Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1153–1157
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Absorption spectral change of peripheral-light harvesting complexes 2 induced by magnesium protoporphyrin IX monomethyl ester association Huiying Yue, Chungui Zhao ⇑, Kai Li, Suping Yang ⇑ Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen 361021, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
MPE was additionally contained in
the LH2 with 423 nm absorption peak. The absorption spectral change of LH2 was ascribed to MPE association. MPE binded to LH2 in vitro, but did not bind to the BChl a binding sites in LH2. MPE accumulation affected the conformation of integral LH2.
a r t i c l e
i n f o
Article history: Received 10 June 2014 Received in revised form 15 August 2014 Accepted 31 August 2014 Available online 22 September 2014 Keywords: LH2 Magnesium protoporphyrin monomethyl IX ester Carotenoid Bacteriochlorophyll Rhodobacter azotoformans
a b s t r a c t Several spectrally different types of peripheral light harvesting complexes (LH) have been reported in anoxygenic phototrophic bacteria in response to environmental changes. In this study, two spectral forms of LH2 (T-LH2 and U-LH2) were isolated from Rhodobacter azotoformans. The absorption of T-LH2 was extremely similar to the LH2 isolated from Rhodobacter sphaeroides. U-LH2 showed an extra peak at 423 nm in the carotenoid region. To explore the spectral origin of this absorption peak, the difference in pigment compositions of two LH2 was analyzed. Spheroidene and bacteriochlorophyll aP were both contained in the two LH2. And magnesium protoporphyrin IX monomethyl ester (MPE) was only contained in U-LH2. It is known that spheroidene and bacteriochlorophyll aP do not produce 423 nm absorption peak either in vivo or in vitro. Whether MPE accumulation was mainly responsible for the formation of the 423 nm peak? The interactions between MPE and different proteins were further studied. The results showed that the maximum absorption of MPE was red-shifted from 415 nm to 423 nm when it was mixed with T-LH2 and its apoproteins, nevertheless, the Qy transitions of the bound bacteriochlorophylls in LH2 were almost unaffected, which indicated that the formation of the 423 nm peak was related to MPE-LH2 protein interaction. MPE did not bind to sites involved in the spectral tuning of BChls, but the conformation of integral LH2 was affected by MPE association, the alkaline stability of ULH2 was lower than T-LH2, and the fluorescence intensity at 860 nm was decreased after MPE combination. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding authors. Tel.: +86 592 6166178. E-mail addresses: [email protected] (H. Yue), [email protected] (C. Zhao), [email protected] (K. Li), [email protected] (S. Yang). http://dx.doi.org/10.1016/j.saa.2014.08.132 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Bacteriochlorophyll (BChl)-protein complexes, such as light harvesting complexes (LH) and reaction center of anoxygenic phototrophic bacteria (APB), represent the simplest photosynthetic
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systems and have been intensively investigated [1,2]. The BChl aggregates anchored within membrane protein frame are responsible for harvesting and converting sunlight into biochemical energy. Porphyrins, as precursors of (B)Chls, have homologous cyclic tetrapyrrole structure and also are intense absorptions [3–5]. These properties make porphyrins an attractive class of light harvesting materials. It has been reported that in vitro organization of mesoporphyrin dimmers through the LH1 polypeptides from Rhodospirillum rubrum would constitute an efficient, artificial energy transfer system [6]. BChl precursor, magnesium protoporphyrin IX monomethyl ester (MPE), preferentially accumulates inside the cell and becomes membrane bound [7]. Therefore, it would be interesting to know whether MPE could bind to the proteins of BChl-protein complexes in physiological conditions and function in photosynthesis. Generally, LH and reaction center exhibit a set of characteristic absorption peaks originating from specific pigment–pigment or pigment–protein interactions. These spectral features can be used for taxonomic purposes. For example, the absorption maxima at 470, 500 and 530 nm were often assigned to spirilloxanthin. Similarly, there are several characteristic spectra were reported for spheroidene (450, 480 and 512 nm), okenone (521 nm), BChl a (380, 590, 800–880 nm). Moreover, the various photosynthetic complexes are named and classified according to the absorption spectra characteristic of the Qy bands of BChl a, including LH1 (875 nm), LH2 (800 and 850 nm), LH3 (800 and 820 nm), LH4 (800 nm) and reaction center (760, 802 and 865 nm) [8,9]. An extra absorption peak at 423 nm appeared in the in vivo spectrum of Rhodobacter azotoformans R7 grown in acetate– glutamate medium but not in acetate–yeast medium [10]. Accordingly, the LH2 isolated from acetate–glutamate