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Modi ?cation of the esterifying farnesyl chain in light-harvesting bacteriochlorophylls in green sulfur photosynthetic bacteria by supplementation of 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol

Yoshitaka Saga a ,b ,*,Keisuke Hayashi a ,Keiya Hirota a ,Jiro Harada c ,Hitoshi Tamiaki d

Department of Chemistry,Faculty of Science and Engineering,Kinki University,Higashi-Osaka,Osaka 577-8502,Japan b

PRESTO,Japan Science and Technology Agency,Kawaguchi,Saitama 332-0012,Japan c

Department of Medical Biochemistry,Kurume University School of Medicine,Kurume,Fukuoka 830-0011,Japan d

Graduate School of Life Sciences,Ritsumeikan University,Kusatsu,Shiga 525-8577,Japan

A R T I C L E I N F O

Article history:

Received 13March 2015

Received in revised form 24April 2015Accepted 7May 2015

Available online 28May 2015

Keywords:Biosynthesis

Bacteriochlorophyll Chlorosome

Green sulfur bacteria Photosynthesis

A B S T R A C T

Green sulfur photosynthetic bacteria Chlorobaculum (Cba .)tepidum and Cba .limnaeum were cultivated with the supplementation of 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol by two methods to biosynthesize unnatural chlorosomal bacteriochlorophyll (BChl)pigments possessing an (un)saturated bond at the terminus of the esterifying chain.The supplementation of the acetone solutions of the exogenous alcohols every 24h to Cba .tepidum (denoted as Method A)successfully produced unnatural BChls c esteri ?ed with the alcohols in vivo .The ratios of unnatural BChls c esteri ?ed with 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol over the total BChls c in Cba .tepidum were not so different from each other,indicating that terminal unsaturated moieties in the exogenous alcohols barely affected the last reaction in the BChl c biosynthesis catalyzed by the BChl c synthase.Cba .tepidum grown in a liquid culture that initially contained 9-decyn-1-ol and 9-decen-1-ol (denoted as Method B)also accumulated BChls c esteri ?ed with these alcohols;the relative ratios of the unnatural BChls c were analogous to those obtained by Method A at the corresponding ?nal concentrations.In contrast,Cba .limnaeum did not synthesize BChl e esteri ?ed with the three exogenous alcohols.The incorporation of unsaturated bonds into the esterifying moieties of BChl c by a biosynthetic reaction will be useful as structural probes for the investigation of pigment interactions and the biogenesis of chlorosomes as well as the scaffolds for bioorthogonal modi ?cations.

1.Introduction

In the early stage of photosynthetic reactions,light-harvesting antenna complexes capture sunlight and transfer its energy to a reaction center complex,in which charge separation takes place.Pigment organization in photosynthetic antenna complexes plays an important role in ef ?cient light-harvesting and excitation energy transferring processes [1–3].The highly ordered pigment assemblies in photosynthetic light-harvesting complexes provide a guide to design arti ?cial photosynthetic devices [4,5].

Most photosynthetic light-harvesting complexes consist of proteins and photofunctional pigments such as chlorophyll(Chl)s,

bacteriochlorophyll(BChl)s,and carotenoids.In these complexes,proteins function as the scaffolds of pigments to regulate their arrangements.The only exception is found in the light-harvesting antenna complexes of green photosynthetic bacteria called chlorosomes [6–9],whose main photofunctional parts are the self-assemblies of 100,000–200,000BChls c ,d ,e ,and f molecules [10,11].These BChl self-assemblies form organized mesoscopic structures in chlorosomes by interactions among BChl pigments [12–17].No protein participates in the BChl assemblies in chlorosomes.Such unique architecture has attracted considerable attention,not only because of its fundamental importance in photobiological and supramolecular chemistry but also because of the developments of arti ?cial photosynthesis.

The molecular structures of BChls c and e in green sulfur photosynthetic bacteria are shown in Fig.1.Both pigments possess a chlorin macrocycle (17,18-dihydroporphyrin)despite the name bacteriochlorophyll,and the 20-methyl group is attached to the chlorin ring [18,19].The structural difference between BChls c and e

*Corresponding author at:Department of Chemistry,Faculty of Science and Engineering,Kinki University,Higashi-Osaka,Osaka 577-8502,Japan.Tel.:+81667305880;fax:+81667232721.

