Analytica Chimica Acta 365 (1998) 199±203
Light-independent isomerization of bacteriochlorophyll g to
chlorophyll a catalyzed by weak acid in vitro
Masami Kobayashia,*, Takehiro Hamanoa, Machiko Akiyamaa, Tadashi Watanabeb,
Kazuhito Inouec, Hirozo Oh-okad, Jan Amesze, Mayu Yamamuraa, Hideo Kisea
a
Institute of Materials Science, University of Tsukuba, Tsukuba 305-8573, Japan
b
Institute of Industrial Science, University of Tokyo, Tokyo 106-8558, Japan
c
Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka 259-1293, Japan
d
Department of Biology, Graduate School of Science, Osaka University, Toyonaka 565-0871, Japan
e
Department of Biophysics, Huygens Laboratory, University of Leiden, Leiden 2300 RA, The Netherlands
Received 17 August 1997; received in revised form 12 October 1997; accepted 15 December 1997
Abstract
Rapid conversion of bacteriochlorophyll g (BChl g) to chlorophyll a (Chl a) was observed in acetone on addition of acid in the
dark. The product, Chl a esteri®ed with farnesol (Chl aF), was identi®ed by liquid chromatography and fast atom
bombardment mass spectrometry. Acid-catalyzed formation of 81-OH-Chl aF, a primary electron acceptor in the heliobacterial
reaction center, was also observed in diethyl ether in the dark. These results suggest that acid-catalyzed isomerization is a
candidate for the chemical evolution of BChl g to the more stable Chl a and that 81-OH-Chl aF can easily be synthesized from
BChl g under weakly acidic conditions in the dark. # 1998 Elsevier Science B.V.
Keywords: Acid-catalyzed reaction; Bacteriochlorophyll g; Chemical evolution; Chlorophyll a; Heliobacteria; Isomerization; Photoisomerization; Photosynthesis
Abbreviations: BChl, bacteriochlorophyll; BPheo, bacteriopheophytin; Chl, chlorophyll; FAB±mass, fast atom bombardment mass; LC,
liquid chromatography; Pheo, pheophytin; PS I, photosystem I; P700, primary electron donor of PS I; P798, primary electron donor of
heliobacteria
1. Introduction
In 1981, Gest and Favinger [1] discovered a photosynthetic bacterium which was placed in a new genus,
Heliobacterium. The heliobacteria have a new type of
bacteriochlorophyll, BChl g (Fig. 1), as the major
pigment [2]. The structure of BChl g resembles that
of BChl b, containing an unusual ethylidene group at
*Corresponding author. E-mail: [email protected]
0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.
PII S0003-2670(98)00088-9
ring II [2], although the esterifying alcohol of BChl g
is not phytol but farnesol, C15H25OH [3].
The heliobacterial reaction center (RC) is very
similar to that of photosystem I (PS I) in higher plants
[4]. The primary electron donor of heliobacteria,
P798, has been proposed to be a dimer of BChl g0 ,
the 132-epimer of BChl g [4±6], while the corresponding epimer of chlorophyll (Chl) a, Chl a0 , has been
implied to constitute P700, the primary electron donor
of PS I [6,7]. The primary electron acceptor, A0, of
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M. Kobayashi et al. / Analytica Chimica Acta 365 (1998) 199±203
Fig. 1. Molecular structure of BChl g and Chl aF, where F stands for farnesyl, according to the IUPAC numbering system. BChl g undergoes
isomerization to yield Chl aF. Replacement of the central Mg2‡ ion with two protons gives BPheo g and Pheo aF, respectively.
heliobacteria was determined to be 81-OH-Chl aF,
where F denotes farnesyl [4,6,8], whereas A0 in PS
I is most probably Chl a. These two RCs do not
possess metal-free chlorophylls, pheophytins, that
function as electron acceptors in RCs of purple bacteria [9] and PS II [6,10,11]. It should be noted that
BChl g and Chl aF, as well as their epimers, are
isomeric to each other (see Fig. 1). In fact, lightinduced isomerization of BChl g to Chl aF and BPheo
g to Pheo aF has already been reported by BeerRomero et al. [12] and Michalski et al. [3], respectively, although the mechanism has not yet been
clari®ed.
In this paper, we report the light-independent
isomerization of BChl g to Chl aF in acetone and
the conversion of BChl g to 81-OH-Chl aF in
diethyl ether, both being catalyzed by weak acid in
the dark.
2. Experimental
2.1. Pigment preparation
Pigments were extracted with acetone from H.
chlorum, Heliobacillus mobilis and H. modesticaldum. Since the experimental results were found to
be essentially species-independent, the results for
H. chlorum alone are presented here. Suf®ciently pure
BChl g was prepared by means of preparative-scale
liquid chromatography (LC) (Senshupak 5251-N,
25020 mm i.d.) with hexane/2-propanol/methanol
(100/2/0.3 by vol.) as an eluent at a ¯ow rate of
7 ml minÿ1 at 277 K, as described elsewhere [5].
