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Chapter 3
Physicochemical Properties of Chlorophylls in Oxygenic

Photosynthesis — Succession of Co-Factors from
Anoxygenic to Oxygenic Photosynthesis
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
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/55460
. Introduction
Chlorophylls are essential components in oxyμenic photosynthesis, and chlorophyll Chl a

Fiμ. is the major chlorophyll in cyanobacteria, alμae and terrestrial plants. However,
primary charμe separation is initiated by a λew specially-tailored chlorophylls in the reaction
centers RCs . Excitation oλ the primary donor reduces the primary and secondary electron
acceptors, which aμain are oλten specially-tailored chlorophylls e.g., the -epimer oλ Chl a,
Chl a' Fiμ. , constitutes the primary electron donor oλ P in PS I as a heterodimer oλ Chl
a/a' Kobayashi et al. Jordan et al. , and a metal-λree Chl a, pheophytin Phe a Fiμ.
, λunctions as the primary electron acceptor in PS II Klimov et al. a,b Zouni et al.
Fiμ. .
In , a unique cyanobacterium, Acaryochloris marina, with Chl d as the dominant chlorophyll

was discovered in colonial ascidians Miyashita et al. . Thouμh P in PS I oλ A. marina
is composed oλ Chl d', pheophytin in PS II is not d-type but a-type “kiyama et al. . One
oλ the diλλiculties in λindinμ Chl d in nature was its overlap with Chl b on a reversed-phase
HPLC trace. To make matters worse, Chl d had been thouμht to have a lower oxidation
potential than Chl a even thouμh no experimental evidence was available, mainly because a
midpoint potential, Em, λor P in A. marina was shown to be + mV Hu et al. ,
© 2013 Kobayashi et al.; licensee InTech. This is an open access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
48 Photosynthesis
marvelously more neμative than that λor P ca. + mV in the Chl a-type cyanobacteria.
The λact that the QY-band oλ Chl d is at the lonμest wavelenμth compared with Chls a and b
seems to have led to some misapprehensions concerninμ the oxidation potential oλ Chl d one
estimated that Chl d had the lowest oxidation potential amonμ Chls a, b and d. In , however,
the Eox value oλ Chl d in vitro was λirst determined and λound to be hiμher than that oλ Chl a
Kobayashi et al. , and hence the Em oλ P was re-examined the value was ca. + mV
+ mV ”enjamin et al. , Telλer et al. , and + mV Tomo et al. , beinμ λar
positive oλ the initial report + mV and almost equal to the Chl a-type P values, around
+ mV ”rettel , Krabben et al. , Ke , Itoh et al. , Nakamura et al. see
Fiμ. in Ohashi et al. .
y x acid base
R2 R3 Phe a Chl a Chl a’
3 5 CH 3
R1 2
4 6
7
Phe b Chl b Chl b’
CH 2
Ϩ ϩ 8 Phe d Chl d Chl d’
1 N N 9
Phe f Chl f Chl f’
20 M 10
M R1 R2 R3 R4 R5
19
11 Chl a Mg CH3 CH=CH2 CH3 H COOCH3
N N Chl a' Mg CH3 CH=CH2 CH3 COOCH3 H
18
H ϫ Ϫ 12 Phe a 2H CH3 CH=CH2 CH3 H COOCH3
H3C 17 15 CH 3 Chl b Mg CH3 CH=CH2 CHO H COOCH3

16 14
17 1 13 Chl b' Mg CH3 CH=CH2 CHO COOCH3 H
Phe b 2H CH3 CH=CH2 CHO H COOCH3
H2 C
H 13 2 Ϭ 13 1 Chl d Mg CH3 CHO CH3 H COOCH3
Chl d' Mg CH 3 CHO CH3 COOCH3 H
2
17 CH R4 O Phe d 2H CH3 CHO CH3 H COOCH3
2 R5 Chl f Mg CHO CH=CH2 CH3 H COOCH3
C Chl f ' Mg CHO CH=CH2 CH3 COOCH 3 H
O O C 20H 39 Phe f 2H CHO CH=CH2 CH3 H COOCH 3
Figure 1. Molecular structure and carbon numbering of chlorophylls, according to the IUPAC numbering system. Nat‐
urally occurring chlorophylls are designated by squares.
In , a red-shiλted chlorophyll was discovered in a methanolic extract oλ Shark ”ay

stromatolites, and was named Chl f Chen et al. a Chl f-containinμ λilamentous cyano‐
4
bacterium was puriλied and named as Halomicronema hongdechloris Chen et al.4 . In ,
hν
a Chl f-like piμment was discovered in a unicellular cyanobacterium, strainhν
- H-
KC , isolated λrom
Δ
hν
Lake ”iwa Ohkubohν et al. , but there were also diλλiculties in its identiλication because
he
there were not systematic physicochemical data as toFe- S a, b, d and
Chls Fe-fSacquired under Fe-the
S same
conditions. For example, the optical peak wavelenμths oλ the red-shiλted chlorophyll puriλied
hν H S
λrom the strain KC Succ

in methanol are almost the same as the reported
yr values oλ Chl f =
nm and nm , whileytthe
c reported ratio oλ Soret/QY-bands exhibited a larμe4 diλλerence the
yt c yt c
piμment puriλied λrom KC showed . , but the reported ratio λor Chl f was surprisinμly hiμh,
. . What is worse, the H-NMR chemical shiλts4 oλ λormyl-H oλ Chl b and f in acetone d at
K beinμ presented in thisHchapter are . and . , respectively, but the correspondinμ
ϫ Ϫ
Physicochemical Properties of Chlorophylls in Oxygenic Photosynthesis… 49

http://dx.doi.org/10.5772/55460
PS II – type RC PS I – type RC
4
4
hν
-1.0 Chl a - H- Chl a F
hν
Chl aΔ2,6P
hν
Em / V vs. SHE
hν he Phe a
Fe- S Fe- S Fe- S
hν H S
Succ yr
0
yt c yt c
yt c 4
(BChl g’)2 (BChl a’)2
BChl 4
Chl a’/a
Chl d’/d
+1.0 H
Chl a 2
Chl a/d
Purple Bacteria Higher Plants, Algae Heliobacteria Green Sulfur Bacteria
Filamentous Green Cyanobacteria
Sulfur Bacteria
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 elec‐
tron 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 BChl b and Zn-BChl a containing purple bacteria, respectively; P865 is the
primary electron donor of green filamentous bacteria. Figure adapted from Akutsu et al. (2011).
values reported were . λor Chl b in CDCl at unknown temperature and . λor Chl f in
CD Cl /d -pyridine / , v/v at K, respectively, where our data are directly counter to the
reported pair. These λacts exhibit that chemical shiλt is hiμhly sensitive to both solvents and
temperature.
In this chapter, we present systematic and essential physicochemical properties oλ chlorophylls
in vitro obtained under as common conditions as possible, introducinμ the detailed experi‐
mental procedures HPLC, absorption, circular dichroism, mass, NMR and redox potential.
We also introduce the succession oλ co-λactors in PS I RCs λrom the viewpoints oλ minor but
key chlorophylls Fiμ. . In the λuture, our basic data will help researchers to identiλy the
molecular structures oλ newly discovered chlorophylls and determine and/or predict their
characteristics.
. Chlorophylls in oxygenic photosynthesis
. . Popular chlorophylls: Chlorophylls a and b
In , the term chlorophyll Chl , the μreen Greek chloros oλ leaλ Greek phyllon , was
introduced λor the piμments extracted λrom leaves with orμanic solvents Pelletier and
Caventou . In , a Russian botanist Tsvet Tswett in Russian meaninμ "colour"
separated leaλ piμments by chromatoμraphy λrom Greek chroma and graphein meaninμ "color"
50 Photosynthesis
アンテナ
e- Fe-S BChl g
e-
OH-Chl aF OH-Chl aF
e-
(BChl g’) 2
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 ar‐
rows.
and "to write", respectively into the blue Chl a, the μreen Chl b and several yellow to oranμe
carotenoids.
. . Recently discovered chlorophylls in algae
. . . Chlorophyll d in a cyanobacterium, Acaryochloris marina
In , Chl d Fiμ. was λirst reported as a minor piμment in several red macroalμae Manninμ
and Strain . In , a unique cyanobacterium, Acaryochloris marina, was isolated λrom
colonial ascidians containinμ Chl d as the dominant chlorophyll Miyashita et al. see
Introduction in a review by Ohashi et al. λor the short history oλ Chl d discovery .
. . . Chlorophyll f in a cyanobacterium, strain KC , isolated from Lake Biwa
“ Chl f-producinμ cyanobacterium, strain KC , was discovered by Miyashita, one oλ the

authors oλ this chapter. The discovery story was a kind oλ λortunate accident, similar to
that oλ Chl d-producinμ cyanobacterium, A. marina Miyashita et al. , Ohashi et al.
. Strain KC was a by-product durinμ the huntinμ oλ chlorophyll d-producinμ
http://dx.doi.org/10.5772/55460
cyanobacteria in λreshwater environment. Chl d had been only produced by cyanobacte‐