E-mail address:saga@chem.kindai.ac.jp (Y.Saga).

Journal of Photochemistry and Photobiology A:Chemistry 313(2015)44–51

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology A:

Chemistry

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j p h o t o c h e

is the substituents at the 7-position;these substituents are the methyl and formyl groups in BChls c and e .The 7-formyl group in BChl e is responsible for the spectral properties of both the monomeric and aggregated forms,which are different from those of BChl c [18–20].

BChls c and e in green sulfur bacteria are majorly esteri ?ed with farnesol at the 17-propionate residue (hereafter denoted as BChls c F and e F ).Hydrophobic interactions among the farnesyl moieties of BChls c F and e F play important roles in the formation of pigment self-assemblies in chlorosomes [21]in addition to interactions via the coordination bond of a 31-hydroxy group in one BChl to a central metal of another BChl and the hydrogen bond between the 31-hydroxy group and a 13-keto group in a third BChl [8,9,22,23].However,little structural information is available on the aggrega-tion of the esterifying chains of BChl pigments in chlorosomes,although the interactions among the three essential parts (central magnesium,31-hydroxy,and 13-keto group)in chlorosomal BChls have been clari ?ed.This enigma is mainly ascribable to the spectroscopic silence of the 174-esterifying chains in

chlorophyllous pigments [21].Therefore,modi ?cation of the esterifying chains from naturally occurring farnesyl to unnatural moieties in chlorosomal BChl pigments will be a clue to investigation of these unclear regions in BChl self-aggregates in chlorosomes.From these viewpoints,the esterifying alcohols of chlorosomal BChl pigments have been substituted in vivo [24–29].Miller and coworkers ?rst reported the composition changes of the esterifying alcohols in BChl c in the ?lamentous anoxygenic phototroph Chloro ?exus (Cfx .)aurantiacus and the green sulfur bacterium Chlorobaculum (Cba .)tepidum by the supplementation of hydrocarbon alcohols such as phytol and geranylgeraniol,which are known to exist intrinsically in these species as minor esterifying alcohols of BChl c ,in their cultures [24,25].In contrast,we successfully biosynthesized BChl c esteri ?ed with unnatural alcohols such as a ,v -diols and v -halogenated alkanols [27,28].Variations in the unnatural esterifying chains of chlorosomal BChl pigments will be helpful to examine the states of these chains and interactions in chlorosomes.In the ?rst part of this paper,we report the in vivo introduction of terminal alkynyl and alkenyl groups,which can function as vibrational probes and/or reactive groups for chemical modi ?cations,in the esterifying chain of chlorosomal BChls in green sulfur photosynthetic bacteria grown by the supplementation of 9-decyn-1-ol and 9-decen-1-ol.

The target of the in vivo esteri ?cation of exogenous alcohols has been limited to bacteriochlorophyllide (BChlide)c ,namely the biosynthesis of BChls c ,in only two kinds of green photosynthetic bacteria Cba .tepidum and Cfx .aurantiacus [24–29].Thus,it remains unclear whether other unnatural chlorosomal BChls (BChls d ,e ,and f )are synthesized in cells by the supplementation of exogenous alcohols.In the second part of this paper,we cultivate Cba.limnaeum ,which produces BChl e F as a major pigment,by the supplementation of 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol,and compare the compositions of BChl e biosynthesized in Cba .limnaeum with those of BChl c in Cba .tepidum .

2.Experimental

2.1.Apparatus

Visible absorption spectra were measured with a Shimadzu UV-2450spectrophotometer.Circular dichroism (CD)spectra were measured with a JASCO J-820spectropolarimeter.HPLC analysis of chlorosomal BChls was carried out with a Shimadzu LC-20AT pump and an SPD-M20A detector by the control of column temperature with a Shimadzu CTO-20AC column oven.Liquid chromatography –mass spectrometry (LC –MS)was done with a Shimadzu LCMS-2020system equipped with an electrospray ionization probe.The optical densities of liquid cultures were monitored with a Taitec colorimeter (model 518).

