Previous Article in Journal
Biological Control of Sclerotinia sclerotiorum on Greenhouse Lettuce Using Trichoderma koningiopsis Agg
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microbiology Research
Volume 16
Issue 2
10.3390/microbiolres16020036
microbiolres-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effect of Carotenoid Composition on Stability and Light-Induced Oxidative Damage of the LH2 Complexes Isolated from Ectothiorhodospira haloalkaliphila

by
Denis V. Yanykin
1,2,*,
Mark O. Paskhin
1,
Sergey A. Shumeyko
1,
Aleksandr A. Ashikhmin
2,* and
Maxim A. Bolshakov
2
1
Prokhorov General Physics Institute of the Russian Academy of Sciences, Vavilov Str. 38, 119991 Moscow, Russia
2
Institute of Basic Biological Problems of the Russian Academy of Sciences, Federal Research Center “Pushchino Scientific Center for Biological Research” FRC PSCBR of the Russian Academy of Sciences, 2 Institutskaya Str., 142290 Pushchino, Russia
*
Authors to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(2), 36; https://doi.org/10.3390/microbiolres16020036
Submission received: 28 November 2024 / Revised: 30 December 2024 / Accepted: 30 January 2025 / Published: 2 February 2025
Browse Figures
Versions Notes

Abstract

:
Earlier, it has been shown that carotenoid-dependent singlet oxygen photogeneration in LH2 of Ectothiorhodospira haloalkaliphila leads to damage to pigments and protein. Present work continues this investigation using LH2 complexes with altered carotenoid composition: carotenoid-less LH2, and LH2 complexes with incorporated neurosporene, spheroidene, or rhodopin (LH2-Neu, LH2-Sph, or LH2-Rho, respectively). This work provides the first data on the products (hydroperoxides of organic molecules, most likely components of the protein matrix of the complexes) of the interaction of singlet oxygen with LH2 components with a modified carotenoid composition; the ability of various carotenoids to both influence the stability of LH2 and participate in oxidative damage to the complexes is assessed. It was shown that inhibition of carotenoid synthesis led to a decrease in LH2 thermal stability and reduced the light-induced oxidative damage to bacteriochlorophyll and protein. Re-incorporation of exogenous carotenoids did not return stability of the complexes but reduced the tendency of complexes to aggregate, and (in the case of LH2-Rho) reactivated both photooxidation of bacteriochlorophyll and photoproduction of organic hydroperoxides. It was concluded that carotenoids play an important role in comple x stability and are capable of inducing oxidative damage to LH2 components through singlet oxygen photogeneration.

1. Introduction

In purple photosynthetic bacteria, the primary processes of photosynthesis involve several steps: (1) photon absorption by a network of antenna-based light-harvesting (LH) complexes, (2) excitation energy transfer from these complexes to reaction centers (RCs), and (3) a primary charge separation process in the photosynthetic membrane, resulting in the formation of a proton gradient used for ATP synthesis [1,2,3]. Antenna complexes are able to harvest light from a wider spectral range than RCs, allowing bacteria to increase their efficiency in using solar energy [4,5,6,7].
In most purple bacteria, two types of LH complexes are usually distinguished according to their in vivo absorption maxima in the near-IR region [1,7,8,9,10,11,12]. The first type includes the LH1 complex (B875), which has a main absorption maximum in the near-IR region at ~865–890 nm and forms the so-called ‘core’ LH1-RC complex with RC. This complex is found in all purple photosynthetic bacteria [2,9]. The second type of LH complex is the LH2 complex (B800-850). It is characterized by two main absorption maxima at ~800 and ~850 nm. The LH2 complex is located more peripherally relative to the LH1 complex and provides energy transfer to the RC via the LH1 complex [5,9]. The above complexes are located in invaginations of the inner membrane and are well-ordered structures built from integral membrane proteins that non-covalently bind several photosynthetic pigments (bacteriochlorophyll and carotenoids) [13,14,15,16]. For a number of purple bacteria, structures of LH2 complexes have been determined, comprising seven [17], eight [18,19,20], and nine pairs of α/β-heterodimers [21,22,23]. For example, the LH2 complex from Rhodoblastus acidophilus (formerly Rhodopseudomonas acidophila) strain 10050 is a nonamer consisting of nine pairs of α/β-heterodimers forming inner and outer rings with diameters of 36 Å and 68 Å, respectively. The double ring of α-β heterodimers serves as a scaffold for two rings of bacteriochlorophyll a molecules (BChl800 and BChl850) and carotenoid molecules. The entire LH2 complex contains 27 bacteriochlorophyll molecules and 9 carotenoid molecules [22,24,25].
Carotenoids in purple bacteria are accessory photosynthetic pigments that perform several important functions [26,27,28,29,30]. They effectively stabilize the structure of LH complexes, collect light in the region of minimal absorption of bacteriochlorophyll (430–570 nm), and protect against potentially dangerous singlet and derivative triplet excited states of bacteriochlorophyll, preventing the formation of reactive oxygen species (ROS). The system of conjugated double bonds of the pigment molecule is capable of quenching the triplet states of bacteriochlorophyll and dissipating the energy received as heat, but if singlet oxygen is still formed, then carotenoids quench it as well. Currently, the hypothesis about the protective function of carotenoids is generally accepted [31,32]. ROS can be represented by a variety of highly reactive and toxic oxygen species that cause damage to proteins, lipids, pigments, carbohydrates, and DNA, ultimately leading to cell death [33,34,35,36]. In purple bacterial cells, ROS can be generated by light exposure through the formation of excited triplet states of bacteriochlorophyll [29,37,38]. It has also been reported that blue-green light absorbed by carotenoids may participate in the formation of singlet oxygen in LH2 complexes of purple sulfur bacteria [39,40,41,42,43,44], as well as by individual carotenoids in model systems [43,44,45,46,47]. We have recently shown that blue-green light can also lead to the formation of hydroperoxides of organic molecules and, as a consequence, to a change in the hydrodynamic radius of LH2 complex proteins of the purple sulfur bacterium Ectothiorhodospira (E.) haloalkaliphila [48].
This work is devoted to the study of the effect of carotenoid composition on photo-induced (375 > λ > 600 nm) processes in carotenoidless light-harvesting complexes LH2 (LH2-DPA) of the purple sulfur bacterium E. haloalkaliphila before and after incorporation of exogenous carotenoids (rhodopin (LH2-Rho), neurosporene (LH2-Neu), and spheroidene (LH2-Sph)). The influence of qualitative carotenoid composition on the photooxidation of bacteriochlorophyll, photoproduction of organic hydroperoxides, and stability protein matrix of LH2 complex are discussed.