cultures also exhibited a 423 nm absorption peak [11]. The 423 nm peak, frequently observed in a wide range of absorption spectra [12–14], was ill-defined. Since this peak is present in the carotenoid region, it is routinely ascribed to carotenoid. Moreover, the carotenoid, such as neurosporene, hydroxyneurosporene or methoxyneurosporene was reported to involve in the formation of a peak at 423 nm because their absorption maxima occurred at 423, 460 and 480 nm, respectively [13]. However, it was surprised that the above carotenoids were not detected in our previous study [11]. The present study was initiated to explore the origin of the 423 nm peak by systematically comparing differences in pigment compositions between the LH2 without (T-LH2) and with (U-LH2) a 423 nm absorption peak. Compared to T-LH2, U-LH2 contained an additional pigment, magnesium protoporphyrin IX monomethyl ester (MPE) besides spheroidene and BChl aP, therefore, the absorption peak at 423 nm was tentatively assigned as MPE. Assembly of purified MPE with LH2 proteins in vitro confirmed the relationship between this pigment component and the 423 nm peak. The binding interaction between MPE and LH2 proteins was discussed. The effects of MPE association on the conformation of the integral LH2 were also investigated. Materials and methods
(pH 8.0), being broken through a French press cell (4.5 kpsi). The suspension was solubilized at 4 °C with 1% (v/v) lauryldimethylamine oxide (LDAO, Fluka) for 40 min in the dark, followed by centrifugation at 12,000 g for 20 min. Crude preparations of the photosynthetic complexes were obtained by subjecting the supernatant to fractional precipitation using ammonium sulfate as the precipitant. Further purification of the complexes was carried out using a DEAE-cellulose column (DEAE52, Whatman) connected to ÄKTA purifier system (Amersham Biosciences). Fractions were eluted using a NaCl gradient (0–0.5 mol/L) in 10 mmol/L Tris–HCl (pH 8.0, 0.1% LDAO). The LH2 fractions were collected and purified again by gel filtration chromatography using a HiPrep 16/60 Sephacryl S-200 HR column (2.6 125 cm, Amersham Biosciences). Elution was carried out with Tris–HCl (10 mmol/L, 0.1% LDAO at pH 8.0) at a flow rate of 0.5 ml/min.
Biochemical analyses SDS–PAGE (12% polyacrylamide gel) was carried out according to the procedure of Laemmli [16,17]. Pigment composition was analyzed by extracting pigment from the isolated LH2 with acetone/methanol mixture (7:2, v/v) [18]. The extracts were dried under a stream of nitrogen gas and then dissolved in methanol for HPLC and MS analyses. HPLC was performed on Shimadzu CTO-20A chromatograph equipped with Shim-Pack VP-ODS column (C18, 4.6 150 mm, Shimadzu, Kyoto) and diode array detector (Model SPD-M10Avp, Shimadzu, Kyoto). Mobile phases were composed of 95% methanol in water (v/v) (phase A) and ethyl acetate (phase B). The gradient elution time program was set as follows: 0–5 min, 0% B; 5–25 min, 0–30% B; 25–40 min, 30–45% B; 40–60 min, 45–60% B. The flow rate was 0.7 ml/min and the detection wavelengths were set at 415, 480 and 770 nm. LC-MS analyses was performed on Agilent 1200 series HPLC system coupled to Agilent 6310 ion-trap mass spectrometer with an ESI source, operated in the positive-ion mode. The ESI capillary voltage was 5500 eV. The nebulizer pressure was set to 30 psi. The drying gas was set to a flow rate of 12 L/min and the temperature of 350 °C. The effects of alkaline treatment on LH2 complexes in suspensions of 10 mmol/L Tris–HCl pH 8.0 (A850 0.24) were studied by adding a convenient volume of NaOH solution to final pH values of 11.0, 12.0 and 13.0, respectively. The samples were incubated for 12 h at 25 °C before spectral analyses.
Spectroscopic analyses Absorption spectra were recorded with a MAPADA UV-3200 PCS spectrophotometer. Fluorescence emission spectra were measured between 800 and 900 nm with a Hitachi F7000 spectrofluorometer using a 1 cm pathlength quartz cuvette. The excitation and emission slit widths were both 5 nm. Excitation was at 480 nm and the A850 of both samples were adjusted to 0.30.
Bacterial strains and growth conditions R. azotoformans R7 was grown anaerobically at 30 °C with a light intensity of 3000 lux in Ormerod [15] medium modified by replacing DL-malic acid and ammonium sulphate with sodium acetate (2.46 g/L) and L-glutamine (1.0 g/L). Preparation of LH2 complexes LH2 complexes were isolated as described in Ref. [11]. Cells were harvested, washed and resuspended in 10 mmol/L Tris–HCl
Interaction of MPE with different proteins MPE was purified from acetate–glutamate cultures by HPLC as mentioned above. The LH2 apoproteins were obtained by extracting all pigments from T-LH2. The T-LH2, apoproteins, and bovine serum albumin were dissolved in 50 mmol/L Tricine-NaOH (pH 8.0, 12.5% dimethylsulfoxide). An appropriate volume of the MPE in methanol was then added. After incubation for 5 min in the dark, absorption spectrum variations were measured with a UV3200PCS spectrophotometer.