2.2.Growth conditions

Green sulfur photosynthetic bacteria Cba .tepidum ATCC 49652and Cba .limnaeum RK-j-1[30]were grown in liquid cultures by irradiation with ?uorescence lamps (13and 4.8m mol m à2s à1)at 43and 30 C,respectively [27,28].Pre-cultured cells in 1/100volume were inoculated into freshly prepared media.Supple-mented 9-decyn-1-ol and 9-decen-1-ol were purchased from Tokyo Chemical Industry,Co.,Ltd.and decan-1-ol was purchased from Wako Chemical Industries,Ltd.The supplementation of these commercially available alcohols was performed by two methods.In one method (hereafter denoted as Method A),1%culture volume of an acetone solution of 9-decyn-1-ol,9-decen-1-ol,or decan-1-ol was added to the liquid culture every 24h for 3–4days.The concentrations of the three alcohols in supplemental acetone solutions were 1,5,and 10mM.The concentrations of the alcohols

N N

R 12

R 7

R 813

(A)

20N N N

R 12

Mg O

R 7

O R 17

R 8

(B)

3117

174

R 17

=Fig.1.(A)Molecular structures of natural BChls c F (R 7=CH 3)and e F (R 7=CHO).

R 8=C 2H 5,n -C 3H 7,iso -C 4H 9.R 12=CH 3,C 2H 5.(B)Molecular structures of unnatural BChls c /e Dy (R 17=(CH 2)8C R CH),c /e De (R 17=(CH 2)8CH ?CH 2),and c /e Da (R 17=(CH 2)9CH 3).

Y.Saga et al./Journal of Photochemistry and Photobiology A:Chemistry 313(2015)44–51

in liquid cultures are hereafter described as?nal concentrations,

which mean the total concentrations of the exogenous alcohols at

the end of the cultivations.Additionally,neat acetone was added in

the same procedure as the supplementation of exogenous alcohols

as control experiments.In the other method(hereafter denoted as

Method B),a small amount of neat alcohols were initially

supplemented to the liquid cultures,followed by cultivation for

3–4days.

2.3.Extraction and analysis of chlorosomal BChls

BChls c and e were extracted from harvested cells with

methanol/acetone(1/1,vol/vol)and acetone,respectively,fol-

lowed by?ltration.The organic solutions containing the extracted

pigments were diluted with diethyl ether,washed with NaCl-

saturated water,and dried over anhydrous Na2SO4,followed by

dryness with nitrogen gas.The compositions of chlorosomal BChls

obtained were analyzed by HPLC using a reverse-phase column

Cosmosil5C18-AR-II(6mm i.d.?250mm).The homologs of

chlorosomal BChls were assigned by LC–MS as well as elution

patterns according to the previous reports[27,28,31–33].

3.Results

3.1.Pigment compositions in cells cultivated by Method A

Fig.2shows the typical HPLC elution patterns of BChl c in the

harvested cells of Cba.tepidum,which were grown by the

supplementation of9-decyn-1-ol,9-decen-1-ol,and decan-1-ol

by Method A as well as in normal and control(acetone-containing)

cultures.Four natural BChl c homologs esteri?ed with farnesol

(denoted as cF1,cF2,cF3,and cF4,respectively)were present in

Cba.tepidum grown under normal and controlled conditions

(Fig.2A).These homologs were assigned as31R-8-ethyl-12-

methyl-(R[E,M]),31R-8-ethyl-12-ethyl-(R[E,E]),8-propyl-12-eth-

yl-([P,E]),and31S-8-isobutyl-12-ethyl-(S[I,E])BChl c F[27,28,31].

The fraction of[P,E]BChl c F homolog consisted of a mixture of31R-

and31S-epimers.The control cultivation of Cba.tepidum barely

affected the homolog compositions of BChl c F and did not induce

additional BChl c derivatives(Fig.2B).