2. Materials and Methods

2.1. Isolation, Purification, and Characterization of LH2-Containing Preparations

Normal or carotenoidless cells (DPA-cells) of purple sulfur bacterium E. haloalkaliphila were grown on Pfenning’s medium under illumination provided by 75 W incandescent lamps (2000 lux) at 26 ± 2 °C [49] in the absence or in the presence of 71 μM diphenylamine (DPA), respectively. Cells were collected in the stationary growth phase on the 4th–6th day of cultivation. The obtained biomass was immediately treated with liquid nitrogen and stored at −18 °C.
To isolate normal or carotenoidless pigment-containing membranes, corresponding cells were resuspended in 10 mM Tris-HCl-buffer (pH 8.0) and disrupted on an ultrasonic disintegrator UZDN-1 (Ultrasonics, Saint-Petersburg, Russia) [50]. Undestroyed cells and fragments of the cell wall were removed by centrifugation using a K24 centrifuge (Janetzki, Germany) for 10 min at 5000 rpm. The pigment-containing membranes isolated from the experiment were treated with liquid nitrogen and stored at −18 °C.
LH2 complexes were isolated by ion-exchange chromatography on a DEAE-TOYOPEARL 650 S column in a linear gradient from 0.06 to 0.22 M NaCl; 2.5% n-Dodecyl β-maltoside (DM) was used for membrane solubilization. The LH2 complexes were eluted at 0.14 M NaCl [51], then desalted and concentrated using Amicon Ultra 50 concentrating tubes (Merck, Darmstadt, Germany) at 2900 rpm on a CM 6M centrifuge (ELMI, Riga, Latvia). Isolated LH2 complexes were suspended (at 1.16 µM) in a medium containing 50 mM Tris-HCl (pH 7.5) and stored at −76 °C.
Incorporation of exogenous carotenoids was performed using the carotenoidless membranes. Preparations were considered as carotenoid-free samples if the total carotenoid content was less than 5% in comparison to control samples. Carotenoids used for incorporation into LH2 of DPA-membranes were isolated from membranes of Allochromatium vinosum, E. haloalkaliphila, and Cereibacter sphaeroides as described previously [52]. Then, 0.15 mL of Tris-HCl buffer (50 mM, pH 7.5) and 0.05 mL of DM (20%) were added to 0.3 mL DPA-membranes (at density of 35–40 optical units at 850 nm). The resulting concentration of bacteriochlorophyll was ≈0.8 μM. In order to avoid carotenoid aggregation, carotenoid solution was added to the resulting mixture fractionally (100 μL at a time). To remove the acetone and methanol from the mixture, the samples were dialyzed in Tris-HCl buffer (50 mM, pH 7.5) after each addition of carotenoids. Up to 10 portions of carotenoids were added in one procedure.
The bacteriochlorophyll concentration was determined in Tris-HCl solution as described in [44]. PG200N Spectral PAR Meter (UPRtek, Zhunan, Miaoli, Taiwan) was used to estimate light spectra and flux density. The ζ-potential of LH2 preparations was determined using Zetasizer Ultra (Malvern Panalytical, Malvern, UK) at 25 °C. Temperature dependence of viscosity of LH2 preparation in solution was obtained using a SmartPave 102 rheometer (Anton Paar GmbH, Germany). The 3D fluorescence spectrum of LH2 preparations was measured using a Jasco FP-8300 spectrofluorimeter (JASCO Applied Sciences, Victoria, BC, Canada) at 25 °C. Absorption spectra were recorded on a Cary 50 spectrophotometer (Agilent Technology, Santa Clara, CA, USA). Pigment analysis of the isolated pigment-protein LH2 complexes was performed using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) using a 4.6 × 250 mm Agilent Zorbax SB-C18 reversed-phase column with particle size of 5 μm (Agilent, Santa Clara, CA, USA). To separate the pigment mixture, solution ”A” (23% ethyl acetate 69% acetonitrile and 8% water) and solution ”B” (pure ethyl acetate) were used. We used the following solvent gradient. The solution “A” was pumped through the column for the first 5 min. The solution “A” was replaced with a linear gradient (0–25%) of solution B for 5–25 min. Then, from 25 to 40 min, the proportion of solution B was gradually increased from 25 to 100%. At the end of the analysis, 100% of the solution B was passed through the column for 3 min. The flow rate of all solvents was one mL/min. Carotenoids were identified based on their absorption spectra and their retention times on the column [50].