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Results Isolation and purification of LH2 complexes The crude proteins precipitated between 30% and 50% saturated ammonium sulfate was further purified by column chromatography. During DEAE ion-exchange chromatography, U-LH2 was eluted between 0.1 and 0.15 mol/L NaCl, whereas T-LH2 was eluted between 0.15 and 0.2 mol/L NaCl (Fig. 1A). Both LH2 fractions showed a monomer peak at the same position on the gel filtration chromatography elution profiles (Fig. 1B). The proteins from both T-LH2 and U-LH2 migrated as a single bright band of 6 kDa on SDS–PAGE (Fig. 1C). The result was consistent with two small (5–7 kDa), hydrophobic apoproteins (a and b) of the light harvesting complexes from Rhodobacter species [19]. Practically no contaminant proteins were observed except the single polypeptide band mentioned above.
Fig. 2. Absorption spectra of T-LH2 (solid line) and U-LH2 (dashed line) from Rhodobacter azotoformans R7. The spectra are normalized to 0.54 at 850 nm.
Absorption spectra of LH2 complexes The absorption spectra obtained for both LH2 were shown in Fig. 2. T-LH2 showed absorption bands at 378, 590, 800 and 850 nm corresponding to BChl a and at 452, 480 and 512 nm corresponding to carotenoid. Therefore, T-LH2 was extremely similar to the LH2 isolated from R. sphaeroides [19]. U-LH2 was spectrally similar to T-LH2 with the curious exception that an intense band at 423 nm and a small band at 555 nm appeared. Pigment compositions The absorption spectra of acetone/methanol extracts from T-LH2 and U-LH2 were compared as shown in Fig. 3A. The pigment extracts from T-LH2 showed absorption bands at 365, 604 and 771 nm for BChl a, and 428, 453 and 483 nm for carotenoid, respectively. The absorption peak at 428 nm vanished in the pigment extracts from U-LH2; instead, a 415 nm peak appeared. The spectral variations indicated there were differences in pigment composition between T-LH2 and U-LH2. The pigment extracts were next characterized by HPLC analysis. As shown in Fig. 3B, three major peaks were monitored in the HPLC elution profiles and named according to the order of elution, e.g., peaks 1, 2 and 3. The absorption spectra analyses (Fig. 3C), retention times and related Refs. [20–22] allowed us to assign peak 1 to be magnesium protoporphyrin IX (MP) or its monomethyl ester (MPE), peak 2 to be BChl a, peak 3 to be spheroidene or spheroidene analogues, respectively. When the pigments components were further analyzed by mass spectrometry technique, pigments from peak 1, 2 and 3 yielded protonated molecular ion [MH]+ at m/z 599.2, 911.4 and 568.4. Thus, pigments of peak 1, 2 and 3 were assigned as MPE, BChl aP and spheroidene, respectively. Besides, two minor components eluting at 22.3 and 34.8 min were assigned as BChl aDHGG and
Fig. 3. Analyses of pigment compositions. (A) Absorption spectra of pigment extracts. (B) HPLC profiles of the extracts. Peak 1, 2 and 3 were detected at 415, 770 and 480 nm, respectively. (C) Absorption spectra of components of peak 1, 2 and 3, recorded with a diode array detector attached to the HPLC system.
spheroidene based on the absorption spectra, retention times and mass spectral analyses (data not shown). On the basis of the above findings, it could be concluded that T-LH2 contained spheroidene as its major carotenoid and BChl aP as its major BChl a, while, U-LH2 contained an additional component, MPE, besides spheroidene and BChl aP.
Interactions between MPE and proteins To explore the relationship between MPE and the 423 nm peak in LH2, the interaction of MPE with different LH2 proteins
Fig. 1. Preparation of the LH2 complexes. (A) Elution profile of the DEAE ion-exchange chromatography. NaCl concentration was indicated by dashed line. (B) Elution profiles of Sephacryl S-200 gel filtration chromatography. (C) SDS–PAGE analyses of protein component from the isolated LH2. Lane 1: T-LH2, Lane 2: molecular weight marker, Lane 3: U-LH2.