ria in the μenus Acaryochloris, and the strains in Acaryochloris had been isolated only λrom
saline environments such as the marine or salty lake but not λrom λreshwater environ‐
ments at all Murakami et al. , Miller et al. , Mohr et al. , ”ehrendt et al. .
Moreover, the strain A. marina M”IC dose not μrow in λreshwater media and requires
sodium chloride λor its μrowth at more than . % w/v in the medium Miyashita et al.
. Chlorophyll d was, however, detected in the sediment at the bottom oλ Lake ”iwa,
the larμest λreshwater lake in Japan Kashiyama et al. , brinμinμ us the idea that Chl
d-containinμ alμae exist in this lake. We collected alμal mats and lake water λrom a shore
zone oλ Lake ”iwa at th, “pr. . The samples were suspended in several media λor
λreshwater alμae, diluted and dispensed into cell culture plate or on aμar plates. Those
culture/aμar plates were kept in an incubator with near inλrared NIR liμht as the sole
liμht source, because we expected that a Chl d-containinμ alμa should μrow λaster than
other alμae under such liμht condition. Colony oλ Acaryochloris cells μrown under NIR liμht
looked yellow-μreen rather than blue-μreen oλ common cyanobacteria, and we also saw
several yellow-μreen colonies Oct. . Our attempt to isolate a Chl d-containinμ
λreshwater Acaryochloris sp. λrom Lake ”iwa turned out to be a success Feb. details
will be reported elsewhere . Miyashita checked the culture/aμar plates, which were leλt λor
a lonμ time in the incubator under NIR liμht, and some unusual cyanobacterial colonies
were λound Oct. . The colonies were diλλerent λrom that oλ Acaryochloris sp. in color
beinμ dark-blue-μreen rather than yellow-μreen. The cells μrew under NIR liμht as the sole
liμht source when we put them in a λreshwater medium. Morpholoμical λeatures oλ the
cells were similar to those oλ Acaryochloris sp. in that the cell was unicellular, spherical to
subspherical and aμμreμated. We expected that the orμanism was a new Chl d-contain‐
inμ cyanobacterium which was closely related to the μenus Acaryochloris, phyloμenetical‐
ly. The results oλ HPLC analysis disappointed us, since the cyanobacterium possessed no
Chl d at all, and Chl a as the major chlorophyll like common cyanobacteria. Immediately
thereaλter, however, came an excitement when an unusual chlorophyll was detected nd.
Jan. . The piμment showed typical two absorption peaks in the Soret nm and
QY nm reμions in MeOH they were clearly diλλerent λrom those oλ known chloro‐
phylls. We concluded that the piμment was a new chlorophyll that should be named Chl
f . We started mass culture oλ the cells λor chemical characterization such as detailed
spectral properties, molecular mass and chemical structure. “t around the same time, Chen
et al. reported the discovery oλ Chl f in a methanolic extract oλ Shark ”ay stromato‐
lites incubated under NIR liμht λor the initial purpose oλ the isolation oλ new Chl d-
containinμ phototrophs. Discoveries are mostly accidental.
Note that in the strain KC Chl f was not induced under white λluorescent liμht even iλ NIR
LED was also used as additional liμht. Further, neither Chl f' nor Phe f was detected, suμμestinμ
that Chl a' and Phe a λunction as P and the primary electron acceptor in PS II, respectively,
as in common cyanobacteria “kutsu et al. . The results indicate that Chl f may λunction
as not an electron transλer component but an antenna part. Chl f is not the major photopiμment,
and may λunction as an accessory chlorophyll, althouμh the λunction oλ Chl f in enerμy storaμe
52 Photosynthesis
is under debate, because uphill enerμy transλer is needed to deliver the excitation enerμy to
Chl a molecules in the RC Chen and ”lankenship .
. . Specially-tailored chlorophylls associated with reaction centers
. . . Prime-type chlorophylls as the primary electron donors in PS I
. . . . Chlorophyll a' and P7
The -epimer oλ Chl a, Chl a' "a-prime" Fiμ. , was λirst reported in Strain and
Manninμ . In , it was proposed that Chl a' constitutes P as a heterodimer oλ Chl
a/a' Fiμ. Kobayashi et al. , and the idea has been conλirmed in Jordan et al.
. “s seen in Fiμ. , it has also been shown that P consists oλ ”Chl g' in the RC oλ
heliobacteria in Kobayashi et al. , and that P consists oλ ”Chl a' in μreen sulλur
bacteria in Kobayashi et al. , , suμμestinμ that prime-type chlorophylls are
essential as the primary electron donors in the PS I-type RCs see Fiμs. and . For more
details, see Chapter in Kobayashi et al. .
. . . . Chlorophyll d' and P7 in Acaryochloris marina
Chl d', the -epimer oλ Chl d Fiμ. , was always detected in A. marina as a minor component,
while Chl a' was absent see Fiμ. C “kiyama et al. . P , the primary electron donor
oλ PS I in A. marina, was initially proposed to be a homodimer oλ Chl d Hu et al. , then a
homodimer oλ Chl d' “kiyama et al. , and λinally a Chl d/d' heterodimer Fiμ. “kiyama
et al. , Kobayashi et al. , Ohashi et al. , just like the Chl a/a' λor P
in other cyanobacteria and hiμher plants Fiμs. and a dimer model λor P was supported
by FTIR spectroscopy Sivakumar et al. . The λindinμ oλ Chl d’ in A. marina appears to
ensure our hypothesis that prime-type chlorophyll is a μeneral λeature oλ the primary electron
donor in the PS I-type RCs see Fiμs. and . The homoloμy oλ Psa“ and Psa” between A.
marina and other cyanobacteria is low Swinμley et al. , which may reλlect the replacement
oλ Chl a by Chl d, also Chl a' by Chl d', in the PS I RC oλ A. marina see Fiμ. .
It is interestinμ to note that the primary electron acceptor, “ , in PS I oλ A. marina is not Chl d
but Chl a Fiμs. and , which was λirst shown by laser photolysis experiment Kumazaki et
al. , and then supported by λlash-induced spectral analysis Itoh et al. . The results
support our hypothesis that Chl a-derivative is a μeneral λeature oλ “ in the PS I-type RCs see
Fiμs. and .
. . . . Evolutionary relationship between chlorophylls and PS-I type reaction centers
Here we introduce our hypothesis about the evolution oλ the PS I-type RCs based on the
structures oλ chlorophylls and quinones Fiμ. . The prime-type chlorophylls, bacteriochlor‐
ophyll ”Chl a' in μreen sulλur bacteria, ”Chl g' in heliobacteria, Chl a' in Chl a-type PS
I, and Chl d' in Chl d-type PS I, λunction as the special pairs, either as homodimers, ”Chl
a' and ”Chl g' in anoxyμenic orμanisms, or heterodimers, Chl a/a' and Chl d/d' in
http://dx.doi.org/10.5772/55460
oxyμenic photosynthesis. ”Chl g/g' may be a convincinμ ancestor oλ Chl a/a', because the
”Chl g/g' → Chl a/a' conversion takes place spontaneously under mild conditions in vitro
Kobayashi et al. . Further, a Chl a/a' → Chl d/d' conversion also occurs with ease in
vitro Kobayashi et al. Koizumi et al. , supportinμ the succession λrom the Chl
a-type cyanobacteria to A. marina Fiμ. . Chl f is produced in very small amounts in a
Chl a-type special cyanobacterium, only when cultivated under NIR liμht, suμμestinμ that
Chl f appeared aλter the birth oλ Chl a. “s mentioned above, the primary electron acceptors,
“ , are Chl a-derivatives even in anoxyμenic PS I-type RCs. The secondary electron
acceptors are naphthoquinones, and the side chains appear to have been modiλied aλter
the birth oλ cyanobacteria, leadinμ to succession λrom menaquinone to phylloquinone in
PS I oλ oxyμenic photosynthetic species Ohashi et al. .
In Fiμ. , ”Chl g/g' and their derivatives λunctioninμ in natural photosynthesis are illustrated.
Chlorophyll a/a' are produced λrom ”Chl g/g' by isomerization, and Chl d/d' are then produced
λrom Chl a/a' by oxidation. These three primed chlorophylls λunction as the primary electron
donors oλ PS I-type RCs. Note that Chls b' and f ' are not λound in natural photosynthesis.
Chlorophylls b and f are produced λrom Chl a by oxidation, but λunction as antenna piμments.
H H H
C H C H C
H H O H C H H
C C CH3 C CH3
5 3 5 CH3
3 CH3 4 6 H 3 5 CH3
4 6
H3C 2 7 H3C 2 7
CH2 2 4 6
7
CH2 8 C CH2
8 8
N 1 N N 9 O
1 N 9 H 1 N N 9
20 10
Mg 10 10
Chl b
20
19
N N 11
Phe a 18
19
N
H
N
11
12
20
18
19
N
Mg
N
11 Chl f
18 H
H 12 H 12
H3C 17 15 CH3
17 15 CH3 1 16 14 H3 C 17 15 CH3
H3C 16 14 17 13 16 14
171 13 171 13
H2C 132
H 2C 132 H 131 H2 C 132
H 131 ox n H 131
tio
172CH H O
172 CH H
2
17 CH
2
H
O C
O id 2 O C
a 2 O C
O
O C a tio
O C
O C20H39 O
id O C
O
ox
O C20H39 O CH3 O C20H39
CH3 n CH3
Mg2+
H
2H+
H
C H H
H C C O
H H C CH3
CH3 H C CH3
3 5 CH3 3 5 CH3
6 3 5 CH3 4 6
4
H3C 2 7
C H 3C 2
4 6
7 H3C 2 7
CH2
CH2 8
8 H 8
N N N N 1 N N 9
1 9 1 9
BChl g 20 Mg 10
Chl a
20
19
Mg 10
oxidation 20
19
Mg 10
Chl d
18
19
N N
11 isomerization 18
N N
11
12 H
18
N N
11
12
12 H
H 17 15 CH3 17 15 CH3
17 CH3 H3 C 1 16 14 H3C 1 16 14
H3C 15 17 13 17 13
16 14
171 13 H 2C 132 H 2C 132
H 131 H 131
H 2C 132
H 131 172CH H O 172 CH H O
2 O C 2
172CH H O O C
2 O C O C O C
O C20H39 O O
O C O C20H39
O CH3 CH3
O C15H25
CH3
H epimerization H epimerization epimerization

C H C H H
H C H C O
CH3 CH3 C
H CH3
3 5 CH3 3 5 CH3
4 6 4 6 3 5 CH3
H3C 2 7
C H 3C 2 7
CH2 H3C 2
4 6
7
8 8 CH2
H 8
1 N N 9 1 N N 9 N N
1 9
BChl g’ 20 Mg 10
Chl a’ 20 Mg 10 oxidation 20 Mg 10
Chl d’
19 19
18
N N
11
12
isomerization 18
N N
11
12
18
19
N N 11
H H H 12
H 3C 17 15 CH3 H3C 17 15 CH3 17 15 CH3
16 14 16 14 H3C 16 14
171 13 171 13 171 13
H2C 132 H2C 132 H2C 132
H 131 H 131 H 131
172CH H 172CH H O 172 CH H O
2
O 2 2 O C
O C O C
O C O C O C
O O O C20H39 O
O C15H25 O C20H39
CH3 CH3 CH3
Figure 4. Bacterlochlorophyll g/g' and their derivatives functioning in natural photosynthesis.

54 Photosynthesis
. . . Pheophytin a as the primary electron acceptor in PS II
In , pheophytin Phe a Fiμ. , a demetallated Chl a, was λirst postulated to be the primary
electron acceptor in PS II Fiμ. van Gorkom , and the idea was experimentally
conλirmed in Klimov et al. b . In , bacteriopheophytin ”Phe a was λound to
λunction as a primary electron acceptor in the RC oλ purple bacteria Fiμ. Parson et al.
Rockley et al. Fajer et al. Kauλmann et al. , and shortly thereaλter ”Phe
b was also λound to perλorm the same λunction Fiμ. Klimov et al. c . In , ”Phe a
was also λound to λunction in μreen λilamentous bacteria Fiμ. Kirmaier et al. Shuvalov
et al. . In , the primary electron acceptor oλ PS II in A. marina was λirst λound to be
Phe a Fiμ. “kiyama et al. and later supported by Tomo et al. . For more details,
see Chapter in Kobayashi et al. and a review by Ohashi et al. .
It is oλ interest to note that Phe a as well as Chl a', d' and Chl d are artiλacts easily produced in
vitro see Fiμ. Phe a can be readily produced λrom Chl a under acidic conditions, primed
chlorophylls λrom non-primed ones by epimerization under basic conditions, and Chl d λrom
Chl a under oxidative conditions. These artiλacts, however, λunction as key components in
natural photosynthesis, while Phes b, d, f and Chls b', f ' are not λound in nature.
. Physicochemical properties of chlorophylls in vitro
. . HPLC
In the late s, the hiμh perλormance liquid chromatoμraphy HPLC technique was applied
to the separation oλ plant piμments. In many cases the reversed-phase HPLC was preλerred
Eskins et al. Shoaλ et al. Schoch et al. , and is still the main option to date. In
that system, however, an eluent μradient is usually required λor simultaneous separation oλ
Chls and Phes and the μradient system is unλavorable λor quantitative analysis, since the molar
absorptivities oλ piμments stronμly depend on solvents. In this context, an isocratic eluent
system is λavorable. In , a simultaneous separation oλ Chls and Phes by normal-phase
HPLC was attained by an isocratic procedure Iriyama et al. . In , the isocratic normal-
phase HPLC was established as a powerλul tool λor chlorophyll analysis Watanabe et al. .
. . . Mixture of chlorophylls and pheophytins
Chls a and b were extracted with acetone/methanol / , v/v mixure at K λrom parsley
Petroselinum crispum Nym. , Chl d λrom A. marina, and Chl f λrom a cyanobacterium strain
KC μrown under near-inλrared LED liμht. The extract was applied to a preparative-
scale HPLC Senshupak -N, mm x mm i.d. and eluted with hexane/ -propanol/
methanol / / . , v/v at a λlow rate oλ mL min- at K, as described elsewhere
Kobayashi et al. . Other authentic piμments, Chl a', Chl f', Phe a and Phe f, were
prepared by epimerization and pheophytinization oλ Chl a and Chl f as described else‐
where Watanabe et al. .
http://dx.doi.org/10.5772/55460
“ mixture oλ Chls and Phes was injected into a silica HPLC column YMC-pak SIL, x .
mm i.d. cooled to K in an ice-water bath. The piμments were eluted isocratically with
deμassed hexane/ -propanol/methanol / . / . , v/v at a λlow rate oλ . mL min- , and were
monitored with a J“SCO UV- detector = nm and a J“SCO Multiwavelenμth MD-
detector = - nm in series.
“s illustrated in Fiμ. F , eiμht Chls and λour Phes are clearly separated. One can easily see
that Synechocystis sp. PCC possesses Phe a and Chl a' as well as Chl a, and that Chlorella
vulgaris has also Chl b.
. . . Pigment extract from A. marina
Piμments were extracted λrom cell suspension ca. L by sonication in a ca. -λold volume
oλ acetone/methanol / , v/v mixture λor min in the dark at room temperature. The extract
was λiltered and dried in vacuo. The whole procedure was completed within min. The solid
material thus obtained was immediately dissolved in L oλ chloroλorm, and injected into a
silica HPLC column.
“s seen in Fiμ. C , A. marina has three minor chlorophylls, Phe a, Chl a and Chl d', in addition
to the major Chl d “kiyama et al. . Pheophytin a λunctions as the primary electron
acceptor in PS II, Chl a as the primary electron acceptor in PS I, and Chl d' as the primary
electron donor P as a heterodimer oλ Chl d/d', like the Chl a/a' in P Fiμ. .
. . . Pigment extract from strain KC
Cells oλ the cyanobacterium strain KC were μrown in ”G- medium in a μlass cell culture
λlask L at K with continuous air-bubblinμ. Cells were incubated under continuous white
λluorescent liμht mol photons/m /s or near-inλrared LED liμht see Fiμ. “, Tokyorika,
Tokyo . Cells at the early stationary phase were harvested by centriλuμation. See “kutsu et al.
λor more details.
Typical HPLC traces λor acetone/methanol extracts λrom cells oλ the cyanobacterium strain
KC cultivated under white λluorescent liμht or NIR LED liμht are shown in Fiμs. D and E,
respectively. “ larμe amount oλ Chl a, as well as small amounts oλ Chl a' and Phe a, were
detected in both cells. We should note that only the strain KC μrown under NIR LED liμht
showed the presence oλ Chl f as a minor piμment and that Chl f' and Phe f were not detected
at all Fiμ. E .
. . Absorption spectra in four solvent varieties
The absorption spectrum is the simplest, most useλul and extensively used analytical property
to characterize chlorophylls. “bsorption spectra oλ Chls show the electronic transitions alonμ
the x axis oλ the Chl runninμ throuμh the two nitroμen N atoms oλ rinμs II and IV, and alonμ
the y-axis throuμh the N atoms oλ rinμs I and III see Fiμ. . The two main absorption bands
in the blue and red reμions are called Soret and Q bands, respectively, and arise λrom π→π*
transitions oλ λour λrontier orbitals Weiss Petke et al. Hanson .
56 Photosynthesis
(A)
Absorbance (a.u.)
(B)
400 500 600 700 800