In contrast,new fractions can be detected in addition to natural

BChl c F in the HPLC chromatograms of BChl c extracted from cells

grown by the supplementation of9-decyn-1-ol,9-decen-1-ol,and

decan-1-ol by Method A(Fig.2C–E).The newly appeared fractions

by cultivation with9-decyn-1-ol,9-decen-1-ol,and decan-1-ol are

hereafter denoted as cDy2–cDy4,cDe2–cDe4,and cDa2–cDa4,

respectively.The on-line visible absorption spectra of these novel

pigments were the same as those of natural BChl c F homologs

(Fig.S1in the Supplementary data),indicating that their chlorin p-macrocycles are identical to those of BChl c F.Fractions cDy2–cDy4,which newly appeared by the supplementation of9-decyn-

1-ol,exhibited molecular ion peaks at m/z739.40,753.45,and

767.45,respectively,in their LC–MS analysis.These values

correspond to the calculated values for the protonated forms (MH+)of[E,E],[P,E],and[I,E]BChl c esteri?ed with9-decyn-1-ol (739.40,753.42,and767.43,respectively).These results indicate that unnatural BChl c esteri?ed with9-decyn-1-ol(denoted as BChl c Dy)was biosynthesized in Cba.tepidum.Fig.3shows a HPLC elution pattern of BChl c in chlorosomes isolated from Cba.tepidum by the supplementation of9-decyn-1-ol by Method A.BChl c Dy was also observed(the fractions cDy2–cDy4)in addition to natural BChl c F(the fractions cF1–cF4),indicating the presence of BChl c Dy in the chlorosomes.

The grown cells supplied with9-decen-1-ol and decan-1-ol also produced new sets of BChl c homologs.The molecular ion peaks of fractions cDe2–cDe4produced by the supplementation of9-decen-1-ol were741.40,755.45,and769.45,respectively,by LC–MS analysis,indicating that these pigments are[E,E]-,[P,E]-,and[I, E]-homologs of BChl c esteri?ed with9-decen-1-ol(denoted as BChl c De).In the case of the supplementation of decan-1-ol,the HPLC eluent was changed from those of the other analysis,since new fractions were eluted close to those of BChl c F.As a result,new fractions cDa2–cDa4can be detected and assigned as BChl c homologs esteri?ed with decan-1-ol(denoted as BChl c Da),based on their molecular ion peaks observed at743.35,757.05,and 771.30,respectively,by LC–MS analysis.Note that the amounts of [E,M]-homologs of BChl c Dy,c De,and c Da were signi?cantly small, since their precursor[E,M]-homolog of BChlide c is generally scarce in cells.Three kinds of supplemented alcohols,therefore,

200

150

100

Retention time / min

(E) cF2

cF3

cF4

cDa2

cF1

cDa3

cDa4

(D)

cDe2

cDe3

cDe4

(C)

cDy2

cDy3

cDy4

(B)

(A)

cF1

cF2

cF3

cF4

Fig.2.HPLC elution patterns of BChl c derivatives in Cba.tepidum under normal(A) and the control conditions(B),and by the supplementation of9-decyn-1-ol(C),9-decen-1-ol(D),and decan-1-ol(E)by Method A.The?nal concentrations of9-decyn-1-ol,9-decen-1-ol,and decan-1-ol were30m M.The pigments were eluted on a reverse-phase column5C18-AR-II(6mm i.d.?250mm)at a?ow rate of1.0mL/ min with methanol/water(94/6,vol/vol)in the chromatograms(A)–(D)and methanol/water(85/15,vol/vol)in the chromatogram(E).The chromatograms were recorded at435nm and normalized at the peaks of the fractions cF2([E,E]BChl c F).

46Y.Saga et al./Journal of Photochemistry and Photobiology A:Chemistry313(2015)44–51

were successfully incorporated into BChl c in Cba .tepidum .The homolog compositions of these unnatural BChls c ,namely BChl c Dy ,BChl c De ,and BChl c Da ,were analogous to those of coexisting BChl c F in the harvested cells (Fig.2C –E).