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was studied. As shown in Fig. 4, the soret band in absorption spectrum of MPE was red-shifted from 415 to 423 nm after mixing MPE with T-LH2 and its apoproteins; the other two smaller peaks at 550 and 590 nm were also red-shifted to 554 and 593 nm, respectively. In all cases, the intensity of 270 nm band increased, though no extra protein was added to the mixture. Meanwhile, the Qy absorption bands of BChl a in T-LH2 remained unchanged after mixing with MPE. Our observation indicated: (1) MPE binded to T-LH2 and apoproteins; (2) the 423 nm absorption peak was caused by the interaction of MPE with LH2 proteins; (3) MPE did not bind to sites involved in the spectral tuning of BChls. Fluorescence spectroscopy We were interested in possible effects upon the structural and functional properties of LH2 resulting from MPE accumulation. As shown in Fig. 5, due to energy transfer among carotenoid and BChls within LH2, fluorescence from BChl a were observed upon excitation into the carotenoid absorption region. The minor 806 nm peak was due to emission by B800-BChls, while the major 860 nm peak was due to emission by B850-BChls. The fluorescence intensity of U-LH2 at 860 nm was lower than that of T-LH2, showing the energy transfer within LH2 was affected by MPE association. Although the detailed molecular mechanism underlying this phenomenon had yet to be elucidated, it remained possible that change in fluorescence intensity might also, in part, reflect an altered conformation of integral LH2. Effects of MPE association on the structural stability of LH2 A comparison was made of the change in alkaline-stability between U-LH2 and T-LH2, as shown in Fig. 6. The U-LH2 and T-LH2 were quite stable over a broad pH range (pH from 8.0 to 12.0). At pH 13.0, the absorbance at 850 nm in U-LH2 was attenuated and blue-shifted to 826 nm, the absorbance at 800 nm decreased, while that at 780 nm increased. In contrast, the intensity of the 850 nm band in T-LH2 decreased but without obvious position change, the relative intensity of the 800 nm band also decreased slightly, while a broad absorption peak around 780 nm loomed. The result suggested that the quaternary structure of U-LH2 was less stable to alkaline than that of T-LH2. Discussion The observation of altered absorption spectral properties of LH2 raises the question of what caused the formation of the absorption peak at 423 nm. The pigment compositions of U-LH2 and T-LH2 were investigated and compared systematically. In both LH2, spheroidene was maintained as the major carotenoid, and BChl aP was the major BChl a. However, a remarkable difference in the pigment
Fig. 5. Fluorescence spectra of T-LH2 (solid line) and U-LH2 (dashed line). The excitation wavelength was at 480 nm.
composition between the two LH2 is that MPE was detected in U-LH2 but not in T-LH2. Since both spheroidene and BChl aP can not generate a 423 nm absorption peak either in vivo or in vitro, and no any neurosporene, hydroxyneurosporene or methoxyneurosporene was detected, thus ruling out the possibility that the absorption peak at 423 nm in U-LH2 was contributed by carotenoid. Therefore, the absorption peak at 423 nm in U-LH2 may be explained as being caused by the accumulation of MPE. This assignment was consistent with the observations of Ouchane et al. [20] that Rubrivivax gelatinosus BChE mutant accumulated large amounts of MPE when grown under low oxygenation conditions, and its membrane exhibited an intensive absorption peak at 423 nm. To ensure that the origin of the 423 nm peak was indeed related to MPE, we estimated the effects of MPE-protein interactions on the absorption spectra of MPE. The absorption band at 415 nm of MPE was, as expected, red-shifted to 423 nm upon mixing with LH2 and its apoproteins. These results suggested that the 423 nm peak in LH2 derived from MPE association. Sawicki and Willows [23] reported that the soret band of MP could be red-shifted from 415 nm to 422 nm when they were added to S-adenosyl-methionine: MP O-methyltransferase (BChM) and speculated MP carrier (BChJ). The authors believed that the spectral changes implied that MP bound in a hydrophobic environment on these proteins, because the shift in the soret peak is often characteristic of a nonplanar distortion of the porphyrins [24]. In our work, the similar red-shift in soret peak of MPE indicated that MPE binded to both LH2 and its apoproteins in vitro. The absorption at 270 nm characteristic of proteins was increased after MPE addition in our in vitro assembly experiment. We also noted that the absorption at 270 nm in isolated U-LH2 was slightly higher than in isolated T-LH2 (Fig. 2). Such an increase may be attributed to MPE-protein interaction. It has been long known that BChls precursors porphyrins bind to proteins, but the mode of
Fig. 4. Absorbance spectral changes of magnesium protoporphyrin IX monomethyl ester (MPE) after interaction with bovine serum albumin (A), apoproteins (B) or T-LH2 (C). Line 2 represents the spectra of MPE alone; line 1, 4 and 6 represent the spectra of bovine serum albumin (BSA), apoproteins and T-LH2; line 3, 5 and 7 represent the spectra of the interactions of BAS, apoproteins and T-LH2 with MPE.
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the BChl a molecules in LH2. MPE combination influenced the conformations of integral LH2. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 31270106 and 31070054), by National Natural Science Foundation of Fujian Province (No. 2012J01136), by National Marine Public Industry Research (No. 201505026). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
Fig. 6. Spectral changes in the near infrared regions of T-LH2 and U-LH2 induced by alkaline treatment.