2.2. Registration of Organic Hydroperoxides Photoproduced in LH2 Preparations

To determine the photoproduction of organic hydroperoxides in LH2 preparations, the previously described approach was used [53,54]. Spy-LHP (a low-fluorescent compound, which can be oxidized with hydroperoxides to form a high-fluorescent compound) was used to detect them. According to the manufacturer’s description, Spy-LHP is highly specific for lipophilic organic hydroperoxides and does not react with hydroxyl radicals, superoxide anion, nitric oxides, peroxynitrite, and alkyl peroxyl radicals, and other species. Two peroxide types (highly lipophilic (LP-OOH) and relatively hydrophilic (HP-OOH)) were determined based on its different reaction rate with Spy-HP. The LP-OOH peroxide oxidized Spy-LHP in 5 min; the reaction with HP-OOH occurred very slowly and did not end after 3 h [54].
The samples were illuminated in a thermostatted cylindrical cell (with a diameter of one cm) with liquid cooling. The cell temperature was maintained at 25 °C with continuous stirring. For the experiment, 2 mL of the sample (0.9 optical units at 850 nm) was used. An ETU LETI illuminator with a KGM-500 lamp (500 W) was used as a light source. The light was passed through a combination of SZS-22 + ZhS-12 light filters (375 > λ > 600 nm). After illumination or dark incubation for the specified time, 400 μL of the sample was added to 3600 μL of SPY-LHP solution in ethanol and the kinetics of fluorescence changes (λex = 524 nm, λem = 538 nm) were measured at 37 °C. The remaining volume of the sample was used for other measurements: absorption and fluorescence spectra, ζ-potential, and viscosity.
The quantity of peroxides (LP-OOH and HP-OOH) was determined using a m-Chloroperbenzoic acid (MCPBA, a standard of lipophilic peroxide) and tert-Butyl hydroperoxide (TBHP, a standard of hydrophilic peroxide) in the presence of LH2 preparations as previously described [48,53,54].

2.3. Statistical Analysis

Statistical significance of differences between groups was determined by one-way analysis of variance (ANOVA), followed by post hoc comparison using Tukey’s test and Student’s t-test for independent means. The normality and homoscedasticity requirements were checked using the Shapiro–Wilk test and Goldfeld–Quandt test, respectively. The difference was considered statistically significant if p ≤ 0.05. All measurements were repeated at least 3 times.