Wavelength / nm
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).
http://dx.doi.org/10.5772/55460
(A) Chl a Synechosystis
×20
Vertical magnification
Phe a Chl a’ ×20
(B) Chl a
×20 Chlorella
Phe a Chl a’ ×20

Chl b
(C) Acariyochloris Chl d
Phe a Chl a
×20
Chl d’
(D) Chl a Strain KC1

×20 White FL.
×20
Phe a Chl a’
(E) Chl a
×20 Strain KC1
NIR LED
×20
Phe a Chl a’
Chl f
(F) Chl a
Chl f ’ Standards
Phe f Chl f
Phe d
Chl b’
Phe b Chl d’ Chl d
Chl b
Phe a Chl a’
0 10 20 30 40 50
Retention time / min
Figure 6. Normal-phase HPLC profiles for acetone/methanol extracts of (A) Synechocystis sp. 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.
58 Photosynthesis
. . . Chlorophylls a, b, d and f
“bsorption spectra oλ Chls a, b, d and f in λour kinds oλ solvent measured at room temperature
are shown in Fiμ. top . “s compared to Chl a, Chl b shows red-shiλted Soret bands and blue-
shiλted weak QY bands, while the QY bands oλ Chls d and f are intensiλied and shiλted to lonμer
wavelenμths. The Qx exhibits practically no intensity. The ratios oλ Soret/QY band intensities
show remarkable diλλerences, e.g., in diethyl ether ca. . in Chl a, ca. . in Chl b, ca. . in
Chl d, and ca. . in Chl f Table . Note that the Soret/QY-band ratios in Chl b is more than
and those in Chls d and f are below in all solvents in Fiμ. , while the ratios oλ Chl a in three
solvents excludinμ methanol are around . , but sliμhtly below in methanol. So one can easily
distinμuish Chl b λrom Chls a, d and f also Chl a λrom Chls d and f, by their absorption spectra
in any solvents used here.
diethyl ether acetone methanol benzene

429.0 660.9 (A) 431.3 662.2 (B) 432.5 665.8 (C) 432.5 665.3 (D)
454.0 (A’) 458.7 (B’) 469.4 (C’) 458.3 (D’)

644.0 646.0 652.2 646.5
452.4 686.2 (A”) 451.7 691.4 (B”) 400.8 455.5

698.1 (C”) 450.2 692.7 (D”)
394.2
695.2 (A”’) 398.2 701.0 (B”’) 406.7 708.3 (C”’) 700.9 (D”’)
395.6 440.5 400.9 444.0
400 500 600 700 400 500 600 700 400 500 600 700 400 500 600 700
diethyl ether acetone methanol benzene
408.4 408.9 (F) 409.2 (G) 414.5 (H)
(E)
667.3 665.4 665.7 670.8
433.4 (E’) 434.4 (F’) 436.0 (G’) 440.0 (H’)
654.4 653.3 653.9 656.9
(E”) (F”) (G”) (H”)

384.0
421.0 383.7 697.3

383.3 691.9 421.5 691.0 410.7 387.8 428.8
693.1
696.9(E”’) (F”’) (G”’) (H”’)

409.3
409.3 409.3 410.0 415.0 701.9

697.9 699.8
400 500 600 700 400 500 600 700 400 500 600 700 400 500 600 700
Figure 7. Comparison of the absorption spectra of Chls a, b, d, f (top), and Phes a, b, d, f (bottom) in diethyl ether,
acetone, methanol and benzene at room temperature. Spectra were scaled to the Soret- or QY-band maximum.
http://dx.doi.org/10.5772/55460
Compound λmax,blue [nm] λmax,red [nm]
(ε[103 M-1cm-1]) (ε[103 M-1cm-1]) Ref
Chl a 428.4 660.3 Watanabe et al. (1984)
(115) (89.8) ibid.
429.1b 661.6b ibid.
(100)b (81.3)b ibid.
432.5d 665.4d ibid.
(101)d (79.7)d ibid.
429.0 660.9 Kobayashi et al.(2006)
(1.00)a (0.775)a ibid.
431.3b 662.2b This work
(1.00)a,b (0.828)a,b ibid.
432.5c 665.8c ibid.
(0.944)a,c (1.00)a,c ibid.
432.5d 665.3d ibid.
(1.00)a,d (0.785)a,d ibid.
Phe a 408.4 667.9 Watanabe et al. (1984)
(107) (52.6) ibid.
409.2b 665.9b ibid.
(104)b (46.0)b ibid.
414.8d 671.6d ibid.
(108)d (53.1)d ibid.
408.4 667.3 Kobayashi et al.(2006)
(1.00)a (0.497)a ibid.
(1.00)a,b (0.440)a,b ibid.
409.2c 665.7c ibid.
(1.00)a,c (0.464)a,c ibid.
414.5d 670.8d ibid.
(1.00)a,d (0.798)a,d ibid.
Chl b 451.9 641.9 Watanabe et al. (1984)
(159) (56.7) ibid.
455.8b 644.6b ibid.
(136)b (47.6)b ibid.

60 Photosynthesis
(ε[103 M-1cm-1]) (ε[103 M-1cm-1]) Ref
457.9d 646.2d ibid.
(152)d (56.2)d ibid.
452.4 642.5 Kobayashi et al.(2006)
(1.00)a (0.355)a ibid.
(1.00)a,b (0.355)a,b ibid.
469.4c 652.2c ibid.
(1.00)a,c (0.355)a,c ibid.
458.3d 646.5d ibid.
(1.00)a,d (0.364)a,d ibid.
Phe b 432.7 654.6 Watanabe et al. (1984)
(172) (34.8) ibid.
433.8b 653.3b ibid.
(153)b (29.3)b ibid.
439.5d 656.7d ibid.
(152)d (4.718)d ibid.
433.2 654.5 Kobayashi et al.(2006)
(1.00)a (0.202)a ibid.
(1.00)a,b (0.195)a,b ibid.
436.0c 653.9c ibid.
(1.00)a,c (0.245)a,c ibid.
440.0d 656.9d ibid.
(1.00)a,d (0.216)a,d ibid.
Chl d 447 688 Smith and Benitez (1955)
(87.6) (98.9) ibid.
447 688 French (1960)
(87.6) (98.5) ibid.
390, 445 686 Miyashita et al. (1997)
392b, 447b 688b ibid.
400c, 455c 697c ibid.
445.6 686.2 Kobayashi et al.(2006)

http://dx.doi.org/10.5772/55460
(ε[103 M-1cm-1]) (ε[103 M-1cm-1]) Ref
(0.853)a (1.00)a ibid.
394.3b, 451.7b 691.4b This work
(0.559)a,b(0.826)a,b (1.00)a,b ibid.
400.8c, 455.5c 698.1c ibid.
(0.735)a,c(0.706)a,c (1.00)a,c ibid.
394.2d, 450.2d 693.7d ibid.
(0.532)a,d, (0.885)a,d (1.00)a,d ibid.
Phe d 421 692 Smith and Benitez (1955)
(84.9) (72.2) ibid.
421 692 French (1960)
(84.9) (72.2) ibid.
382.7, 421.3 692.0 Kobayashi et al.(2006)
(0.881)a,(1.00)a (0.911)a ibid.
383.7b, 421.5b 691.0b This work
(0.888)a,b, (1.00)a,b (0.761)a,b ibid.
384.0c, 410.7c 693.1c ibid.
(1.00)a,c, (0.964)a,c (0.637)a,c ibid.
387.8d, 428.8d 697.3d ibid.
(0.802)a,d, (1.00)a,d (0.915)a,d ibid.
Chl f 395.6, 440.5 695.2 This work
(0.657)a (0.648)a (1.00)a ibid.
398.2b, 442.0b 701.0b ibid.
(0.780)a,c, (0.576)a,b (1.00)a,b ibid.
400.9d, 444.0d 700.9d ibid.
(0.668)a,d, (0.658)a,d (1.00)a,d ibid.
406.7c 708.3c Akutsu et al. (2011)
(0.904)a,c (1.00)a,c ibid.
406 706 Chen et al. (2010)
(1.00)a,d (0.527)a,d ibid.
Phe f 409.3 696.9 This work
(1.00)a (0.727)a ibid.
409.3b 697.9b ibid.