[15] [16] [17] [18] [19]
binding remains unsolved [25,26]. Our results showed that there was no obvious change in the Qy absorption bands of the BChl a in T-LH2, indicating that MPE did not bind to sites involved in the spectral tuning of BChls. However, the spectroscopic data from fluorescence measurements and alkaline-stability demonstrated that the MPE association influenced the conformation of the integral LH2. In conclusion, the 423 nm peak in U-LH2 was caused by MPE association. MPE could bind to LH2 proteins, but did not replace
[20] [21] [22] [23] [24] [25] [26]
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Absorption spectral change of peripheral-light harvesting complexes 2 induced by magnesium protoporphyrin IX monomethyl ester association Huiying Yue, Chungui Zhao ⇑, Kai Li, Suping Yang ⇑ Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen 361021, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
MPE was additionally contained in
the LH2 with 423 nm absorption peak. The absorption spectral change of LH2 was ascribed to MPE association. MPE binded to LH2 in vitro, but did not bind to the BChl a binding sites in LH2. MPE accumulation affected the conformation of integral LH2.
a r t i c l e
i n f o
Article history: Received 10 June 2014 Received in revised form 15 August 2014 Accepted 31 August 2014 Available online 22 September 2014 Keywords: LH2 Magnesium protoporphyrin monomethyl IX ester Carotenoid Bacteriochlorophyll Rhodobacter azotoformans
a b s t r a c t Several spectrally different types of peripheral light harvesting complexes (LH) have been reported in anoxygenic phototrophic bacteria in response to environmental changes. In this study, two spectral forms of LH2 (T-LH2 and U-LH2) were isolated from Rhodobacter azotoformans. The absorption of T-LH2 was extremely similar to the LH2 isolated from Rhodobacter sphaeroides. U-LH2 showed an extra peak at 423 nm in the carotenoid region. To explore the spectral origin of this absorption peak, the difference in pigment compositions of two LH2 was analyzed. Spheroidene and bacteriochlorophyll aP were both contained in the two LH2. And magnesium protoporphyrin IX monomethyl ester (MPE) was only contained in U-LH2. It is known that spheroidene and bacteriochlorophyll aP do not produce 423 nm absorption peak either in vivo or in vitro. Whether MPE accumulation was mainly responsible for the formation of the 423 nm peak? The interactions between MPE and different proteins were further studied. The results showed that the maximum absorption of MPE was red-shifted from 415 nm to 423 nm when it was mixed with T-LH2 and its apoproteins, nevertheless, the Qy transitions of the bound bacteriochlorophylls in LH2 were almost unaffected, which indicated that the formation of the 423 nm peak was related to MPE-LH2 protein interaction. MPE did not bind to sites involved in the spectral tuning of BChls, but the conformation of integral LH2 was affected by MPE association, the alkaline stability of ULH2 was lower than T-LH2, and the fluorescence intensity at 860 nm was decreased after MPE combination. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding authors. Tel.: +86 592 6166178. E-mail addresses: [email protected] (H. Yue), [email protected] (C. Zhao), [email protected] (K. Li), [email protected] (S. Yang). http://dx.doi.org/10.1016/j.saa.2014.08.132 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Bacteriochlorophyll (BChl)-protein complexes, such as light harvesting complexes (LH) and reaction center of anoxygenic phototrophic bacteria (APB), represent the simplest photosynthetic
1154
H. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1153–1157
systems and have been intensively investigated [1,2]. The BChl aggregates anchored within membrane protein frame are responsible for harvesting and converting sunlight into biochemical energy. Porphyrins, as precursors of (B)Chls, have homologous cyclic tetrapyrrole structure and also are intense absorptions [3–5]. These properties make porphyrins an attractive class of light harvesting materials. It has been reported that in vitro organization of mesoporphyrin dimmers through the LH1 polypeptides from Rhodospirillum rubrum would constitute an efficient, artificial energy transfer system [6]. BChl precursor, magnesium protoporphyrin IX monomethyl ester (MPE), preferentially accumulates inside the cell and becomes membrane bound [7]. Therefore, it would be interesting to know whether MPE could bind to the proteins of BChl-protein complexes in physiological conditions and function in photosynthesis. Generally, LH and reaction center exhibit a set of characteristic absorption peaks originating from specific pigment–pigment or pigment–protein interactions. These spectral features can be used for taxonomic purposes. For example, the absorption maxima at 470, 500 and 530 nm were often assigned to spirilloxanthin. Similarly, there are several characteristic spectra were reported for spheroidene (450, 480 and 512 nm), okenone (521 nm), BChl a (380, 590, 800–880 nm). Moreover, the various photosynthetic complexes are named and classified according to the absorption spectra characteristic of the Qy bands of BChl a, including LH1 (875 nm), LH2 (800 and 850 nm), LH3 (800 and 820 nm), LH4 (800 nm) and