3. Results

HPLC analysis (Figure 1, curve 1) and absorption spectrum (Figure 2A, curve 1) of LH2 preparations isolated from untreated cells’ purple sulfur bacteria E. haloalkaliphila (LH2-control preparations) showed that the LH2 complexes used in present investigation contained bacteriochlorophyll, didehydrorhodopin, rhodopin, spirilloxanthin, anhydrorhodovibrin, and lycopene. The preparations contained 7.98 ± 0.01 molecules of carotenoid per one LH2 complex.
Inhibition of carotenoid synthesis with DPA-treatment of E. haloalkaliphila cells led to the assembly of LH2-complexes with almost complete loss of colored carotenoids (LH2-DPA preparations; Figure 1, curve 2; Figure 2A, curve 2) which is consistent with the previously published data [55,56]. The carotenoid content in LH2 preparations are presented in Table 1. LH2-DPA preparations contained trace amounts of neurosporene, ζ—carotene, phytoene, and phytofluene. Incubation of LH2-DPA preparations in the presence of purified neurosporene, spheroidene, or rhodopin resulted in the incorporation of the carotenoids into the light-harvesting complexes and formation of corresponding LH2-carotenoid complexes (LH2-Neu, LH2-Sph, or LH2-Rho, respectively). Figure 1 and Figure 2 (curves 3–5) confirm the incorporation of the carotenoids into the LH2. It was shown that the efficiency of incorporation of neurosporene, spheroidene, or rhodopin was 86%, 52%, or 72%, respectively. The carotenoid composition of the obtained preparations was as follows. LH2-Neu: 0.07, 6.78, and 0.02 molecules of lycopene, neurosporene, and ζ—carotene per one LH2 complex, respectively; LH2-Sph: 4.16 molecules of spheroidene per one LH2 complex; LH2-Rho: 0.22, 5.46, and 0.07 molecules of didehydrorhodopine, rhodopin, and spirilloxanthin per one LH2 complex, respectively (Table 1).
Illumination (20 min, 375 > λ >600 nm, the carotenoid absorption region) of LH2 preparations led to changes in absorption spectrum. Photobleaching of the bacteriochlorophyll band and the appearance of a 3-acetyl-chlorophyll band in LH2-control preparations were demonstrated (Figure 2B, curve 1). Bacteriochlorophyll photobleaching and the appearance of a 3-acetyl-chlorophyll band reflect the formation of 3-acetyl-chlorophyll as a result of bacteriochlorophyll photooxidation. The absorption spectrum of carotenoidless LH2 preparations does not undergo significant changes after illumination (Figure 2B, curve 2). A slight photobleaching of the bacteriochlorophyll band is observed in LH2-Neu and LH2-Sph preparations (Figure 2B, curves 3 and 4). This may indicate that the incorporation of these carotenoids activates processes leading to the photooxidation of bacteriochlorophyll in the control samples. However, compared to the LH2-control, the efficiency of these processes in LH2-Neu and LH2-Sph is extremely low. The incorporation of rhodopin into the LH2-DPA led to the increase in the photosensitivity of the samples. The amplitude of the negative bacteriochlorophyll band in the differential spectrum was about 40% in comparison with the amplitude of the bacteriochlorophyll band in the LH2-control preparations (Figure 2B, curve 5).
It is known that damage to proteins is usually accompanied by a change in their fluorescent properties. Depending on the target for damage, fluorescence can either increase or decrease [57,58,59,60,61,62]. Figure 3 shows the fluorescence spectra of LH2-control preparations before and after illumination. It was revealed that illumination led to an increase in fluorescence intensity. The positive changes in the protein region of difference “light minus dark” fluorescence spectra of LH2 preparations may reflect damage to the protein matrix of the LH2 complexes (Figure 4A). However, light-induced changes in fluorescence intensity of the protein component of the LH2-DPA preparations were negligible, as were LH2-Neu and LH2-Sph (Figure 4B–D). LH2-Rho demonstrated a small increase in the fluorescence intensity of the protein component. It should be noted that the fluorescence intensity of unilluminated preparations of all types was practically the same.