Table 1shows the relative ratios of unnatural BChls c in the cells grown by the supplementation of 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol over the total BChl c .The relative ratios of BChl c Dy ,c De ,and BChl c Da were 15.4,22.4,and 20.3%at an alcohol concentration of 30m https://www.wendangku.net/doc/2f8570771.html,rger amounts of 9-decyn-1-ol were connected with BChlide c in Cba .tepidum grown at concentrations of 150and 300m M (the relative ratios were 25.0and 24.3%,respectively),although the growth slightly retarded with an increase of the concentration of 9-decyn-1-ol (Fig.4A).The growth pro ?le of Cba .tepidum with 9-decyn-1-ol at 30m M was analogous to that under the control conditions (Fig.4E).In contrast,the supplementation of 9-decen-1-ol and decan-1-ol at 150and 300m M inhibited the growth of Cba .tepidum (Fig.4B and C),which did not allow us to estimate the amounts of BChl c De and BChl c Da in the cells grown under these conditions.These results suggest that the terminal bonds of the supplemented alcohols barely affect the accumulation amounts of these unnatural BChls c ,although the toxicity depends on the terminal groups.The three kinds of alcohols were also supplemented to the liquid cultures of Cba .limnaeum by Method A.Fig.4D shows typical growth pro ?les of Cba .limnaeum by the supplementation of 9-decyn-1-ol by Method A.Cba .limnaeum hardly grew by the supplementation of this alcohol by Method A under the conditions in which Cba .tepidum can grow.The supplementation of 9-decen-1-ol and decan-1-ol to Cba .limnaeum by Method A also inhibited its growth (data not shown).In addition,Cba .limnaeum did not grow by the supplementation of neat acetone (Fig.4F),although the reason for the different growth pro ?les between Cba .limnaeum and Cba .tepidum by this method is unclear at present.Therefore,Method A cannot be applied to the substitution of the esterifying chain in BChl e of Cba .limnaeum ,

although this method is useful for the biosynthesis of unnatural

BChls c in Cba .tepidum .

3.2.Pigment compositions in cells cultivated by Method B

Cba .tepidum and Cba .limnaeum were grown by Method B,in which neat 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol were initially supplemented into their liquid cultures.Cba .tepidum grew by the supplementation of 9-decyn-1-ol and 9-decen-1-ol at concentrations of 50–300m M and 30m M,respectively,by this method,although the addition of decan-1-ol even at 30m M signi ?cantly retarded the growth (Fig.5A),which did not allow us to analyze BChl c in the cells grown with this alcohol.Fig.6depicts the HPLC elution patterns of BChl c from Cba .tepidum cells grown by the supplementation of 9-decyn-1-ol and 9-decen-1-ol by Method B.In addition to fractions cF1–cF4,which were BChl c F homologs,fractions cDy2–cDy4and cDe2–cDe4were observed in these chromatograms and assigned as BChls c Dy and c De ,as described above.Therefore,the cultivation of Cba .tepidum with 9-decyn-1-ol and 9-decen-1-ol by Method B induced the biosynthe-sis of these unnatural BChls c .The relative ratios of BChl c Dy over the total BChl c in Cba .tepidum cells grown in liquid cultures containing 9-decyn-1-ol at 50,100,and 300m M by this method were 15.9,18.6,and 27.3%,respectively (Table 2),which are analogous to those by Method A.BChl c De was also accumulated in cells grown with 9-decyn-1-ol by Method B (the relative ratio,15.6%,Table 2),whose level was not so different from that in the cells grown by Method A.

Cba .limnaeum was successfully cultivated in the presence of 9-decyn-1-ol and 9-decen-1-ol at 100and 30m M,respectively,by Method B,although this species did not grow by the supplemen-tation of decan-1-ol by this method (Fig.5B).BChl e was extracted from the cells grown with these two alcohols by Method B,followed by HPLC analysis (Fig.7).Cba .limnaeum ,grown under normal conditions,possessed three BChl e homologs esteri ?ed with farnesol (denoted as eF1,eF2,and eF3in Fig.7A),which were assigned as [E,E],[P,E],and [I,E]BChl e F ,respectively,based on LC-MS analysis and the elution patterns reported previously [31,33].Cba .limnaeum grown by the supplementation of 9-decyn-1-ol also had three BChl e F isomers with quite similar isomer ratio to that in cells under normal conditions (Fig.7B).Additional fractions were observed around 5min,but they were mainly ascribable to BChlide e and its demetalated pigment bacteriopheophorbide (BPheide)e ,judged from their on-line absorption spectra and the comparison of retention times with BChlide e and BPheide e prepared from isolated BChl e F .No fraction derived from BChl e esteri ?ed with 9-decyn-1-ol (denoted as BChl e Dy )was observed around 10min in this chromatogram under the present detection conditions.When 9-decen-1-ol was supplemented to Cba .limnaeum by Method B,unnatural BChl e esteri ?ed with this alcohol cannot be detected around 10min whereas natural BChl e F and BChlide/BPheide e were

A b s o r b a n c e a t 435 n m

Retention time / min

cDy2

cDy3

cDy4

cF1cF2

cF3

cF4

Fig.3.A HPLC elution pattern of BChl c derivatives in chlorosomes isolated from Cba.tepidum by the supplementation of 9-decyn-1-ol by Method A.The ?nal concentration of 9-decyn-1-ol in the liquid culture was 300m M.The pigments were eluted on a reverse-phase column 5C 18-AR-II (6mm i.d.?250mm)at a ?ow rate of 1.0mL/min with methanol/water (94/6,vol/vol).