reaction center (760, 802 and 865 nm) [8,9]. An extra absorption peak at 423 nm appeared in the in vivo spectrum of Rhodobacter azotoformans R7 grown in acetate– glutamate medium but not in acetate–yeast medium [10]. Accordingly, the LH2 isolated from acetate–glutamate cultures also exhibited a 423 nm absorption peak [11]. The 423 nm peak, frequently observed in a wide range of absorption spectra [12–14], was ill-defined. Since this peak is present in the carotenoid region, it is routinely ascribed to carotenoid. Moreover, the carotenoid, such as neurosporene, hydroxyneurosporene or methoxyneurosporene was reported to involve in the formation of a peak at 423 nm because their absorption maxima occurred at 423, 460 and 480 nm, respectively [13]. However, it was surprised that the above carotenoids were not detected in our previous study [11]. The present study was initiated to explore the origin of the 423 nm peak by systematically comparing differences in pigment compositions between the LH2 without (T-LH2) and with (U-LH2) a 423 nm absorption peak. Compared to T-LH2, U-LH2 contained an additional pigment, magnesium protoporphyrin IX monomethyl ester (MPE) besides spheroidene and BChl aP, therefore, the absorption peak at 423 nm was tentatively assigned as MPE. Assembly of purified MPE with LH2 proteins in vitro confirmed the relationship between this pigment component and the 423 nm peak. The binding interaction between MPE and LH2 proteins was discussed. The effects of MPE association on the conformation of the integral LH2 were also investigated. Materials and methods
(pH 8.0), being broken through a French press cell (4.5 kpsi). The suspension was solubilized at 4 °C with 1% (v/v) lauryldimethylamine oxide (LDAO, Fluka) for 40 min in the dark, followed by centrifugation at 12,000 g for 20 min. Crude preparations of the photosynthetic complexes were obtained by subjecting the supernatant to fractional precipitation using ammonium sulfate as the precipitant. Further purification of the complexes was carried out using a DEAE-cellulose column (DEAE52, Whatman) connected to ÄKTA purifier system (Amersham Biosciences). Fractions were eluted using a NaCl gradient (0–0.5 mol/L) in 10 mmol/L Tris–HCl (pH 8.0, 0.1% LDAO). The LH2 fractions were collected and purified again by gel filtration chromatography using a HiPrep 16/60 Sephacryl S-200 HR column (2.6 125 cm, Amersham Biosciences). Elution was carried out with Tris–HCl (10 mmol/L, 0.1% LDAO at pH 8.0) at a flow rate of 0.5 ml/min.
Biochemical analyses SDS–PAGE (12% polyacrylamide gel) was carried out according to the procedure of Laemmli [16,17]. Pigment composition was analyzed by extracting pigment from the isolated LH2 with acetone/methanol mixture (7:2, v/v) [18]. The extracts were dried under a stream of nitrogen gas and then dissolved in methanol for HPLC and MS analyses. HPLC was performed on Shimadzu CTO-20A chromatograph equipped with Shim-Pack VP-ODS column (C18, 4.6 150 mm, Shimadzu, Kyoto) and diode array detector (Model SPD-M10Avp, Shimadzu, Kyoto). Mobile phases were composed of 95% methanol in water (v/v) (phase A) and ethyl acetate (phase B). The gradient elution time program was set as follows: 0–5 min, 0% B; 5–25 min, 0–30% B; 25–40 min, 30–45% B; 40–60 min, 45–60% B. The flow rate was 0.7 ml/min and the detection wavelengths were set at 415, 480 and 770 nm. LC-MS analyses was performed on Agilent 1200 series HPLC system coupled to Agilent 6310 ion-trap mass spectrometer with an ESI source, operated in the positive-ion mode. The ESI capillary voltage was 5500 eV. The nebulizer pressure was set to 30 psi. The drying gas was set to a flow rate of 12 L/min and the temperature of 350 °C. The effects of alkaline treatment on LH2 complexes in suspensions of 10 mmol/L Tris–HCl pH 8.0 (A850 0.24) were studied by adding a convenient volume of NaOH solution to final pH values of 11.0, 12.0 and 13.0, respectively. The samples were incubated for 12 h at 25 °C before spectral analyses.
Spectroscopic analyses Absorption spectra were recorded with a MAPADA UV-3200 PCS spectrophotometer. Fluorescence emission spectra were measured between 800 and 900 nm with a Hitachi F7000 spectrofluorometer using a 1 cm pathlength quartz cuvette. The excitation and emission slit widths were both 5 nm. Excitation was at 480 nm and the A850 of both samples were adjusted to 0.30.
Bacterial strains and growth conditions R. azotoformans R7 was grown anaerobically at 30 °C with a light intensity of 3000 lux in Ormerod [15] medium modified by replacing DL-malic acid and ammonium sulphate with sodium acetate (2.46 g/L) and L-glutamine (1.0 g/L). Preparation of LH2 complexes LH2 complexes were isolated as described in Ref. [11]. Cells were harvested, washed and resuspended in 10 mmol/L Tris–HCl
Interaction of MPE with different proteins MPE was purified from acetate–glutamate cultures by HPLC as mentioned above. The LH2 apoproteins were obtained by extracting all pigments from T-LH2. The T-LH2, apoproteins, and bovine serum albumin were dissolved in 50 mmol/L Tricine-NaOH (pH 8.0, 12.5% dimethylsulfoxide). An appropriate volume of the MPE in methanol was then added. After incubation for 5 min in the dark, absorption spectrum variations were measured with a UV3200PCS spectrophotometer.