62 Photosynthesis
(ε[103 M-1cm-1]) (ε[103 M-1cm-1]) Ref
(1.00)a,b (0.610)a,b ibid.
410.0c 699.8c ibid.
(1.00)a,c (0.561)a,c ibid.
415.0d 701.9d ibid.
(1.00)a,d (0.776)a,d ibid.
a: relative values
b: in acetone
c: in methanol
d: in benzene
Table 1. Absorption properties of chlorophylls in diethylether at room temperature
It is somewhat diλλicult to distinμuish Chl f λrom Chl d, when one rouμhly compares the
absorption spectrum oλ Chl f in diethyl ether Fiμ. “"' with that oλ Chl d in methanol Fiμ.
C" , because their spectral shapes are very similar. In contrast, in diethyl ether one can easily
distinμuish Chl f λrom Chl d without spectrophotometer, because Chl f looks blue-μreen as Chl
a, while Chl d liμht-μreen as Chl b, indicatinμ that the naked eye is oλten powerλul λor colour
judμement.
The Soret bands include several intense bands. In diethyl ether and benzene, the Soret band
oλ Chl f is clearly split into two bands, most probably the so-called ”-bands lonμer wavelenμth
and η-bands shorter wavelenμth , while Chl d shows such a split not in those solvents but in
methanol, and hence one can easily distinμuish them by comparinμ their optical spectra in the
same solvents, e.g., diethyl ether Fiμs. “" and “"' .
In Fiμ. are shown the absorption spectra oλ the strain KC μrown under white λluores‐
cent liμht and NIR LED liμht. The cells μrown under NIR LED liμht show a clear should‐
er over nm, extendinμ up to almost nm Fiμ. “ . “bsorption spectra in acetone
solution oλ acetone/methanol extracts λrom the KC cells cultivated under NIR LED also
exhibit a lonμer wavelenμth peak in the ranμe oλ about to nm Fiμ. ” , due to the
presence oλ Chl f. We had better pay attention that the NIR LED emission spectrum seen
in Fiμ. “ overlaps the absorption spectrum oλ the strain KC cells μrown under white
λluorescent liμht, indicatinμ that the cells without Chl f can absorb NIR LED liμht, where
some Chl a molecules possessinμ lonμer wavelenμth absorption may act as a triμμer λor
Chl f biosynthesis under NIR LED liμht. Iλ this hypothesis holds, a much lonμer wave‐
lenμth LED could not induce Chl f biosynthesis. In such a study, one should μive a lot oλ
care to the shorter wavelenμth λoot oλ emission spectrum oλ NIR LED not to overlap the
absorption oλ cells at all.
http://dx.doi.org/10.5772/55460
We should note that inductive eλλects on the absorption wavelenμths and intensities oλ QY-
bands oλ chlorophylls stronμly depend on the nature and position oλ substituent s on the
macrocycle, due to the presence oλ two diλλerent electronic transitions polarized in the x
and y directions the axes oλ transition moments are depicted in Fiμ. Gouterman ,
Gouterman et al. Weiss Petke et al. Hanson , Kobayashi et al. b.
Replacement oλ the electron-donatinμ μroup, -CH , on rinμ II oλ Chl a by the electron-
withdrawinμ μroup, -CHO, yieldinμ Chl b, causes the blue-shiλt and siμniλicant intensity
reduction oλ the QY-band Fiμ. . In contrast, replacement oλ -CH on rinμ I oλ Chl a by -
CHO, yieldinμ Chl f, causes the red-shiλt and intensity increase oλ the QY-band Fiμ. . “
similar phenomenon is clearly seen in Chl d, where -CH=CH on rinμ I oλ Chl a is replaced
with -CHO. These results indicate it is a μeneral λeature that substitution by the electron-
withdrawinμ μroup on rinμ II causes the blue-shiλt and intensity reduction oλ the QY-
band and that the same substitution on rinμ I leads the opposite, namely, the red-shiλt and
intensity increase oλ the QY-band. Moreover, it looks that substitution on rinμ I by the
electron-withdrawinμ μroup μenerates the well-split Soret band, while showinμ heavy
dependence on solvent as described above.
. . . Pheophytins a, b, d and f
The λree base related to Chl is called Phe. First oλ all, we emphasize that in natural photosyn‐
thesis only Phe a λunctions, and Phes b, d and f are not λunctional. In μeneral, the more
structured shape and red shiλted Soret band oλ Chls distinμuishes them λrom the correspondinμ
Phes. In contrast to Chls, the η bands in the Soret band was poorly resolved in any Phes except
Phe d Fiμ. . Removal oλ the central Mμ increases deviation λrom planarity and reduces the
molecular symmetry, thus increasinμ Soret and QX transition. The Soret/QY-band ratios
noticeably increases by pheophytinization in diethyl ether Phe b shows the hiμhest value oλ
around , Phe a the secondary hiμhest about , and Phe d the lowest near compare bottom
with top in Fiμ. , see also Table . Thereλore, contamination oλ pheophytins in a Chl sample
is oλten noticed λrom the optical spectra.
“s seen in Fiμ. , Phe b can be easily distinμuished λrom Phe a by its blue shiλted QY-band, red
shiλted Soret band, and by the marμinally hiμher Soret/QY band ratio. Phes d and f can be
distinμuished λrom Phes a and b by their red shiλted QY band and intense QY bands, i.e., the
Soret/QY-band ratios in Phes d and f are not hiμh 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 Fiμs. G with G"' , althouμh they can be distinμuished by the QY wavelenμth
diλλerence. We must emphasize aμain that Phes possess relatively stronμ and characteristic
QX-bands in the reμion oλ - nm the QX bands in Phes a and d are better resolved to the
QX , and QX , transitions. Phe d also shows siμniλicant splittinμ oλ the Soret and QX bands
in all solvents as illustrated in Fiμ. . In contrast, the Qx band correspondinμ to the QX ,
transition shorter wavelenμth oλ Phe f looks unclear. It is oλ interest to note that in diethyl
ether Phe d assumes a pale pink color, while both Phes b and f show a dull color. These
characteristics will help us to discern Phes λrom Chls, and amonμ Phes.
64 Photosynthesis
. . Circular dichroism spectra
Circular dichroism CD spectra are very useλul λor distinction between the primed chloro‐
phyll, e.g., Chl a', and the correspondinμ non-primed one, Chl a, althouμh the absorption
characteristics oλ the primed derivatives Chls a', b', d', f', Phes a', b', d' and f' are identical
with those oλ the non-primed ones Wolλ and Scheer Weiss Watanabe et al.
Kobayashi et al. b.
“ spectropolarimeter Model FDCD- J“SCO was used λor CD measurements. ”enzene

was chosen as the solvent, in view oλ the suλλiciently slow interconversion between epimeric
species in this medium Watanabe et al. . The spectra were recorded λrom nm to
nm at a scan rate oλ nm/min with scans at room temperature time λor measurement
was ca. one hour.
The CD spectra oλ Chl a/a', b/b', d/d' and f/f' in benzene are illustrated in Fiμ. . It is immediately
seen that, λor a μiven pair oλ epimers, the CD spectra are considerably diλλerent, althouμh the
absorption spectra are practically identical with each other. For each oλ Chls a', b', d' and f', an
intense neμative CD is associated with QY , and a well-deλined weakly neμative satellite
with QY , . On the other hand, the non-primed species, Chls a, b, d and f, show complicated,
very weak neμative and/or positive activities at these transitions. In Fiμ. , all pheophytins
show neμative activities, and primed ones reveal stronμer and red-shiλted siμnals compared
to the non-primed ones, althouμh the absorption spectra oλ the primed derivatives are also
identical with those oλ the non-primed ones. The λindinμs suμμest that the QY maximum
transition consists oλ at least two bands, and shorter wavelenμth band shows stronμer activity
in primed Phe and lonμer wavelenμth band stronμer in non-primed Phe.
“ series oλ QX transitions occur in the "valley" oλ the absorption spectrum as described in section
. The positive CD activities derived λrom the QX , absorption called bands III, see Fiμ. in
Petke et al. appear at , , , , , , and nm λor Chl a', Phe a', Chl
d', Phes d, d', Chl f', Phes f and f', respectively, while Chl f and Phe a exhibit weakly neμative
activities at nm and nm, respectively. Such deλinite activities are not observed in Chls
a, b/b', d and Phe b/b' optical activities oλ b-type piμments, Chl b/b' and Phe b/b', are extremely
weak, which may be related to the very diλλuse λeature oλ their absorption spectra. The CD
activity associated with the QX , absorption satellites band IV at the shorter wavelenμth
also is very weak and vaμue in all the piμments examined.
The Chl and Phe Sorets contain many π-π* transitions characterized by a complex mixture oλ
conλiμurations. “ccordinμ to the results oλ molecular orbital calculations Weiss Petke et
al. Hanson , band ” in the Soret absorption consists oλ two nearly deμenerate
electronic transitions, ”X , and ”Y , . “ll the primed derivatives μave sinμle and stronμly
positive CD spectra at this absorption peak, suμμestinμ that the two transitions contribute to
CD spectra in a similar manner Watanabe et al. . In contrast, the CD spectra oλ non-
primed species, except Chl f and Phes, apparently reλlect the existence oλ the two transitions
they show a maximum and a minimum with the center wavelenμth rouμhly coincidinμ with
the Soret absorption maximum. Diλλerent λeature oλ CD spectrum λor Chl f amonμ Chls may
come λrom its characteristically splitted Soret absorption arisinμ λrom ”-bands and so-called
http://dx.doi.org/10.5772/55460
435.5
(A) (B)
461.0
10 10
θ ×10-4 / deg M-1 cm-1
θ ×10-4 / deg M-1 cm-1

578.5
5 5
0 0
438.5
452.5
-5 -5
384.5
Chl a Chl b
646.0
Chl b’
382.5
-10 -10
Chl a’
665.5
432.5
458.3
665.3
(A’) (B’)
Absorbance ( a.u. )
Absorbance ( a.u. )
Chl b,b’
646.5
Chl a, a’
400 500 600 700 400 500 600 700

Wavelength / nm Wavelength / nm
(C) 10 Chl f
(D)
10
393.0
θ ×10-4 / deg M-1 cm-1
444.0
448.5
θ ×10-4 / deg M-1 cm-1
593.5
5
604.0
0 0
-5 -5
459.5
609.0
Chl d
Chl d’
395.0
398.0
-10 -10
692.5
692.5
Chl f’
692.7
700.9
450.2
(C’) (D’)
Absorbance ( a.u. )
Absorbance ( a.u. )
400.9
440.0
Chl f ,f’
Chl d,d’
400 500 600 700

400 500 600 700
Wavelength / nm
Wavelength / nm
Figure 8. CD spectra for

θ (A) Chls a/a', (B) Chls b/b', (C) Chls d/d', (D) Chls f/f', and (A')-(D') the corresponding absorp‐
tion spectra in benzene at room temperature. [θ] denotes the molar ellipticity.
66 Photosynthesis
η-bands in benzene. Primed Chls exhibit relatively intense neμative CD spectra in the near
ultraviolet at η-bands oλ the Soret reμion Welss Petke et al. , whereas non-primed
ones exhibit positive activities. Phes a and f also show positive CD spectra at η-bands in the
near ultraviolet reμion, but such a tendency is not clear in Phe b, and Phe d exhibits neμative
activity Fiμ. , althouμh all the primed Phes show neμative activity and Phe d' shows the
most intense activity.
414.2
(A) (B)
438.0
10 10
θ ×10-4 / deg M-1 cm-1

θ ×10-4 / deg M-1 cm-1
419.0
Phe a
397.0
449.5
535.2
5 5 Phe b
423.0
0 0
658.5
378.5
674.0
374.1
650.4
-5 -5
Phe b’
663.0
Phe a’
-10 -10
440.0
414.5
(A’) (B’)
Absorbance ( a.u. )
Absorbance ( a.u. )
Phe a, a’ Phe b, b’
670.8
656.9
507.2
537.2
400 500 600 700 400 500 600 700

(C) (D)
10
429.5
547.9
429.8
10 Phe d
416.0
θ ×10-4 / deg M-1 cm-1
θ ×10-4 / deg M-1 cm-1
556.5
422.0
567.3
5
559.3
5 Phe f
0
0
385.2
702.5
-5
698.5
Phe f’
691.5
-5
Phe d’
389.5
693.9
383.9
-10
-10
415.0
428.8
(C’) (D’)
701.9
697.3
Absorbance ( a.u. )
Absorbance ( a.u. )
387.8
Phe d, d’ Phe f, f ’
562.7
552.4
519.9
523.0
400 500 600 700 400 500 600 700