Table 1

Relative ratios of unnatural BChl c esteri ?ed with supplemental alcohols to total BChl c in Cba .tepidum cells grown by Method A.

Supplemental alcohol

Final concentration/m M

Ratio of BChl c esteri ?ed with supplemental alcohol/%a

9-Decyn-1-ol

3015.415025.030024.39-Decen-1-ol

3022.4150n.d.b 300n.d.b Decan-1-ol

3020.3150n.d.b 300

n.d.b

a Averaged values of 3–4independent measurements.

Relative ratios cannot be measured due to little growth of cells.

Y.Saga et al./Journal of Photochemistry and Photobiology A:Chemistry 313(2015)44–51

eluted around 13–16min and 5min (Fig.7C).These results indicate that no exogenous alcohols were attached to BChlide e in Cba .limnaeum .

3.3.Spectral properties of Cba .tepidum containing unnatural BChls c

Fig.8depicts the visible absorption spectra of Cba .tepidum cells grown by the supplementation of three kinds of alcohols by Method A as well as those grown under normal and control

conditions.Cba .tepidum cells grown in normal and control liquid cultures had Soret and Q y bands at 461and 748nm (Fig.8A and B).The peak positions of the two major absorption bands of the cells grown under the control conditions were the same as those under the normal conditions.In contrast,cultivation with the exogenous alcohols induced slight blue-shifts of the Q y absorption bands of the cells.Cba .tepidum cells grown with 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol exhibited Q y absorption bands at 746,745,and 746nm,respectively (Fig.8C –E).The Q y absorption bands of the BChl c monomers around 670nm were barely detected in the spectra of the cells grown with the three alcohols (Fig.8C –E).

Fig.9depicts the CD spectra of Cba .tepidum cells grown by the supplementation of three kinds of alcohols by Method A.Cells

100

O D 660

0100

8060

4020

Cultivation time / h

0Fig.4.Growth pro ?les of Cba.tepidum by the supplementation of 9-decyn-1-ol (A),9-decen-1-ol (B),and decan-1-ol (C),and Cba.limnaeum by the supplementation of 9-decyn-1-ol (D)at the ?nal concentrations of 30(open circle),150(open square),and 300m M (open triangle)by Method A.Growth pro ?les of Cba.tepidum and Cba.limnaeum under normal (closed circle)and control conditions (closed triangle)are shown in (E)and (F),respectively.Growth curves were obtained by monitoring optical density (OD)at 660nm.

3.02.01.00.0

O D 660

100

80604020

Cultivation time / h

(B)

3.02.01.0

0.0

6050403020100(A)

Fig.5.Growth pro ?les of Cba.tepidum (A)and Cba.limnaeum (B)in the presence of 9-decyn-1-ol (open circle),9-decen-1-ol (open square),and decan-1-ol (open triangle)at 100m M as well as 9-decen-1-ol at 30m M (open diamond)by Method B.Growth curves were obtained by monitoring optical density (OD)at 660nm.

A b s o r b a n c e a t 435 n m

1050cF1

cF2

cF3

cF4 cDy2

cDy3

cDy4 (A)

2520151050Retention time / min

(B)

cDe2

cDe3

cDe4 Fig. 6.HPLC elution patterns of BChl c derivatives in Cba.tepidum by the supplementation of 9-decyn-1-ol (A)and 9-decen-1-ol (B)by Method B.The ?nal concentrations of 9-decyn-1-ol and 9-decen-1-ol were 100and 30m M,respectively.The pigments were eluted on a reverse-phase column 5C 18-AR-II (6mm i.d.?250mm)with methanol/water (94/6,vol/vol)at a ?ow rate of 1.0mL/min.The chromatogram was recorded at 435nm.