A change in the temperature dependence of the sample viscosity before and after illumination is known may reflect light-induced changes in the stability of the complexes. It was previously shown that illumination of LH2 preparations led to changes in thermal stability of the protein matrix of the samples [48]. The data presented in Figure 5 repeat this observation (compare curve 1 and curve 2 in Figure 5A,B). Thermal stability of the carotenoidless LH2 complexes is significantly reduced. If in the LH2-control preparations, the heat-induced transition started at ≈50 °C, then in LH2-DPA preparations such a transition was observed already at 31–33 °C, indicating the instability of the complexes. Surprisingly, the incorporation of rhodopin as well as neurosporene and spheroidene did not lead to an increase in the temperature of the state transition of the complexes (Figure 4). Pre-illumination of the LH2-DPA, LH2-Neu, LH2-Sph and LH2-Rho preparations did not result in any additional shift of the transition point to lower temperatures.
Table 2 shows the influence of illumination of LH2 complexes on the ζ-potential, which reflects the tendency of the components of a colloidal solution to aggregate. It was shown that the illumination of samples did not lead to the changes in ζ-potential (except for the control samples, in which some decrease in the parameter was observed). However, carotenoid composition affects the ζ-potential of LH2 preparations. So, ζ-potential of LH2-control, LH2-Neu, and LH2-Sph was significantly higher in comparison with LH2-Rho and LH2-DPA. Present data may indicate that carotenoid composition of LH2 strongly affect the electrical charge of the complex surface and stability of the LH2 complexes in the colloid.
Earlier, it has been shown that illumination of LH2 preparations isolated from E. haloalkaliphila led to singlet oxygen-dependent formation of organic hydroperoxides (R-OOH) on the protein matrix [48]. The results of the present work are consistent with these data. Two types of organic hydroperoxides formed under illumination (20 min, 375 > λ >600 nm) of LH2-control preparations were revealed: highly lipophilic (LP-OOH, 11.4 molecules per one LH2) and relatively hydrophobic (HP-OOH, 70 per one LH2) (Figure 6, curve 1). However, photoformation of R-OOH in LH2-DPA preparations was not detected (curve 2).
The effect of the incorporation of exogenous carotenoids in LH2 on R-OOH photoproduction was dependent on carotenoid species. While the incorporation of neurosporene or spheroidene into LH2 did not lead to R-OOH photoformation (curves 3 and 4), the incorporation of rhodopin resulted in photoformation of both LP-OOH and HP-OOH (curve 5). The quantity of photoproduced LP-OOH and HP-OOH in LH2-Rho reached almost 30% compared to LH2-control preparations.
The addition of Rose Bengal (RB, singlet oxygen-generating photosensitizer) before illumination of the LH2-control samples did not lead to an increase in the photoproduction of R-OOH (Figure 7). RB reduced the photoproduction of LP without affecting HP, which is consistent with previously obtained data. Moreover, RB did not increase R-OOH photoformation in other (LH2-DPA, LH2-Neu, LH2-Sph, or LH2-Rho) preparations. On the one hand, this may indicate that hydroperoxide precursor molecules are not formed in LH2 due to the action of DPA. On the other hand, the incorporation of carotenoids into LH2 does not promote their appearance.
Thus, our data suggest that carotenoids both stabilize the LH2 complexes (Figure 5 and Table 2) and promote the light-induced oxidative damage to the LH2 (Figure 2, Figure 6 and Figure 7).

