This work was partially supported by Grants-in-Aid for Scientific Research (C) (no. 23550201) (to YS) and (A) (no. 22245030) (to HT) from the Japan Society for the Promotion of Science.

References

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
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