Y.Saga et al./Journal of Photochemistry and Photobiology A:Chemistry 313(2015)44–51

grown with them exhibited reverse S-shaped CD signals around the Q y region (Fig.9C –E).These spectral shapes were analogous to those of the cells grown under normal and control conditions (Fig.9A and B),although the negative signal of the cells possessing BChl c Dy around 770nm was small.These CD signals are characteristic of BChl c self-aggregates in chlorosomes.Thus,chlorosomes containing unnatural BChl c Dy ,BChl c De ,and BChl c Da retain the native supramolecular structures of BChl self-aggregates in chlorosomes.Note that Cba .tepidum cells grown with 9-decyn-1-ol by Method B exhibited almost the same spectral tendencies as those by Method A (data not shown).The spectral properties of Cba .tepidum grown with the exogenous alcohols indicate that their supplementation barely interferes with the in vivo self-aggregation of BChl c .

Table 2

Relative ratios of unnatural BChl c esteri ?ed with supplemental alcohols to total BChl c in Cba.tepidum cells grown by Method B.

Supplemental alcohol

Final concentration/m M

Ratio of BChl c esteri ?ed with supplemental alcohol/%a

9-Decyn-1-ol

5015.910018.630027.39-Decen-1-ol 3015.6100n.d.b Decan-1-ol

30n.d.b 100

n.d.b

a Averaged values of 2independent measurements.

Relative ratios cannot be measured due to little growth of cells.

A b s o r b a n c e a t 435 n m

2520151050eF1 eF2 eF3

(A) 30

Retention time / min

(C)

*Fig.7.HPLC elution patterns of BChl e derivatives in Cba.limnaeum under normal condition (A)and by the supplementation of 9-decyn-1-ol (B)and 9-decen-1-ol (C)by Method B.The ?nal concentrations of 9-decyn-1-ol and 9-decen-1-ol were 100and 30m M,respectively.The pigments were eluted on a reverse-phase column 5C 18-AR-II (6mm i.d.?250mm)with methanol/water (94/6,vol/vol)at a ?ow rate of 1.0mL/min.The chromatograms were recorded at 435nm and normalized at the peaks of the fractions eF1([E,E]BChl e F ).The fractions denoted by *and #in the chromatograms (B)and (C)were mainly ascribable to BChlide e and BPheide e ,respectively.

0.60.4

0.20.0900

800700600500400300

Wavelength / nm

(E)

746

461

1.0

0.5

0.0(D)

745

460

0.4

0.30.20.10.0A b s o r b a n c e

(C)

746

460

0.80.60.40.2

0.0(B)

748

461

1.0

0.5

0.0(A)

748

461

Fig.8.Visible absorption spectra of Cba.tepidum cells grown under normal (A)and control conditions (B),and by the supplementation of 9-decyn-1-ol (C),9-decen-1-ol (D),and decan-1-ol (E)by Method A.The ?nal concentrations of 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol were 30m M.The spectra were measured after 10-fold dilution of cell cultures with 50mM Tris –HCl buffer (pH 8.0).

Y.Saga et al./Journal of Photochemistry and Photobiology A:Chemistry 313(2015)44–51

4.Discussion

The farnesyl moiety in BChls c /d /e /f in green sulfur photosyn-thetic bacteria plays important roles in the self-aggregation in chlorosomes,but the interactions among the farnesyl groups in BChl pigments as well as those with fatty groups in the surrounding lipids of chlorosomes remain unclear.In vivo substitution of the farnesyl group with unnatural hydrocarbon chains is one strategy for solving these questions.This study ?rst demonstrates that straight alcohols possessing ààC R CH and ààCH ?CH 2moieties at the terminus can be attached to BChlide c in vivo and the resulting pigments participate in the chlorosomes in Cba .tepidum .The in vivo relative ratios of the unnatural BChls c over the total BChls c reached to 27%.Rough estimation from these

relative ratios and the pigment amounts in the harvested cells suggests that the supplemental alcohols are partially used for esteri ?cation with BChlide c .The visible absorption and CD spectra of Cba .tepidum containing unnatural BChls c esteri ?ed with the three exogenous alcohols indicate that these novel BChls c participate in self-aggregates in chlorosomes and the terminal unsaturated bonds in BChl c barely affect the interactions among the pigments and between the pigments and lipids on the chlorosome envelope.The successful incorporation of unsaturated bonds into BChl c by the biosynthetic reaction without decompo-sition of the chlorosomal supramolecular structures will be a clue in the investigation of such interactions by the utilization of these moieties as structural probes and/or scaffolds for bioorthogonal modi ?cations.It is noted that the present HPLC conditions did not allow us to detect the incorporation of the exogenous alcohols to minor chlorophyllous pigments,BChl a and Chl a .