Figure 9. CD spectra for (A) Phes

θ
a/a', (B) Phes b/b', (C) Phes d/d', (D) Phes f/f', and (A')-(D') the corresponding absorp‐
tion spectra in benzene at room temperature. [θ] denotes the molar ellipticity.
http://dx.doi.org/10.5772/55460
. . Mass spectra
Chlorophylls in natural photosynthesis are sometimes present in very small amounts, and
hence the use oλ mass spectrometry MS can be advantaμeous since only minute samples are
required. MS can provide accurate and useλul inλormation not only on molecular weiμhts and
elemental compositions but also on the nature oλ λunctional μroups attached to the macrocycle
e.g. phytol and oλ the central metal see reviews by Smith Hunt and Michalski
Porra and Scheer Kobayashi et al. .
LC/MS experiments were perλormed on an LCQ mass spectrometer Thermo Fisher Scientiλic
Inc., M“, U.S.“. equipped with an HPLC system HP , “μilent, C“, U.S.“. connected
with a diode array detector. Each sample dissolved in dichloromethane beλore analysis was
applied on a J“SCO Finepak SIL C S column mm x . mm i.d. cooled to K in an
ice-water bath, and separated usinμ a mixture oλ ethanol/methanol/ -propanol/water
/ / / , v/v at a λlow rate oλ L min- . The eluate was monitored by the UV-Vis
absorption in a ranμe oλ - nm, and was introduced into the mass spectrometer λrom
to min aλter sample injection. “tmospheric pressure chemical ionization “PCI mass and
MS/MS spectra were recorded in the positive-ion mode in the mass ranμe oλ m/z - , .
Helium was used as collision μas λor MS/MS experiments, λollowed by the isolation oλ ions
over a selected mass window oλ Da. The mass spectrometer was initially tuned usinμ a
standard Chl a solution as λollows “PCI vaporizer temp., K spray voltaμe, kV capillary
temperature, K capillary voltaμe, V sheath μas nitroμen λlow rate, arbitrary unit
auxiliary μas λlow rate, arbitrary unit .
“s illustrated in Fiμ. leλt , Chl a C H MμN O , monoisotopic mass . . Hereaλter the

value in the bracket shows the monoisotopic mass oλ the molecule or the ion μives the
protonated molecule [M+H]+ at m/z . producinμ the dominant λraμment ion at m/z . .
The mass diλλerence between [M+H]+ and the product ion corresponds to C H . This
shows the presence oλ a phytyl chain in Chl a. The other product ions at m/z . and m/z .
correspondinμ to [M+H- - ] and [M+H- - ] , respectively, are supposed to be the
+ +
results oλ the loss oλ carboxymethyl μroup λollowed by the cleavaμe oλ phytol. The losses oλ
, and λrom the precursor ion in MS/MS spectra are seen in all the piμments examined
here reveals the presence oλ a phytyl chain.
It is interestinμ to note that a typical λast atom bombardment F“” -mass spectrum oλ Chl a
shows two intense molecular ion peaks, [M]+ at m/z . and [M + H]+ at . , as well as the
dominant λraμment ion [M-C H +H] at m/z +
. see Fiμ. in Kobayashi et al. . ”oth
spectra, however, reveal that Chl a has a phytyl chain C H . We should make sure that the
mass spectra oλ chlorophyll molecular ion peak s and the λraμment ion peak s vary by m/z
. accordinμ to the ionization methods.
Chlorophyll d C H MμN O , . μives the protonated molecule [M+H]+ at m/z .

and the prominent λraμment ion at m/z . . Thouμh chlorophylls b and f both
C H MμN O , . are eluted at diλλerent LC retention times, they show the same mass
and MS/MS spectral patterns, [M+H]+ at m/z and the dominant product ion at m/z ,
which correspond to [C H MμN O ] +
. and [M-C H +H] +
. . These results
68 Photosynthesis
[M+H‐C20H38]+ [M+H]+ [M+H‐C20H38]+ [M+H]+

Chl a 615.1 893.2 Phe a 593.1 871.3
-278 -278
-60
-32 -60
533.2
555.2 583.0
850 900 950 850 900 950
500 600 700 800 900 500 600 700 800 900
Chl d 617.1 895.2 Phe d 595.2 873.4

-278 -278
-60 -60
-32
535.3
557.2
585.1 850 900 950 850 900 950
500 600 700 800 900 500 600 700 800 900
Chl b 629.1 907.2 Phe b 607.2 885.4

-278 -278
-60 -60
-32
547.2
597.2
569.2 850 900 950 850 900 950
500 600 700 800 900 500 600 700 800 900
Chl f 629.0 907.1 Phe f 607.0 885.3

-278 -278
-60
-32 -60
547.1
569.1 597.1 850 900 950 850 900 950
500 600 700 800 900 500 600 700 800 900
m/z m/z
Figure 10. MS/MS spectra of Chls a, b, d, f (left column) and Phes a, b, d and f (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 Chls a,
b, d and f give product ions of [M+H-278]+, [M+H-278-32] + and [M+H-278-60] +. Pheophytins a, b, d and f give product
ions of [M+H-278]+ and [M+H-278-60]+.
suuμest that Chl f also possesses a phythyl lonμ chain in such molecules as Chls a, b and d, and
that most probably one -CH moiety oλ Chl a is substituted λor -CHO μroup in Chl f like Chl
b, yieldinμ [ -λormyl]-Chl a, [ -λormyl]-Chl a or [ -λormyl]-Chl a.
“s seen in Fiμ. riμht , the correspondinμ pheophytins prepared by acid treatment clearly
showed the absence oλ maμnesium Fiμ. . For example, [M+H]+ oλ Phe a is observed at m/z
. which is Da smaller than that oλ Chl a, showinμ the substitution oλ Mμ with two H
atoms by pheophytinization see Fiμ. . Pheophytins b and f C H N O , . and Phe d
C H NO . show the similar pattern, supportinμ that all oλ them do not possess Mμ
as central metal.
. . Nuclear magnetic resonance spectra
Nuclear maμnetic resonance NMR spectroscopy can oλλer ample inλormation about the
molecular structure. Coupled use oλ NMR with HPLC, absorption-, CD- and mass-spectro‐
metries has not only deλinitively identiλied the structures oλ several major naturally-occurrinμ
Chls but has also assisted recent studies oλ minor Chl piμments, present in minute quantities,
such as electron donors and acceptors in the RC.
http://dx.doi.org/10.5772/55460
The NMR spectra were recorded on a ”ruker “vance spectrometer ”ruker ”iospin,
Karlsruhe, Germany , with a λrequency oλ H at MHz and C at MHz, equipped with
TCI CryoProbe usinμ a microtube Shiμemi Inc., Tokyo and about . mμ oλ sample in . mL
oλ acetone-d with tetramethylsilane TMS as an internal standard. The chemical shiλts are
μiven in δ-scale [ppm] downλield λrom TMS. The measurements were perλormed at K. The
typical experimental conditions λor the H NMR spectra were scans, a spectral width oλ
ppm, k data points. The C spectra were acquired usinμ a power μated decouplinμ with
k scans. The spectral width oλ ppm was acquired in k data points. The D-homonuclear
Nuclear Overhauser and Exchanμe Spectroscopy NOESY and D-heteronuclear H,C-
Heteronuclear Sinμle Quantum Coherence H,C-HSQC and H,C-Heteronuclear Multiple
”ond Correlation H,C-HM”C experiments were perλormed λor the structural assiμnments
oλ the H and C siμnals usinμ standard D-NMR pulse sequences oλ ”ruker soλtware.
. . . H-NMR
“s observed in one-dimensional H-NMR spectra Fiμ. , Table , marked diλλerences are

seen in the siμnals arisinμ λrom the λormyl μroup. Each low-λield sinμlet siμnal characteristic
oλ the λormyl moiety observed around ppm in the spectra oλ Chls b ,d and f is
absent λrom the spectrum oλ Chl a. Similarly, double doublet siμnal oλ -H vinylic proton at
- . ppm in the spectra oλ Chls a, b and f is not seen in the spectrum oλ Chl d. These results
reconλirm that Chl d is -desvinyl- -λormyl Chl a [ -λormyl]-Chl a .
Here we note that the -H vinylic proton shows a larμe downλield shiλt in Chl f . ppm
and that is sliμhtly upλield shiλted in Chl b . ppm , as compared to Chl a . ppm ,
suμμestinμ that Chl f should be λormylated alonμ the y-axis and that interaction between -
CH=CH and -CHO in Chl f is rather stronμ than that in Chl b. The -CHO substitution position
in Chl f is hence most probably at C , next to the -vinyl μroup.
The pair siμnals oλ - and '-H vinylic protons are well resolved in the spectra oλ Chls a .
ppm and . ppm and b . ppm and . ppm , while the correspondinμ pair siμnals
in Chl f show low resolution . ppm and . ppm , suμμestinμ that the environment oλ
-vinyl moiety in Chls a and d is very similar to each other, but is proλoundly diλλerent λrom
that in Chl f, most probably due to the presence oλ λormyl moiety at the neiμhborinμ C in Chl
f. The H sinμlet siμnals λor -CH in Chls a, d and f are at . ppm and absent λrom the spectrum
oλ Chl b, while the H siμnal λor -CHO in Chl b appears on a much lower λield at . ppm.
“nother marked diλλerence seen in Chl f spectrum is the disappearance oλ the sinμlet siμnal oλ
-CH proton the correspondinμ siμnals are observed at . - . ppm in the spectra oλ Chls
a, b and d, implyinμ that -CH oλ Chl a is substituted in Chl f λor some other moiety, most
probably -CHO. No other chanμe is clear in the one-dimensional H-NMR spectra.
. . . C-NMR
In the C-NMR spectra Fiμ. , Table , marked diλλerences are noted in the ranμe oλ ppm
to ppm, ppm to ppm, and ppm to ppm, relatinμ to the -CH , -CH=CH and
-CHO moieties. Compared to Chl a, in the spectrum oλ Chl f, the C siμnal oλ -CH is absent,
70 Photosynthesis
the -CH and , -CH=CH carbon siμnals appear at ppm, ppm and ppm, respec‐
tively, similar to Chl a ppm, ppm and ppm . Further, a new carbon siμnal appears
at ppm, close to the siμnals oλ -CHO in Chls b ppm and d ppm , supportinμ the
presence oλ -CHO at C in Chl f.
71 s
Chl a 31 d d 134
121
21
71
82
P1 171 181
132 81
10 5 20 32 P2 17 172
32
31 18
Chl b 71 s 31 d d 121
134 21
181
82
P1 171172
132
71 32 32 81
5 10 20 17
31 P2 18
134
Chl d 31
71 s
81
21
121 7
1
s 82
17 171 2 181
31 P1 P1 17
5 10 20 132
P2 18
71 s 134
Chl f 21 81
121
71
s 82
181
P1
21 5 31 d d 132 17 172
32 171
10 20
31
P2 18
Chemical Shifts (ppm)