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Results Isolation and purification of LH2 complexes The crude proteins precipitated between 30% and 50% saturated ammonium sulfate was further purified by column chromatography. During DEAE ion-exchange chromatography, U-LH2 was eluted between 0.1 and 0.15 mol/L NaCl, whereas T-LH2 was eluted between 0.15 and 0.2 mol/L NaCl (Fig. 1A). Both LH2 fractions showed a monomer peak at the same position on the gel filtration chromatography elution profiles (Fig. 1B). The proteins from both T-LH2 and U-LH2 migrated as a single bright band of 6 kDa on SDS–PAGE (Fig. 1C). The result was consistent with two small (5–7 kDa), hydrophobic apoproteins (a and b) of the light harvesting complexes from Rhodobacter species [19]. Practically no contaminant proteins were observed except the single polypeptide band mentioned above.
Fig. 2. Absorption spectra of T-LH2 (solid line) and U-LH2 (dashed line) from Rhodobacter azotoformans R7. The spectra are normalized to 0.54 at 850 nm.
Absorption spectra of LH2 complexes The absorption spectra obtained for both LH2 were shown in Fig. 2. T-LH2 showed absorption bands at 378, 590, 800 and 850 nm corresponding to BChl a and at 452, 480 and 512 nm corresponding to carotenoid. Therefore, T-LH2 was extremely similar to the LH2 isolated from R. sphaeroides [19]. U-LH2 was spectrally similar to T-LH2 with the curious exception that an intense band at 423 nm and a small band at 555 nm appeared. Pigment compositions The absorption spectra of acetone/methanol extracts from T-LH2 and U-LH2 were compared as shown in Fig. 3A. The pigment extracts from T-LH2 showed absorption bands at 365, 604 and 771 nm for BChl a, and 428, 453 and 483 nm for carotenoid, respectively. The absorption peak at 428 nm vanished in the pigment extracts from U-LH2; instead, a 415 nm peak appeared. The spectral variations indicated there were differences in pigment composition between T-LH2 and U-LH2. The pigment extracts were next characterized by HPLC analysis. As shown in Fig. 3B, three major peaks were monitored in the HPLC elution profiles and named according to the order of elution, e.g., peaks 1, 2 and 3. The absorption spectra analyses (Fig. 3C), retention times and related Refs. [20–22] allowed us to assign peak 1 to be magnesium protoporphyrin IX (MP) or its monomethyl ester (MPE), peak 2 to be BChl a, peak 3 to be spheroidene or spheroidene analogues, respectively. When the pigments components were further analyzed by mass spectrometry technique, pigments from peak 1, 2 and 3 yielded protonated molecular ion [MH]+ at m/z 599.2, 911.4 and 568.4. Thus, pigments of peak 1, 2 and 3 were assigned as MPE, BChl aP and spheroidene, respectively. Besides, two minor components eluting at 22.3 and 34.8 min were assigned as BChl aDHGG and
Fig. 3. Analyses of pigment compositions. (A) Absorption spectra of pigment extracts. (B) HPLC profiles of the extracts. Peak 1, 2 and 3 were detected at 415, 770 and 480 nm, respectively. (C) Absorption spectra of components of peak 1, 2 and 3, recorded with a diode array detector attached to the HPLC system.
spheroidene based on the absorption spectra, retention times and mass spectral analyses (data not shown). On the basis of the above findings, it could be concluded that T-LH2 contained spheroidene as its major carotenoid and BChl aP as its major BChl a, while, U-LH2 contained an additional component, MPE, besides spheroidene and BChl aP.
Interactions between MPE and proteins To explore the relationship between MPE and the 423 nm peak in LH2, the interaction of MPE with different LH2 proteins
Fig. 1. Preparation of the LH2 complexes. (A) Elution profile of the DEAE ion-exchange chromatography. NaCl concentration was indicated by dashed line. (B) Elution profiles of Sephacryl S-200 gel filtration chromatography. (C) SDS–PAGE analyses of protein component from the isolated LH2. Lane 1: T-LH2, Lane 2: molecular weight marker, Lane 3: U-LH2.