The connection of a farnesyl moiety to the 172-carboxy group in BChlide c in Cba .tepidum is catalyzed by the BChl c synthase,BchK,in the ?nal step of the BChl c biosynthesis [7,9,34].This study and previous reports show that this enzyme in Cba .tepidum displays variation for long hydrocarbon substrates that connect to BChlide c .Such variation is consistent with the promiscuous recognition of isoprenoid by prenyltransferase UbiA [35–37],which belongs to the superfamily including BChl synthases [38].The enzyme UbiA attaches various lengths of isoprenoid moieties to p -hydroxyben-zoate.The structural basis of loose isoprenoid recognition was indicated by the three-dimensional crystal structure of UbiA,in which an isoprenoid chain stuck out from the cavity of this enzyme [39].BChl c synthase (BchK)of Cba .tepidum would have a binding site of farnesyl diphosphate similar to that of UbiA.

The present study,however,indicates that Cba .limnaeum cannot biosynthesize unnatural BChls e esteri ?ed with the supplemented alcohols.One possible reason is the difference between the permissive range in the speci ?city of the long hydrocarbon substrates of BchK in Cba .tepidum and that in Cba.limnaeum ,although the sequence homology of BchK of Cba .limnaeum and Cba.tepidum is 79.9%.One other possible reason is the substrate speci ?city of the diphosphorylation enzymes of the exogenous alcohols in green sulfur bacteria.Since long hydrocar-bon alcohols are utilized by BchK after their diphosphorylation in vivo ,the enzymatic activities for the diphosphorylation of the exogenous alcohols should be considered.The in vivo localization and/or the penetration of the exogenous alcohols should also be considered to discuss the difference in the biosynthesis of unnatural chlorosomal BChls between the two species.

5.Conclusion

Unnatural BChls c possessing unsaturated triple and double bonds at the terminus of esterifying chains were successfully biosynthesized in the green sulfur photosynthetic bacterium Cba .tepidum .The terminal unsaturated bonds of the exogenous alcohols hardly affected in vivo enzymatic reactions by BChl c synthase.In contrast,Cba .limnaeum produced no unnatural BChl e esteri ?ed with the supplemented alcohols.Terminal unsaturated bonds in the esterifying chains of BChls c will be helpful to investigate the biosynthesis of chlorosomal BChl pigments and their self-aggregation in vivo as well as developments of pigment-assembling nano-devices for arti ?cial photosynthesis by the functional regulation and chemical modi ?cation of chlorosomes via the esterifying moieties of light-harvesting BChls.

Acknowledgements

This work was partially supported by a Grant-in-Aid for Scienti ?c Research on Innovative Areas ‘Arti ?cial Photoshynthesis

3020100-10E l l i p t i c i t y / m d e g

(C)

40200-20800

700600500400300

Wavelength / nm

(E)

-200

0200(A)

-50

050(B)

100500-50(D)

Fig.9.CD spectra of Cba.tepidum cells grown under normal (A)and control conditions (B),and by the supplementation of 9-decyn-1-ol (C),9-decen-1-ol (D),and decan-1-ol (E)by Method A.The ?nal concentrations of 9-decyn-1-ol,9-decen-1-ol,and decan-1-ol were 30m M.The spectra were measured after 10-fold dilution of cell cultures with 50mM Tris –HCl buffer (pH 8.0).

Y.Saga et al./Journal of Photochemistry and Photobiology A:Chemistry 313(2015)44–51

(AnApple)’(No.25107525)from the Japan Society for the Promotion of Science(JSPS).

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.wendangku.net/doc/2f8570771.html,/10.1016/j. jphotochem.2015.05.002.

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