4. Discussion

Previously, the generation of singlet oxygen was shown upon illumination of LH2 preparations with light, which is effectively absorbed by carotenoids but practically does not excite bacteriochlorophyll molecules, which was accompanied by oxidation of bacteriochlorophyll to 3-acetylchlorophyll [39,40,41,62,63] and photoformation of R-OOH with damage to protein molecules included in the complexes [48]. Similar conclusions can be drawn from the data presented in this work. Indeed, singlet oxygen is capable of oxidizing bacteriochlorophyll and interacting with protein molecules, damaging them [33,34]. Degradation of plant LHC2 proteins under the influence of singlet oxygen is known [64,65]. Amino acids of the protein (tyrosine, tryptophan, and histidine) are capable of forming hydroperoxides when interacting with singlet oxygen [33,34,66,67,68,69,70,71,72,73,74]. The photogeneration of singlet oxygen associated with photosynthetic pigments has been known for a long time. For example, in addition to isolated antenna complexes of bacteria, singlet oxygen was detected during illumination of plant antenna complexes [75,76,77,78] and in model pigment solutions [79,80,81,82]. In this case, photogeneration of singlet oxygen is usually associated with pigments of chlorophyll nature. Nevertheless, the possibility of energy transfer from a carotenoid in the triplet state to molecular oxygen with the formation of singlet oxygen was demonstrated previously [83]. The authors indicate that carotenoids with the number of conjugated double bonds (N) ≤ 11 are energetically capable of this. The mechanism of formation of the triplet state of carotenoids as a result of singlet–triplet excitation fission (1Car* + Car → 3Car + 3Car) was proposed earlier [44]. This process is well known in photochemical studies and has been described in the case of light-harvesting complexes of Alc. vinosum [43,44,84,85,86]. Moreover, this process is very fast and can have a fairly high quantum yield (up to 0.32) [87]. Carotenoids of the LH2-control preparation have a sufficient number of conjugated double bonds. In addition, carotenoid-dependent damage to the proteins of the light-harvesting complex and photoformation of R-OOH due to the generation of singlet oxygen were confirmed by inhibitory analysis and the absence of effects upon illumination in the absorption band of bacteriochlorophyll [48]. In this work, we show that illumination of LH2-DPA preparations lacking carotenoids does not result in damage to the proteins of the light-harvesting complex and photoformation of R-OOH, despite the fact that bacteriochlorophyll is retained in their structure. It should be noted that LH2-DPA complexes initially have reduced thermal stability (Figure 6) and a reduced surface charge (Table 2). It has been previously demonstrated that carotenoids play an important role in LH2 complexes, increasing their structural stability [88]. We assumed that the incorporation of carotenoids with different N into LH2 may cause different effects on both the stability of the complexes and the photoproduction of 3-acetyl-chlorophyll and R-OOH. We selected neurosporene (N = 9), spheroidene (N = 10), and rhodopin (N = 11) for incorporation. The choice of carotenoids for incorporation was based on the fact that these carotenoids meet the criteria necessary for incorporation. First, these carotenoids can be obtained in sufficient quantities. Second, these carotenoids can be effectively incorporated into light-harvesting complexes. Most other carotenoids either do not practically incorporate, or their incorporation is very slow and is accompanied by serious destruction of LH2-DPA due to the effect of the solvent. Among the carotenoids selected for incorporation, there are both carotenoids with a relatively short chain of conjugated double bonds and a carotenoid with a chain length of conjugated double bonds sufficient for the generation of singlet oxygen. Indeed, the incorporation of carotenoids with a short chain of conjugated double bonds (neurosporene (N = 9) and spheroidene (N = 10)) did not result in either activation of bacteriochlorophyll photobleaching (Figure 2) or R-OOH photoformation (Figure 4) or an increase in the stability of the complexes (Figure 6). However, these carotenoids significantly shifted the ζ-potential to a more negative region, bringing its value closer to that measured in the control samples. The incorporation of rhodopin (N = 11) also had no effect on the thermal stability of the complexes but activated the photoformation of R-OOH and photobleaching of bacteriochlorophyll (≈30% and ≈40% compared to the control, respectively). At the same time, the restoration of ζ-potential was not as strong as in the case of neurosporene and spheroidene. It is known that oxidative damage to protein particles (including as a result of the action of reactive oxygen species) is accompanied by a change in ζ-potential [89,90,91,92,93]. The difference in the ζ-potential of proteins can reflect the surface charge on proteins. This parameter allows one to estimate the tendency of colloid particles to aggregate or the ability of the colloid to be stable. Table 2 shows that DPA treatment results in a shift of ζ-potential of LH2 colloid from approximately −30 mV to −12 mV. Re-incorporation of carotenoids mitigates this effect. These data indicate a significant effect of carotenoids on the surface charge of the proteins of the complex, which may be of great importance for the functioning of the antenna complex in vivo. Despite this, the incorporation of carotenoids did not lead to the restoration of the thermal stability of the complexes (Figure 6). However, according to other data, carotenoids incorporated into LH2-DPA preparations had a significant effect on the stability of the complexes [88].
There are several possible explanations for why illumination of the LH2-DPA does not result in photoformation of R-OOH, etc. The first possibility is that carotenoids are required for photoformation of R-OOH. The second possibility is that in the presence of DPA, altered complexes without the hydroperoxide precursor molecules are assembled. The addition of an artificial singlet oxygen photosensitizer did not result in an increase in R-OOH production in LH2-DPA preparations. However, given the fact that RB in the control samples surprisingly inhibited R-OOH photoformation, we cannot claim that LH2-DPA preparations do not have sites where singlet oxygen can oxidize. On the contrary, the incorporation of rhodopin showed that the incorporation of a carotenoid with the required number of conjugated double bonds is sufficient for the reactivation of the studied processes. The question of the functional activity of the carotenoids incorporated into LH2-DPA preparations remains debatable. On the one hand, incomplete restoration of the efficiency of bacteriochlorophyll photobleaching and R-OOH photoformation after rhodopin incorporation may indicate that other carotenoids also “work” in the control preparations. On the other hand, the efficiency and physiologicality of exogenous carotenoid incorporation may be incomplete, which leads to a loss of functionality. Third, DPA treatment could lead to the assembly of LH2 complexes with disturbances that are not corrected by carotenoid incorporation.
Thus, our data indicate that carotenoids, on the one hand, stabilized the LH2 complexes and, on the other hand, were involved in the light-induced oxidative damage to the LH2 probably due to redox activity of the singlet oxygen, and rhodopin may be one of the carotenoids that is able to participate in the generation of singlet oxygen in LH2-control preparations. Moreover, this work provides the first data on the products (hydroperoxides of organic molecules, most likely components of the protein matrix of the complexes) of the interaction of singlet oxygen with LH2 components with a modified carotenoid composition; the ability of various carotenoids to both influence the stability of LH2 and participate in oxidative damage to the complexes is assessed.