Figure 11. 1H-NMR spectra of Chls a, b, d and f measured 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.
http://dx.doi.org/10.5772/55460
IUPAC no.of
Chl a Chl b Chl d Chl f
carbon atom
11.215
21 3.343 (3.36)1 (s) 3.316 (3.40)2*(s) 3.724 (3.68)3(s)
(11.35)4**(s)
3 - - - -
31 8.162 (8.18)1(dd) 8.043(7.95)2*(dd) 11.460 (11.40)3(s) 8.534(dd)
6.242 (6.24)1(dd), 6.028 6.302(6.25)2*(dd), 6.055 6.324(dd),
32 -
(6.03)1(dd) (6.04)2*(dd) 6.365(dd)
4 - - - -
5 9.410 (9.40)1(s) 10.192 (10.04)2*(s) 10.294(10.20)3(s) 9.770 (9.79)4**(s)
71 3.300 (3.30)1(s) 11.305 (11.22)2*(s) 3.365 (3.33)3(s) 3.351(s)
8 - - - -
81 3.817 (3.82)1(q) 4.243 3.876 (3.86)3(q) 3.754(q)
82 1.696 (1.69)1(t) 1.815 1.723 (1.73)3(t) 1.705(t)
10 9.749 (9.75)1(s) 9.934 (9.64)2*(s) 9.873 (9.8)3(s) 9.838 (9.86)4**(s)
11 - - - -
12 - - - -
121 3.619 (3.61)1(s) 3.606 (3.65)2*(s) 3.668 (3.65)3(s) 3.637(s)
13 - - - -
132 6.234 (6.24)1(s) 6.189 (6.19)2*(s) 6.335 (6.28)3(s) 6.318(s)
133 - - - -
134 3.829 (3.83)1(s) 3.842 (4.02)2*(s) 3.851 (3.83)3(s) 3.887(s)
17 4.175 (4.16)1 4.128 4.242 (4.25)3 4.230
17 1
2.589 (2.60) , 2.461 (2.45)
1 1
2.43,2.593 2.484,2.622 2.467,2.632
172 2.431 (2.35)1, 2.159 (2.05)1 2.08,2.44 1.98,2.418 2.08,2.47
18 4.572 (4.57)1(q) 4.524(q) 4.660 (4.63)3(q) 4.634(q)
181 1.772 (1.77)1, 1.762(1.76)1(d) 1.768,1.759(1.78)2*(d) 1.812, 1.802(1.82)3(d) 1.800, 1.791(d)
20 8.582 (8.58)1(s) 8.480(8.20)2*(s) 8.867 (8.81)3(s) 9.533 (9.77)4**(s)
4.343 (4.26)3, 4.227
P1 4.342 (4.33)1,4.224 (4.21)1 4.364, 4.247 4.361, 4.263
(4.36)3
P2 4.955 (4.95) 1
4.980 4.944 (5.04) 3
4.987
P3 - - - -
P31 1.509 (1.51)1 1.519 1.505 (1.54)3 1.525
P4 1.822 (1.82)1 1.845 1.832 (1.85)3 1.845
P5 1.31 1.31 1.30 1.195
P6 0.97,1.17 0.98,1.18 1.97,1.16 0.97,1.17
P7 1.31 1.33 1.31 1.324
P71 0.811,(0.81)1, 0.803(0.80)1 0.785, 0.777 0.778, 0.770 (0.79)3 0.785, 0.777
P8 1.01,1.23 1.02,1.22 1.01,1.22 1.01,1.22
P9 1.15,1.28 1.15,1.28 1.14,1.28 1.15,1.27
P10 1.01,1.23 1.02,1.22 1.01,1.22 1.01,1.22
P11 1.31 1.32 1.32 1.32
P111 0.783(0.79)1,0.774 (0.78)1 0.809,0.801 0.806, 0.797 (0.81)3 0.807, 0.798
P12 1.01,1.23 1.02,1.22 1.01,1.22 1.01,1.22
P13 1.23,1.28 1.23,1.28 1.23,1.28 1.23,1.28
P14 1.12 1.12 1.12 1.12
P15 1.500 (1.50)1 1.489 1.497 (1.51)3 1.495
0.850 (0.85)3, 0.842
P151 0.854(0.86)1,0.845 (0.84)1 0.851,0.842 0.849, 0.841
(0.85)3
0.850 (0.85) , 0.842
3
P16 0.854(0.86)1,0.845 (0.84)1 0.851,0.842 0.849, 0.841
(0.85)3
1
Kobayashi 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)
Table 2. 1H-chemcal shifts of Chls a, b, d and f in acetone-d6 at 273K

72 Photosynthesis
172
P8,P10,P12 21 ,121
Chl a P14 P7
P15 1
P7
P11 P5 81
P13
71
13 17 82
32 P4 P6
P2 P1 P31
12 111 132 134 18 181
133
7 3 5 171
16 6 9 P3 10 20
131 19 4 2 15
173 14 1 8 3
P8,P10,P12
Chl b P7
172
P5
P15
P151, P16
P14 P11 81,P71, P111

P13
82 121
31 17 181
P4 P6 21
13 2 P1
3
132 P31
71 11 134 171
173 1 P2 10 20
2
131 133 14 6 9
37 15 5 18
19 P3
16 4 8 12
P15
Chl d 172
P8,P10,P12
P7
P151, P16
P14 P11 P5 81,P71, P111
P13
121 1
82 2
17 18 1
P4 P6 P31 71
132 P1
133 11 P2 134 18
31 3 5 20
7 10 171
131 17 19 14 16 1 92 P3
3
15
6 48 12 13
P8,P10,P12 P7 P15
Chl f P14
P11
P5,P13 1 1
8 ,P7 , P111
P151, P16
82
P9 71
31 P6 1
17 1 12
18
4 P4 P31
1 8 13 P1 13 171 172
32 P2 5 132
7 20
21 133 10 15 18
173 4 P3 12
131 19 1416 11 6 9 3 2
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10
ppm
Chemical Shifts (ppm)
Figure 12. NMR spectra of Chls a, b, d and f measured in acetone-d6 at 273 K. Signals corresponding to 13C atoms of
the molecules are labeled. The peak at 30-32 ppm is acetone.
http://dx.doi.org/10.5772/55460
IUPAC no. of
carbon atom
1 155.47(155.46)1 159.43 151.47(151.81)2 151.02
2 136.28(136.24)1 136.98 147.66(147.33)2 136.49
21 12.65(12.70)1 12.33 12.58(11.75)2 189.27
3 139.76(139.68)1 142.53 134.77(135.12)2 138.17
31 131.30(131.29)1 130.83 189.59(189.54)2 130.22
32 120.33(120.33)1 120.80 - 126.36
4 148.96(148.99)1 150.15 146.00(146.36)2 150.20
5 100.58(100.51)1 103.63 106.24(104.41)2 105.39
6 152.78(152.80)1 157.25 152.27(152.55)2 149.24
7 134.68(134.65)1 140.76 136.19(136.31)2 133.35
71 11.15(11.16)1 188.62 11.25(11.39)2 11.17
8 145.08(145.02)1 148.32 145.02(145.13)2 143.83
81 19.91(19.88)1 19.51 19.94(20.12)2 19.87
82 18.12(18.18)1 19.91 18.12(18.12)2 18.07
9 146.74(146.74)1 149.29 148.28(148.64)2 144.59
10 108.50(108.53)1 111.53 107.68(107.8)2 107.82
11 148.34(148.34)1 149.94 149.97(150.35)2 159.99
12 134.57(134.46)1 138.66 136.50(136.63)2 132.12
121 12.65(12.66)1 12.47 12.79(12.89)2 12.82
13 131.53(131.41)1 132.13 132.76(133.04)2 131.19
131 190.30(190.37)1 190.66 190.49(190.50)2 190.68
132 66.00(65.95)1 65.77 66.19(66.47)2 66.13
133 171.32(171.36)1 171.04 171.20(171.33)2 173.30
134 52.67(52.71)1 52.79 52.79(52.84)2 52.83
14 162.55(162.58)1 164.11 162.30(162.68)2 168.67
15 106.35(106.27)1 105.94 106.82(107.04)2 106.03
16 156.50(156.54)1 160.38 158.12(158.60)2 163.04
17 50.98(50.92)1 51.14 51.54(52.05)2 51.74
171 30.28(30.03)1 30.28 30.66(30.64)2 30.90
172 30.66(30.09)1 30.66 31.27(31.37)2 30.35
173 173.29(173.39)1 173.35 173.26(173.45)2 189.25
18 49.73(49.69)1 49.65 49.44(49.80)2 49.26
181 23.01(23.88)1 23.68 24.13(24.25)2 24.20
19 169.69(167.74)1 168.03 168.09(168.34)2 171.15
20 93.78(93.79)1 93.86 95.26(95.36)2 97.60
P1 61.31(61.32)1 61.42 61.33(61.67)2 61.35
P2 120.17(119.12)1 119.19 119.13(119.53)2 119.18
P3 142.46(142.48)1 143.42 142.49(142.70)2 142.49
P31 16.11(16.10)1 16.16 16.11(16.40)2 16.13
P4 40.19(40.19)1 40.23 40.19(40.46)2 40.20
P5 25.54(25.52)1 25.57 25.53(25.86)2 25.55
P6 37.11 37.14 38.11(37.40)2 37.11
P7 33.43(33.44)1 33.44 33.22(33.47)2 33.50
P71 20.03(20.02)1 20.03 19.98(20.19)2 20.02
P8 37.94 37.95 37.94(38.21)2 37.94
P9 25.04(25.06)1 25.06 25.04(25.25)2 25.04
P10 38.01 38.02 38.01(38.26)2 38.01
P11 33.22(33.23)1 33.25 33.42(33.65)2 33.42
P111 19.98(19.98)1 19.99 20.02(20.23)2 19.98
P12 37.87 37.89 37.88(38.14)2 37.88
P13 25.49(25.52)1 25.49 25.49(25.64)2 25.48
P14 40.00(39.98)1 40.00 40.00(40.27)2 40.00
P15 28.65(28.66)1 28.65 28.65(28.85)2 28.65
P151 23.013 (28.66)1 23.014 23.015 (23.15)2 23.006
P16 22.903 (22.91)1 22.914 22.905 (23.07)2 22.906
1
Kobayashi et al. (2000), 2Miyashita et al. (1997)
3-6
assignment interchangeable
Table 3. 13C-chemcal shifts of Chls a, b, d and f in acetone-d6 at 273K

74 Photosynthesis
The siμnals oλ -CH oλ Chls a, d and f show almost the same chemical shiλts, . ppm,
. ppm and . ppm, respectively, indicatinμ the interaction between the -CHO and
-CH moieties in Chls d and f are neμliμibly small, and hence the -CHO substituent is
not so close to the -CH moiety in Chls d and f. The chemical shiλts oλ -vinyl carbons
in Chls a and b are almost identical . - . ppm , but Chl f shows a sliμht but
siμniλicant downλield shiλt . ppm , suμμestinμ that the λormyl substituent in Chl f is
positioned very close to the -vinyl μroup, most probably on C . The siμnals oλ -CHO
moiety oλ Chls d and f exhibits almost the same chemical shiλt . ppm and .
ppm , but a sliμht upλield C-λormyl siμnal is observed at . ppm in Chl b, indicatinμ
that the environment oλ -CHO in Chls d and f is very similar, supportinμ that the -CHO
moieties oλ Chls d and f are positioned at the same rinμ I, while that oλ Chl b is at rinμ II.
. . . NOESY
Two-dimensional NMR spectra provide λurther inλormation about a molecule than one-
dimesional NMR spectra. NOESY is one oλ several types oλ two-dimensional NMR, where
the nuclear Overhauser eλλect NOE between nuclear spins is used to establish the
correlations. The cross-peaks in the two-dimensional spectrum connect resonances λrom
spins that are spatially close to each other.
To obtain λurther evidence λor the structural identity oλ Chl f, the siμnals were investiμat‐
ed usinμ NOESY spectra. First oλ all, we had better see well-deλined coherent correla‐
tions on the NOESY spectrum oλ Chl a. Here, we will trace the coherent correlations λrom
meso- -H, because Chl f posseses -CHO most probably at C near to C . Coherent
correlations can be easlily traced λrom -H on the NOESY spectrum oλ Chl a Fiμ. ,
where the siμnal oλ -H at . ppm shows three cross peaks with the siμnals oλ -H
at . ppm, -H at . ppm, and -H at . ppm. Good coherent correlations can
also be traced λrom meso- -H and meso- -H the siμnal oλ -H at . ppm shows
three cross peaks with the siμnal oλ -H at . ppm, -H at . ppm, and -H at
. ppm, and the siμnal oλ -H at . ppm shows two cross peaks with the siμnal
oλ -H at . ppm and -H at . ppm. In Chl b and Chl d, similar nice correla‐
tions are seen Fiμ. .
To obtain λurther evidence λor Chl f, a NOESY spectrum is illustrated in Fiμ. . “s

expected, nice coherent correlations can be traced λrom meso- -H, the siμnal oλ -H at
. ppm shows three cross peaks with the siμnal oλ -H at . ppm, -H at .
ppm, and -H, most probably assiμned to -CHO moiety, at . ppm. The siμnal oλ
meso- -H at . ppm shows three cross peaks with the siμnal oλ -H at . ppm, -
H at . ppm, and -H at . ppm. Similarly, coherent correlations oλ meso- -H with
-H . ppm and -H . ppm are clearly seen. These results indicate that the
C- methine, amonμ the three methines, is nearest to the -CHO moiety, supportinμ the
substitution position by -CHO is most likely at C in Chl f.
http://dx.doi.org/10.5772/55460
Chl a Chl b Chl d 5 10 Chl f 10 5 20