Pigment analyses were carried out using Senshupak
1251-N (2504.6 mm i.d.), cooled to 277 K, with
degassed hexane/2-propanol/methanol (100/1.5/0.2
by vol.) at a ¯ow rate of 1.3 ml minÿ1. Pigment elution
was monitored by means of a Jasco UV-970 detector
and a Jasco multiwavelength MD-915 detector in
series.
2.2. Isomerization and pheophytinization
All the procedures were conducted in darkness. To
initiate isomerization or pheophytinization, 5 ml of
water containing HCl (0.005±2 M) or acetic acid
(0.005±2 M) was added to 3 ml of a solution of BChl
g at a concentration of ca. 310ÿ6 M. The solvents
used here were acetone, diethyl ether, toluene, ethanol
and aqueous acetone (acetone/H2Oˆ2/1, v/v). The
progress of the reaction was monitored spectrophotometrically by means of a Jasco Ubest-50 at 293 K.
After washing with toluene/water until the solution
exhibited neutral pH, the toluene solution was evaporated under vacuum, and the residue was analyzed by
M. Kobayashi et al. / Analytica Chimica Acta 365 (1998) 199±203
LC and fast atom bombardment mass spectrometry
(FAB±mass).
2.3. FAB±mass
FAB±mass of BChl g and acid-treated BChl g was
performed on a JEOL JMS-SX-102A in a m-nitrobenzyl alcohol matrix at a resolution of 3000. The
acceleration potential was 10 keV.
3. Results and discussion
3.1. Absorption spectral changes in acetone and
aqueous acetone
Fig. 2(A) shows the absorption spectral changes of
BChl g in acetone in the dark after addition of HCl to a
®nal concentration of ÿlog[HCl]ˆ4.4. The spectral
change of BChl g to one resembling that of Chl a with
good isosbestic points indicates that the reaction
Fig. 2. Temporal evolution of absorption spectra for BChl g (A) by
isomerization in acetone at ÿlog [HCl]ˆ4.4 and (B) by demetalation in aqueous acetone (acetone/H2Oˆ2/1, v/v) at ÿlog[HCl]ˆ
3.0. Spectra were traced in a 3 min cycle. Both reactions were
carried out at 293 K in the dark.
201
essentially yielded a single product. The absorption
spectrum suggests that the product possesses a chlorin
macrocycle with a double bond between C7 and 8 (see
Fig. 1). Essentially, no spectral change took place
when an acetone solution of BChl g was allowed to
stand for a few hours without addition of HCl in the
dark. It should be noted that no demetalation of BChl g
proceeded despite the presence of acid. In sharp
contrast, demetalation of BChl g to BPheo g with a
half-time of 4 min occurred in aqueous acetone (acetone/waterˆ2/1, v/v) on addition of HCl to a ®nal
concentration of ÿlog[HCl]ˆ3.0, again exhibiting
isosbestic points (Fig. 2(B)).
These results indicate that the isomerization is
much faster than demetalation in acetone, while the
opposite holds in aqueous acetone. Thus, the isomerization and demetalation behaviors of BChl g are
strongly solvent-dependent. The half-time of the isomerization of BChl g is 13 min in acetone at
ÿlog[HCl]ˆ4.4 (Fig. 2(A)). In contrast, the half-time
of the demetalation of BChl g in acetone is estimated
to be ca. 400 days at the same ÿlog[HCl] value, if it is
assumed that the demetalation rate of BChl g is the
same as that of BChl a in acetone where only demetalation takes place because of the lack of an ethylidene group. This assumption is, in part, supported by
an observation that the demetalation rates of BChl a
and BChl g are almost the same in aqueous acetone
[13]. The large difference between the rates of isomerization and demetalation in acetone is one of the
reasons why only isomerization was observed in
Fig. 2(A). For the moment, a similar comparison is
hampered in aqueous acetone, since an estimation of
the isomerization half-time of BChl g in aqueous
acetone has not yet been successful.
The acid-catalyzed isomerization is observed even
at ÿlog[HCl]ˆ5.0 with a half-time of 2 h, where
demetalation of BChl g hardly occurs (t1/220 years,
estimated in the manner mentioned above) [13]. The
acid-catalyzed isomerization of BChl g is faster than
that of BChl b (t1/2ˆ6 h at ÿlog[HCl]ˆ5.0) [14].
When ÿlog[HCl] was lowered to 4.0, rapid isomerization and subsequent demetalation to yield Pheo
aF were observed, indicating that the demetalation of
Chl aF is much faster than that of BChl g in acetone.