Author Contributions

Conceptualization, D.V.Y. and M.A.B.; methodology, D.V.Y. and M.A.B.; formal analysis, D.V.Y. and M.A.B.; investigation, D.V.Y., M.O.P., S.A.S., A.A.A. and M.A.B.; data curation, D.V.Y., M.O.P. and M.A.B.; writing—original draft preparation, D.V.Y., A.A.A. and M.A.B.; writing—review and editing, D.V.Y., A.A.A. and M.A.B.; funding acquisition, M.A.B. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the Russian Science Foundation (grant 23-24-00362, https://www.rscf.ru/project/23-24-00362/ accessed on 13 June 2023).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors thank Shared Core Facilities of the Pushchino Scientific Center for Biological Research (http://www.ckp-rf.ru/ckp/670266/).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Hu, X.; Ritz, T.; Damjanović, A.; Autenrieth, F.; Schulten, K. Photosynthetic apparatus of purple bacteria. Q. Rev. Biophys. 2002, 35, 1–62. [Google Scholar] [CrossRef]
  2. Cogdell, R.J.; Gall, A.; Köhler, J. The architecture and function of the light-harvesting apparatus of purple bacteria: From single molecules to in vivo membranes. Q. Rev. Biophys. 2006, 39, 227–324. [Google Scholar] [CrossRef] [PubMed]
  3. Miller, L.C.; Martin, D.S.; Liu, L.-N.; Canniffe, D.P. Composition, organisation and function of purple photosynthetic machinery. In Microbial Photosynthesis; Springer: Singapore, 2020; pp. 73–114. [Google Scholar]
  4. Thornber, J.P.; Sokoloff, M.K. Photochemical reactions of purple bacteria as revealed by studies of three spectrally different carotenobacteriochlorophyll-protein complexes isolated from Chromatium, strain D. Biochemistry 1970, 9, 2688–2698. [Google Scholar] [CrossRef] [PubMed]
  5. Sener, M.; Strumpfer, J.; Singharoy, A.; Hunter, C.N.; Schulten, K. Overall energy conversion efficiency of a photosynthetic vesicle. Elife 2016, 5, e09541. [Google Scholar] [CrossRef]
  6. Lokstein, H.; Renger, G.; Götze, J.P. Photosynthetic light-harvesting (antenna) complexes—Structures and functions. Molecules 2021, 26, 3378. [Google Scholar] [CrossRef] [PubMed]
  7. Kimura, Y.; Tani, K.; Madigan, M.T.; Wang-Otomo, Z.-Y. Advances in the spectroscopic and structural characterization of core light-harvesting complexes from purple phototrophic bacteria. J. Phys. Chem. B 2023, 127, 6–17. [Google Scholar] [CrossRef] [PubMed]
  8. Moskalenko, A.; Erokhin, Y.E. Isolation of the pigment–lipoprotein complexes from purple bacteriaby preparative polyacrylamide gel electrophoresis. Mikrobiologiya 1974, 43, 654–657. [Google Scholar]
  9. Gabrielsen, M.; Gardiner, A.T.; Cogdell, R.J. Peripheral complexes of purple bacteria. In The Purple Phototrophic Bacteria; Springer: Dordrecht, The Netherlands, 2009; pp. 135–153. [Google Scholar]
  10. Gardiner, A.T.; Nguyen-Phan, T.C.; Cogdell, R.J. A comparative look at structural variation among RC–LH1 ‘Core’ complexes present in anoxygenic phototrophic bacteria. Photosynth. Res. 2020, 145, 83–96. [Google Scholar] [CrossRef] [PubMed]
  11. Swainsbury, D.J.; Qian, P.; Hitchcock, A.; Hunter, C.N. The structure and assembly of reaction centre-light-harvesting 1 complexes in photosynthetic bacteria. Biosci. Rep. 2023, 43, BSR20220089. [Google Scholar] [CrossRef] [PubMed]
  12. Liu, L.-N.; Bracun, L.; Li, M. Structural diversity and modularity of photosynthetic RC− LH1 complexes. Trends Microbiol. 2024, 32, 38–52. [Google Scholar] [CrossRef] [PubMed]
  13. Bahatyrova, S.; Frese, R.N.; Siebert, C.A.; Olsen, J.D.; van Der Werf, K.O.; Van Grondelle, R.; Niederman, R.A.; Bullough, P.A.; Otto, C.; Hunter, C.N. The native architecture of a photosynthetic membrane. Nature 2004, 430, 1058–1062. [Google Scholar] [CrossRef]
  14. Şener, M.K.; Olsen, J.D.; Hunter, C.N.; Schulten, K. Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle. Proc. Natl. Acad. Sci. USA 2007, 104, 15723–15728. [Google Scholar] [CrossRef]