10 5 20 31 ppm 510 20 20 31 ppm
ppm ppm
0.0 0.0 82 1.5
0.5 0.5 0.5
82-10 2.0
1.0 1.0
1.0 181 181-20 2.5
181-20 1.5 181-20 1.5
82-10 1.5 1812 71 3.0 181
82 8 2.0 71-5 2.0
181 82-10 121-10
2.0 121 3.5
181-20 2.5 2.5
2.5 4.0 3.0
1 3.0
81-10 21-20 71-5
21 12 -10 21 4.5 71 3.5
711
71-5 3.0
121 21-20
3.5 81 18 18-20 121 121-10
4.0 5.0 81 4.0
21 3.5 1 81-10
12 21-20 8 4.5 4.5
4.0 18 81-10 5.5 18
121-10
1H-NMR
1H-NMR
1H-NMR
1H-NMR
18-20 5.0 18-20 5.0
4.5 6.0
18 5.5 5.5
18-20 5.0 6.5
6.0 6.0
32 7.0 32-31
5.5
32-5 6.5 32 6.5
6.0 7.0 7.5 32-5 7.0
32 32-31 8.0
32 6.5 7.5 7.5
32-5 31 8.0 8.5 8.0
7.0 31-5
31-5 8.5 8.5
7.5 9.0 31
9.0 9.0
8.0 9.5
9.5 9.5
31 10.0
31-5 8.5 10.0 5
5-31 10.0
9.0 10.5 10.5
10.5
9.5 11.0 11.0
5 71 71-5 11.0
5-31 11.5 31 31-5 11.5 21
10.0 21-20 11.5
12.0 12.0
10 9 8 ppm 10 9 ppm 12.0
10 9 ppm 10 9 ppm
1H-NMR 1H-NMR 1H-NMR 1H-NMR
Figure 13. 1H-1H-NOESY spectra of Chls a, b, d and f measured in acetone-d6 at 273 K.
. . . HSQC
HSQC is a two-dimensional inverse correlation technique that allows λor the determination oλ
connectivity between two diλλerent nuclear species, and HSQC is selective λor direct couplinμ.
“s illustrated in the H- C HSQC spectra oλ Chls a, b, d and f Fiμ. , all substituents on the
macrocycle show the correspondinμ cross peaks. The results support that one methyl μroup
oλ Chl a is replaced with a λormyl one in Chl f.
. . . HMBC
HM”C is also a two-dimensional inverse correlation method that allows λor the determination
oλ connectivity between two diλλerent nuclear species like HSQC, but HM”C μives lonμer ranμe
couplinμ - bond couplinμ than HSQC.
Three meso-Hs in Chl a exhibit one to three cross peaks as seen in Fiμ. “. The λormyl-H in
Chls b, d and f shows two to λour cross peaks. For example, in the H- C HM”C spectrum oλ
Chl f, the H-siμnal λor -CHO has λour cross peaks, one is with the siμnal λor -vinylic carbons,
one is with the siμnal λor C carbon, and the other two are with the siμnal λor -CHO carbon,
where the cross peak split is due to the direct couplinμ. Conclusively, the Chl f-like piμment
isolated λrom the strain KC has been identiλied as Chl f, namely, -desmethyl- -λormyl-Chl a
[ -CHO]-Chl a .
76 Photosynthesis
1 1
81 12 2 71 181 82 121 21 82181
132 Chl a 31 132 32 81 17 Chl b
20 31 32 171
32
10 5 32 18 17 71 5 10 20 18
ppm ppm
71
121-121 71-71 10 121 21 21-21
10
21 121 121-121 82-82
21-21 82-82 20 81 82 20
82 81-81 181-181 30
81 17 1 81-81 181-181 30 181 18 18-18 40
181 171-171 40 50
17-17 60
18 17 132
50 70
17 132-132
13C-NMR
13C-NMR
18-18 17-17 60 80
20 5-5 90
132 70 32-32 100
132-132 5 20-20
32-32 110
80
10 32 10-10 120
90 31 130
20 20-20
5 100 31-31 140
5-5 150
10 110 160
10-10 170
32 32-32 32-32 120 71-71 180
31 31-31 130 71 190
200
9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm
1
H-NMR
13 12 11 10 9 8 7 6 5 4 3 2 1 ppm
1H-NMR
81 21 121 181 82 81 121 71 181 82

18 Chl d 132
Chl f
71 21 10 5 20 1 171
31 510 20 132 3 32 1817
71 17 12171 ppm
21 ppm 121-121 71-71 2 2
21-21 71-71 82 8 -810
121 10 81 181 20
8 2 121-121 82-82
81 1 171 81-81 30
20 18-18
1
17 -17 1
40
18 81-81 18
181-181 30 181-181 50
17 60
18-18 40 132 17-17
18 70
132-132 80
13C-NMR
13C-NMR
17 50 20-20
20 90
17-17 60 100
132 5
70 10 5-5 110
132-132 10-10 120
80 32
130
31 31-31 32-32 140
90
20 5-5 20-20 150
100 160
5
10 110 170
10-10 21-21 180
120 21 190
12 11 10 9 8 7 6 5 4 3 2 1 ppm
1H-NMR 11 10 9 8 7 6 5 4 3 2 1 ppm
1H-NMR
Figure 14. 1H-13C-HSQC spectra of Chls a, b, d and f measured in acetone-d6 at 273 K. □ in Chl d: δC189.59 (folded
from outside the spectral window)
71 31 21
10 5 20
ppm ppm ppm ppm
130
10 7 110
7-71 3 135 115
20
140
3-31
30 140 120
40 4-31 125
145
6 150 4 130
18 50 6-71 2 31
18-20 2-31 150 31-21 135
60
13C-NMR
13C-NMR
13C-NMR
13C-NMR
160 140
70 155
145
80 160
170 1 150
90 1-21 155
165
100 160
180 170
110 165
120 175 170
190
130 175
180
7,12 7-5 180
140
200 185 185
12-10 150 21-21
31-31 21 190
160 31 190
9.5 9.0 8.5 ppm
12.0 11.0 ppm 12.0 11.5 11.0 ppm 12.0 11.5 11.0 ppm
1H-NMR 1H-NMR 1H-NMR 1H-NMR
Figure 15. 1H-13C-HMBC spectra of Chls a, b, d and f measured in acetone-d6 at 273 K.

http://dx.doi.org/10.5772/55460
. . Redox potentials
To understand the charμe separation in the RC, electrochemical characterization oλ chloro‐

phylls is oλ crucial importance. In this section, the redox potentials oλ Chls and Phes in vitro
are presented.
“cetonitrile “ldrich, anhydrous μrade water < ppm was deoxyμenated and dried beλore
use. The solvent was subjected to λreeze-pump-thaw cycles at least three times under about
-
torr. Under a nitroμen atmosphere, the deoxidized solvent was then dried λor h with
activated molecular sieves “ / , Wako , pretreated in vacuo at K over h. Tetra-n-
butylammonium perchlorate ”u NClO , T”“P “ldrich, Electrochemical μrade > . % ,
was used as the supportinμ electrolyte, which had been recrystallized λrom methanol solution
and then dried in vacuo at K over h.
The redox potentials oλ chlorophylls were measured by square wave voltammetry SWV . The
siμnal-to-noise ratio oλ SWV is μenerally better than that oλ CV, especially λor measurinμ redox
couples at such low concentration ca. . mM as in the present case Cotton et al. ,
Wasielewski et al. . Measurements were done with an “LS model “ electrochemical
analyzer. Parameters λor SWV were Vstep = . mV, “C siμnal Vpulse = mV, and p-p at Hz.
The measurements were carried out in an air-tiμht electrochemical cell containinμ a small
compartment λor a sample solution ca. . mM equipped with a μlass λilter that can be
deμassed and λilled with dry N . “ platinum disk electrode with . mm in diameter outer
diameter mm was used as the workinμ electrode, and a platinum black wire λabricated in
the small compartment internal diameter . mm as the counter electrode. “n “μ/“μCl
electrode, chosen λor μood reproducibility despite possibility oλ junction potential, was
connected throuμh a salt bridμe to the outer electrolytic solution oλ the small components. “λter
measurement, the redox potentials oλ the λerrocene-λerrocinium were measured as + . V vs.
“μ/“μCl in acetonitrile.
Typical square wave voltammoμrams SWVs λor Chls a, b, d and f in acetonitrile are illustrated
in Fiμ. . Four peaks are observed in each voltammoμram and the potentials in anodic sweep
and cathodic sweep data not shown are identical to each other, indicatinμ that the λour redox
reactions are reversible. Similar trends are observed λor Phes a, b and d data not shown . The
measurement λor Phe f is now underway.
In Table are summarized the redox potentials λor Chls a, b, d, f, Phes a, b and d. Chl d
shows hiμher oxidation potentials than Chl a, lower than Chl b, and much lower than Phe
a, Phe b and Phe d. Chl f exhibits hiμher oxidation potentials than Chls a, d, and lower
than Chl b. The results can be explained by invokinμ the inductive eλλect oλ substituent
μroups on the macrocycle, because the redox potentials oλ chlorophylls are sensibly aλλected
by the nature oλ substituent μroups on the π-electron system Fuhrhop , Watanabe
and Kobayashi , Kobayashi et al. .
The -CHO substituent on Chls b, d and f is an electron-withdrawinμ μroup →CHO , and hence
reduces the electronic density in the π-system oλ chlorophyll. The replacements oλ -CH at C
or C oλ Chl a by →CHO to yield Chl b or Chl f cause the macrocycle to be electron poor, thus
78 Photosynthesis
renderinμ the molecule less oxidizable E ox Chl b, f > Chl a . Similarly, replacement oλ -
CH=CH at C oλ Chl a by →CHO to yield Chl d makes the λirst oxidation potential, E ox, more
positive than that oλ Chl a E ox Chl d > Chl a . Thereλore, the E ox order becomes Chls b, d, f >
Chl a see Fiμs and . When one pays attention to the μroup oλ -CH at C oλ Chl d and the
μroup oλ -CH=CH at C oλ Chl b or C oλ Chl f, the -CH μroup is more electron-donatinμ
←CH , thus makinμ the macrocycle oλ Chl d more electron rich, and hence its oxidation
potential less positive Chls b, f > d the E ox order results in Chls b, f > Chl d > Chl a. “s expected
λrom the inductive eλλect oλ substituent μroups, Chls b and f show the almost the same E ox
values. Consequently, as seen in Fiμ. , the E ox order results in Chl b > Chl f > Chl d > Chl a
a little hiμher oxidation potential oλ Chl b than that oλ Chl f, mV, cannot be explained λrom
the primitive way used here. π
Figure 16. Square wave voltammograms of Chls a, b, d and f in acetonitrile.
E2red E1red E1ox E2ox E1ox - E1red