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was studied. As shown in Fig. 4, the soret band in absorption spectrum of MPE was red-shifted from 415 to 423 nm after mixing MPE with T-LH2 and its apoproteins; the other two smaller peaks at 550 and 590 nm were also red-shifted to 554 and 593 nm, respectively. In all cases, the intensity of 270 nm band increased, though no extra protein was added to the mixture. Meanwhile, the Qy absorption bands of BChl a in T-LH2 remained unchanged after mixing with MPE. Our observation indicated: (1) MPE binded to T-LH2 and apoproteins; (2) the 423 nm absorption peak was caused by the interaction of MPE with LH2 proteins; (3) MPE did not bind to sites involved in the spectral tuning of BChls. Fluorescence spectroscopy We were interested in possible effects upon the structural and functional properties of LH2 resulting from MPE accumulation. As shown in Fig. 5, due to energy transfer among carotenoid and BChls within LH2, fluorescence from BChl a were observed upon excitation into the carotenoid absorption region. The minor 806 nm peak was due to emission by B800-BChls, while the major 860 nm peak was due to emission by B850-BChls. The fluorescence intensity of U-LH2 at 860 nm was lower than that of T-LH2, showing the energy transfer within LH2 was affected by MPE association. Although the detailed molecular mechanism underlying this phenomenon had yet to be elucidated, it remained possible that change in fluorescence intensity might also, in part, reflect an altered conformation of integral LH2. Effects of MPE association on the structural stability of LH2 A comparison was made of the change in alkaline-stability between U-LH2 and T-LH2, as shown in Fig. 6. The U-LH2 and T-LH2 were quite stable over a broad pH range (pH from 8.0 to 12.0). At pH 13.0, the absorbance at 850 nm in U-LH2 was attenuated and blue-shifted to 826 nm, the absorbance at 800 nm decreased, while that at 780 nm increased. In contrast, the intensity of the 850 nm band in T-LH2 decreased but without obvious position change, the relative intensity of the 800 nm band also decreased slightly, while a broad absorption peak around 780 nm loomed. The result suggested that the quaternary structure of U-LH2 was less stable to alkaline than that of T-LH2. Discussion The observation of altered absorption spectral properties of LH2 raises the question of what caused the formation of the absorption peak at 423 nm. The pigment compositions of U-LH2 and T-LH2 were investigated and compared systematically. In both LH2, spheroidene was maintained as the major carotenoid, and BChl aP was the major BChl a. However, a remarkable difference in the pigment
Fig. 5. Fluorescence spectra of T-LH2 (solid line) and U-LH2 (dashed line). The excitation wavelength was at 480 nm.
composition between the two LH2 is that MPE was detected in U-LH2 but not in T-LH2. Since both spheroidene and BChl aP can not generate a 423 nm absorption peak either in vivo or in vitro, and no any neurosporene, hydroxyneurosporene or methoxyneurosporene was detected, thus ruling out the possibility that the absorption peak at 423 nm in U-LH2 was contributed by carotenoid. Therefore, the absorption peak at 423 nm in U-LH2 may be explained as being caused by the accumulation of MPE. This assignment was consistent with the observations of Ouchane et al. [20] that Rubrivivax gelatinosus BChE mutant accumulated large amounts of MPE when grown under low oxygenation conditions, and its membrane exhibited an intensive absorption peak at 423 nm. To ensure that the origin of the 423 nm peak was indeed related to MPE, we estimated the effects of MPE-protein interactions on the absorption spectra of MPE. The absorption band at 415 nm of MPE was, as expected, red-shifted to 423 nm upon mixing with LH2 and its apoproteins. These results suggested that the 423 nm peak in LH2 derived from MPE association. Sawicki and Willows [23] reported that the soret band of MP could be red-shifted from 415 nm to 422 nm when they were added to S-adenosyl-methionine: MP O-methyltransferase (BChM) and speculated MP carrier (BChJ). The authors believed that the spectral changes implied that MP bound in a hydrophobic environment on these proteins, because the shift in the soret peak is often characteristic of a nonplanar distortion of the porphyrins [24]. In our work, the similar red-shift in soret peak of MPE indicated that MPE binded to both LH2 and its apoproteins in vitro. The absorption at 270 nm characteristic of proteins was increased after MPE addition in our in vitro assembly experiment. We also noted that the absorption at 270 nm in isolated U-LH2 was slightly higher than in isolated T-LH2 (Fig. 2). Such an increase may be attributed to MPE-protein interaction. It has been long known that BChls precursors porphyrins bind to proteins, but the mode of
Fig. 4. Absorbance spectral changes of magnesium protoporphyrin IX monomethyl ester (MPE) after interaction with bovine serum albumin (A), apoproteins (B) or T-LH2 (C). Line 2 represents the spectra of MPE alone; line 1, 4 and 6 represent the spectra of bovine serum albumin (BSA), apoproteins and T-LH2; line 3, 5 and 7 represent the spectra of the interactions of BAS, apoproteins and T-LH2 with MPE.
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the BChl a molecules in LH2. MPE combination influenced the conformations of integral LH2. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 31270106 and 31070054), by National Natural Science Foundation of Fujian Province (No. 2012J01136), by National Marine Public Industry Research (No. 201505026). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
Fig. 6. Spectral changes in the near infrared regions of T-LH2 and U-LH2 induced by alkaline treatment.
[15] [16] [17] [18] [19]
binding remains unsolved [25,26]. Our results showed that there was no obvious change in the Qy absorption bands of the BChl a in T-LH2, indicating that MPE did not bind to sites involved in the spectral tuning of BChls. However, the spectroscopic data from fluorescence measurements and alkaline-stability demonstrated that the MPE association influenced the conformation of the integral LH2. In conclusion, the 423 nm peak in U-LH2 was caused by MPE association. MPE could bind to LH2 proteins, but did not replace
[20] [21] [22] [23] [24] [25] [26]
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