This is in line with an observation that the demetalation half-time of Chl a at ÿlog[HCl]ˆ4.0 in acetone
(16 min) is short enough to be observed, although that
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M. Kobayashi et al. / Analytica Chimica Acta 365 (1998) 199±203
of BChl g (as estimated from the behavior of BChl a)
is too long (ca. 70 days) [13]. The reason why the
demetalation of Chl aF cannot be observed in
Fig. 2(A) is due to the signi®cantly slower demetalation (t1/290 min) than isomerization (t1/2ˆ13 min).
Isomerization of BChl g was also observed in
acetone on addition of acetic acid. However, the
reaction was very slow, most probably due to the
lesser production of H‡ from acetic acid than
from HCl. So the results for HCl alone are presented
below.
3.2. Analysis by LC
Fig. 3(B) shows the liquid chromatogram of acidtreated BChl g in acetone in the dark. Essentially a
single component is seen at a retention time shorter
than that of BChl g. The retention time of this component is only marginally but de®nitely longer than
that of Chl aP in higher plants, where P denotes phytyl.
The longer retention time of the component is due to
the lower hydrophobicity of farnesyl than that of
phytyl. Note that more polar pigments, being usually
Fig. 3. LC traces for (A) BChl g and (B) acid treated of BChl g in
acetone at ÿlog[HCl]ˆ4.4 for 30 min at 293 K in the dark.
Detection wavelength is 415 nm.
produced by photoisomerization in the presence of air
[15±17] and having longer retention times than BChl
g, are detected in only small amounts. This demonstrates that acid treatment of BChl g in acetone in the
dark promotes a simple isomerization of BChl g to Chl
aF without signi®cant side reactions.
3.3. FAB±mass
The
FAB±mass
spectrum
of
BChl
g
(C50H58N4O5Mg, molecular massˆ819.34) shows a
dominant peak at m/z 818 due to the fragment (M±
H)‡, together with intense peaks at m/z 613 and 614
assignable to (M±C15H25±H)‡ and (M±C15H25)‡ due
to the loss of farnesyl (C15H25) at C17 (Fig. 4(A)). The
product formed in Fig. 2(A) also showed the same
mass peaks at m/z 818 and 614 (Fig. 4(B)). This
con®rmed our conclusion that the product formed in
Fig. 2(A) is Chl aF, resulting from a novel lightindependent isomerization of BChl g catalyzed by
weak acid.
Fig. 4. FAB-mass spectra of (A) BChl g and (B) a purified main
peak product in Fig. 3(B).
M. Kobayashi et al. / Analytica Chimica Acta 365 (1998) 199±203
3.4. Oxidative isomerization
In sharp contrast to the results mentioned above, the
use of diethyl ether as solvent for the reaction led to
the formation of a large amount of oxidation products
of BChl g even in the dark, showing mass fragments at
m/z 834 and 850 (data not shown). These larger masses
indicate addition of one or two oxygen atoms to Chl
aF. The ef®ciency of this reaction depended heavily on
the nature of the solvent, the order being: diethyl
ether>tolueneethanol>acetone. The small amounts
of oxidation products with longer retention times seen
in Fig. 3(B) may have been produced during washing
with toluene/water; the amount of oxidation products
was higher when diethyl ether was used for washing.
The candidates for the singly and doubly oxygenated
products are 81-OH-Chl aF and 81-OOH-Chl aF [17],
respectively, although other oxidation forms may also
be possible. The molecular structural determination of
the products is now under way, and the mechanisms
for the acid-catalyzed isomerization will be discussed
elsewhere [14].
3.5. Implication for the chemical evolution of
pigments
The facile isomerization from BChl g to Chl aF, in
combination with the well-known much higher stability of Chl a as compared to BChl g, may suggest that
this reaction may represent a step in the evolution of
photosynthetic organisms. It is easily seen that at one
stage when the environment became slightly acidic to
an extent where heliobacteria can still continue to
grow, they tried to adapt themselves to such an environmental change by converting the major pigment into
Chl a.
We should note that heliobacteria grown in darkness
synthesize BChl g [18] and are photoactive [19],
indicating that the biosynthesis of 81-OH-Chl aF is
also most likely taking place in the dark in vivo.
As yet, these remain mere hypotheses and need to
be substantiated by further work.
4. Conclusions
The isomerization of BChl g to Chl aF is effectively
promoted by weak acid in acetone under air in dark-
203
ness. This route may be one of the possible candidates
for the chemical evolution of BChl g to Chl a in the
history of life. The oxidative conversion of BChl g into
81-Oh-Chl aF catalyzed by acid in diethyl ether is also
of interest in connection with its biosynthesis in
heliobacteria in the dark.
Acknowledgements
We thank Dr. M. Ogawa (Fuji Photo Film Co., Ltd.)
for the FAB±Mass measurements. This work was
supported in part by a grant-in-aid from the Ministry
of Education, Science, Sport and Culture of Japan, and
by the Kurata Foundation.
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