V vs. SHE
Chl a -1.46 -1.12 0.81 1.04 1.93
Chl b -1.41 -1.02 0.94 → 1.15 1.96
π →
Chl d -1.27 -0.91 0.88 1.09 1.79
→
Chl f -1.12 -0.75 0.92 1.13 1.67
←
Phe a -1.00 -0.75 1.14 1.49 1.89
Phe b -1.05 -0.64 1.25 1.58 1.89
Phe d -0.87 -0.63 1.21 1.50 1.84
Table 4. Redox potentials of Chls a, b, d, f, Phes a, b and d in acetonitrile

http://dx.doi.org/10.5772/55460
-1.5
Chl a /Chl a -
-1120mV Chl b /Chl b -
-1.0 Chl d /Chl d -
Potential E / V vs. SHE
-1020mV
-910mV -
Chl f /Chl f
-750mV
-0.5
1.93 V
1.96 V
1.79 V
0 1.67 V
+0.5
+810mV
+880mV
Chl a /Chl a + +920mV +940mV
Chl d /Chl d +
+1.0 Chl f /Chl f + Chl b /Chl b +
Figure 17. Schematic comparison of redox potentials of Chls a, b, d and f in acetonitrile.
The redox behavior oλ a compound is related to the enerμy levels oλ its molecular orbitals E ox
is intimately related to the hiμhest occupied molecular orbital HOMO and E red to the lowest
unoccupied molecular orbital Watanabe and Kobayashi Hanson . The order oλ
HOMO enerμy levels well parallels the E ox values, while the correlation between the LUMO
levels and the E red values is less conspicuous see Fiμ. in Watanabe and Kobayashi .
“s clearly seen in Fiμ. , the order oλ absolute values oλ the λirst reduction potentials, E red, oλ
Chls a, d and f is Chl a > Chl d > Chl f, which can be well explained by substituent inductive
eλλect, like E ox as mentioned above. This simple rule, however, does not hold λor Chls d and
f their values oλ E red are remarkably more positive than those oλ Chls a and b compared to
Chl a, Chl b is harder to oxidize by mV, and easier to reduce by mV, namely, similar
in deμree, while Chls d and f peculiarly easier to reduce by mV and mV. The λindinμs
indicate that the inductive eλλect oλ substituent μroups at rinμ II conspicuously appears λor
E red. “nyway, this irreμularity may come λrom the λact that the LUMO enerμy levels are to a
lesser extent correlated with the E red values.
The primary redox potential diλλerence, ΔE = E ox - E red, seen in Fiμ. can be taken as an index
λor the QY excitation enerμy Watanabe and Kobayashi Hanson . For example, ΔE
λor Chl a is . eV in Fiμ. , which corresponds to the QY excitation wavelenμths to a certain
extent, - nm λor Chl a in Fiμ. . Similarly. ΔE = . eV, . eV and . eV λor Chls b,
80 Photosynthesis
d and f also correlate to the QY wavelenμths, - nm, - nm. and - nm.

Pheophytins also behave in a similar λashion.
In , the domination oλ inductive eλλects oλ the central metal over a conjuμative macrocycle
has λirst been λormulated Gouterman . The redox potential oλ chlorophyll shows a
systematic shiλts with the electroneμativity oλ the central metal, and such a trend is rationalized
in terms oλ an electron density decrease in the chlorin π-system by the presence oλ an electron
neμative metal in the center oλ chlorophylls Watanabe and Kobayashi Hanson
Noy et al. . Inspection oλ Table demonstrates that such a trend is essential λor the pair
oλ Chls and Phes the electroneμativity oλ . λor H is siμniλicantly hiμher than that oλ . λor
Mμ, which renders Phes more diλλicult to oxidize than the correspondinμ Chls.
. Evolutionary and ecological aspects of chlorophylls
. . Diversification of chlorophylls during the evolution of photosynthetic organisms
Chlorophylls are distributed amonμ oxyμenic photosynthetic orμanisms, includinμ cyanobac‐

teria, alμae and terrestrial plants Falkowski et al. . It is μenerally accepted that plastids
λirst arose by endosymbiosis between photosynthetic prokaryotes ancestral to present
cyanobacteria and non-photosynthetic eukaryotic hosts Fiμ. . There are two diλλerent types
oλ hypothesis λor the evolution oλ Chl b. One is based on vertical transλer oλ the μene λor Chl b
biosynthesis, which includes the lost oλ the μene. The other is based on the lateral μene transλer
λor Chl b biosynthesis. In the hypothesis oλ the λormer one, the ancestral cyanobacteria
contained both Chls a and b, and the μene λor Chl b biosynthesis had been lost. In many
cyanobacterial linaμe as well as the linaμes oλ the red alμae and μlaucophytes, the μene had
been lost, whereas a λew linaμe oλ cyanobacteria, Prochloron and Prochlorothrix and the μreen
alμae and terrestrial plants retained it. This hypothesis is partly supported by the hiμh
homoloμy oλ the enzyme λor Chl b biosynthesis C“O Tomitani et al. . In the latter
hypothesis, the μene λor Chl b biosynthesis was λirstly evolved in the ancestor oλ μreen alμae
or cyanobacteria, and the μene was transλerred into diλλerent linaμes. This is supported by the
λact that one μene transλer was enouμh λor the acquisition oλ Chl b biosynthesis in cyanobace‐
rium. The secondary endosymbioses oλ μreen alμae μave rise to euμlenophytes, chlorarach‐
niophytes and "μreen" dinoλlaμellates, which were containinμ Chls a and b. The secondary or
tertiary plastids oλ cryptophytes, haptophytes, heterokonts and dinoλlaμellates contain Chl c
in addition to Chl a. Most oλ present-day cyanobacteria contain only Chl a, and only a λew
μenera are known to keep Chl b Prochloron and Prochlorothrix . This suμμests that Chl b had
been lost in polyphyletic lineaμe durinμ the evolution oλ cyanobacteria. Some cyanobacteria
contain divinyl chlorophylls DVChls a and b Prochlorococcus , Chl d Acaryochloris and Chl
f strain KC and Halomicronema hongdechloris . These chlorophylls are only λound in cyano‐
bacteria, and it has yet to be revealed when and how they acquired these piμments.
http://dx.doi.org/10.5772/55460
Figure 18. The distribution of various chlorophylls among oxygenic photosynthetic organisms.
. . Ecology of the red-shifted chlorophylls
“cquisition oλ new or additional chlorophylls by photosynthetic orμanisms is thouμht to be

an adaptation to the liμht quality oλ their niches. The two red-shiλted chlorophylls, Chls d and
f, are also thouμht be acquired and selected by a part oλ cyanobacteria λor their μrowth and
survival by sustaininμ oxyμenic photosynthesis usinμ the liμht available at their niches.
Chl d-containinμ cyanobacterium, Acaryochloris marina, was λirstly λound as a minor symbionts
in colonial ascidians which had larμe numbers oλ cyanobacterial symbionts, Prochloron, which
82 Photosynthesis
contain Chls a and b as their photosynthetic chlorophylls Miyashita et al. . Subsequently,

Acaryochloris spp. has been λound on the surλace oλ coastal macroalμae Murakami et al. ,
under those colonial ascidians K(hl et al. , and in coastal microbial mats at a saline lake
Miller et al. . In all oλ those niches, Acaryochloris spp. competes λor liμht λor their oxyμenic
photosynthesis. However, due to the possession oλ Chl d as primary chlorophyll, Acaryochlo‐
ris spp. can absorb and utilized λar-red liμht λrom nm to nm λor oxyμenic photosynthesis
Miyashita et al. . The ability has advantaμe λor their μrowth and survival, since another
oxyμenic phototrophs which contain Chl a as their primary chlorophyll cannot absorb and
utilize the liμht.
Chl f-containinμ cyanobacterium was λirstly reported in microbial mat, where the competition
λor the liμht also occurs amonμ the phototrophs Chen et al. . While the distribution and
detailed niche oλ Chl f-containinμ cyanobacteria have not been elucidated yet, Chl f must
contribute to the survival oλ those cyanobacteria. Since Chl f in cyanobacteria also absorbed
λar-red liμht around nm and Chl f-possessinμ cyanobacteria can μrow under λar-red LED
liμht as a sole liμht source Chen and ”lankenship .
Acknowledgements
We thank Dr. Nobuaki Ishida Ishikawa Preλectural Univ. , Dr. Yoshihiro Shiraiwa and Dr.
Koji Iwamoto Univ. Tsukuba λor their invaluable help. This work was supported in part by
Special Project oλ Orμanization λor the Support and Development oλ Strateμic Initiatives Green
Innovation Univ. Tsukuba to M.K.
Author details
Masami Kobayashi , Shinya “kutsu , Daiki Fujinuma , Hayato Furukawa ,

Hirohisa Komatsu , Yuichi Hotota , Yuki Kato , Yoshinori Kuroiwa , Tadashi Watanabe ,
Mayumi Ohnishi-Kameyama , Hiroshi Ono , Satoshi Ohkubo and Hideaki Miyashita
Institute oλ Materials Science, University oλ Tsukuba, Tsukuba, Japan
Institute oλ Industrial Science, University oλ Tokyo, Tokyo, Japan
Research Center λor Math and Science Education, Orμanization λor “dvanced Education,
Tokyo University oλ Science, Tokyo, Japan
National Food Research Institute, Tsukuba, Japan
Graduate School oλ Human and Environmental Studies, Kyoto University, Kyoto, Japan
http://dx.doi.org/10.5772/55460
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Physicochemical Properties of Chlorophylls in Oxygenic

Masami Kobayashi, Shinya Akutsu, Daiki Fujinuma,

Additional information is available at the end of the chapter

Chlorophylls are essential components in oxyμenic photosynthesis, and chlorophyll Chl a

In , a unique cyanobacterium, Acaryochloris marina, with Chl d as the dominant chlorophyll

H3C 17 15 CH 3 Chl b Mg CH3 CH=CH2 CHO H COOCH3

In , a red-shiλted chlorophyll was discovered in a methanolic extract oλ Shark ”ay

λrom the strain KC Succ

Physicochemical Properties of Chlorophylls in Oxygenic Photosynthesis… 49

. Chlorophylls in oxygenic photosynthesis

. . Popular chlorophylls: Chlorophylls a and b

. . Recently discovered chlorophylls in algae

. . . Chlorophyll d in a cyanobacterium, Acaryochloris marina

. . . Chlorophyll f in a cyanobacterium, strain KC , isolated from Lake Biwa

“ Chl f-producinμ cyanobacterium, strain KC , was discovered by Miyashita, one oλ the

cyanobacteria in λreshwater environment. Chl d had been only produced by cyanobacte‐

. . Specially-tailored chlorophylls associated with reaction centers

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

. . . . Chlorophyll a' and P7

. . . . Chlorophyll d' and P7 in Acaryochloris marina

. . . . Evolutionary relationship between chlorophylls and PS-I type reaction centers

H epimerization H epimerization epimerization

Figure 4. Bacterlochlorophyll g/g' and their derivatives functioning in natural photosynthesis.

. . . Pheophytin a as the primary electron acceptor in PS II

. Physicochemical properties of chlorophylls in vitro

. . . Mixture of chlorophylls and pheophytins

. . . Pigment extract from A. marina

. . . Pigment extract from strain KC

. . Absorption spectra in four solvent varieties

400 500 600 700 800

(A) Chl a Synechosystis

Phe a Chl a’ ×20

(C) Acariyochloris Chl d

(D) Chl a Strain KC1

diethyl ether acetone methanol benzene

454.0 (A’) 458.7 (B’) 469.4 (C’) 458.3 (D’)

452.4 686.2 (A”) 451.7 691.4 (B”) 400.8 455.5

433.4 (E’) 434.4 (F’) 436.0 (G’) 440.0 (H’)

654.4 653.3 653.9 656.9

(E”) (F”) (G”) (H”)

421.0 383.7 697.3

696.9(E”’) (F”’) (G”’) (H”’)

409.3 409.3 410.0 415.0 701.9

Compound λmax,blue [nm] λmax,red [nm]

(ε[103 M-1cm-1]) (ε[103 M-1cm-1]) Ref

Chl a 428.4 660.3 Watanabe et al. (1984)

(115) (89.8) ibid.

429.1b 661.6b ibid.

(100)b (81.3)b ibid.

432.5d 665.4d ibid.

(101)d (79.7)d ibid.

429.0 660.9 Kobayashi et al.(2006)

(1.00)a (0.775)a ibid.