  15. Niederman, R.A. Structure, function and formation of bacterial intracytoplasmic membranes. In Complex Intracellular Structures in Prokaryotes; Springer: Berlin/Heidelberg, Germany, 2006; pp. 193–227. [Google Scholar]
  16. Sturgis, J.N.; Niederman, R.A. Organization and assembly of light-harvesting complexes in the purple bacterial membrane. In The Purple Phototrophic Bacteria; Springer: Berlin/Heidelberg, Germany, 2009; pp. 253–273. [Google Scholar]
  17. Gardiner, A.T.; Naydenova, K.; Castro-Hartmann, P.; Nguyen-Phan, T.C.; Russo, C.J.; Sader, K.; Hunter, C.N.; Cogdell, R.J.; Qian, P. The 2.4 Å cryo-EM structure of a heptameric light-harvesting 2 complex reveals two carotenoid energy transfer pathways. Sci. Adv. 2021, 7, eabe4650. [Google Scholar] [CrossRef] [PubMed]
  18. Koepke, J.; Hu, X.; Muenke, K.; Schulten, K.; Michel, H. The crystal structure of the light-harvesting complex II (B800-850) from Rhodospirillum molischianum. Structure 1996, 4, 581. [Google Scholar] [CrossRef]
  19. Leiger, K.; Linnanto, J.M.; Rätsep, M.; Timpmann, K.; Ashikhmin, A.A.; Moskalenko, A.A.; Fufina, T.Y.; Gabdulkhakov, A.G.; Freiberg, A. Controlling Photosynthetic Excitons by Selective Pigment Photooxidation. J. Phys. Chem. B 2019, 123, 29–38. [Google Scholar] [CrossRef]
  20. Burtseva, A.; Baymukhametov, T.; Ilyasov, I.; Bolshakov, M.; Moskalenko, A.; Boyko, K.; Ashikhmin, A. Structural Study of the Light-Harvesting Complex LH2 from the Purple Sulfur Bacteria Ectothiorhodospira haloalkaliphila by Cryoelectronic Microscopy. Crystallogr. Rep. 2023, 68, 879–885. [Google Scholar] [CrossRef]
  21. McLuskey, K.; Prince, S.; Cogdell, R.; Isaacs, N. The crystallographic structure of the B800-820 LH3 light-harvesting complex from the purple bacteria Rhodopseudomonas acidophila strain 7050. Biochemistry 2001, 40, 8783–8789. [Google Scholar] [CrossRef]
  22. McDermott, G.; Prince, S.; Freer, A.; Hawthornthwaite-Lawless, A.Á.; Papiz, M.; Cogdell, R.; Isaacs, N. Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 1995, 374, 517–521. [Google Scholar] [CrossRef]
  23. Qian, P.; Swainsbury, D.J.; Croll, T.I.; Castro-Hartmann, P.; Divitini, G.; Sader, K.; Hunter, C.N. Cryo-EM structure of the Rhodobacter sphaeroides light-harvesting 2 complex at 2.1 Å. Biochemistry 2021, 60, 3302–3314. [Google Scholar] [CrossRef] [PubMed]
  24. Papiz, M.Z.; Prince, S.M.; Howard, T.; Cogdell, R.J.; Isaacs, N.W. The structure and thermal motion of the B800–850 LH2 complex from Rps. acidophila at 2.0 Å resolution and 100 K: New structural features and functionally relevant motions. J. Mol. Biol. 2003, 326, 1523–1538. [Google Scholar] [CrossRef] [PubMed]
  25. Prince, S.; Howard, T.; Myles, D.; Wilkinson, C.; Papiz, M.; Freer, A.; Cogdell, R.; Isaacs, N. Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050. J. Mol. Biol. 2003, 326, 307–315. [Google Scholar] [CrossRef] [PubMed]
  26. Foote, C.S.; Chang, Y.C.; Denny, R.W. Chemistry of singlet oxygen. X. Carotenoid quenching parallels biological protection. J. Am. Chem. Soc. 1970, 92, 5216–5218. [Google Scholar] [CrossRef] [PubMed]
  27. Moskalenko, A.A.; Karapetyan, N.V. Structural role of carotenoids in photosynthetic membranes. Z. Naturforschung C 1996, 51, 763–771. [Google Scholar] [CrossRef]
  28. Theiss, C.; Leupold, D.; Moskalenko, A.A.; Razjivin, A.P.; Eichler, H.J.; Lokstein, H. Femtosecond spectroscopy of native and carotenoidless purple-bacterial LH2 clarifies functions of carotenoids. Biophys. J. 2008, 94, 4808–4811. [Google Scholar] [CrossRef] [PubMed]
  29. Telfer, A.; Pascal, A.; Gall, A. Carotenoids in photosynthesis. In Carotenoids: Volume 4: Natural Functions; Birkhäuser: Basel, Switzerland, 2008; pp. 265–308. [Google Scholar]
  30. Hashimoto, H.; Uragami, C.; Cogdell, R.J. Carotenoids and photosynthesis. In Carotenoids in Nature: Biosynthesis, Regulation and Function; Springer: Cham, Switzerland, 2016; pp. 111–139. [Google Scholar]
  31. Frank, H.; Cogdell, R. Carotenoids in photosynthesis. Photochem. Photobiol. 1996, 63, 257–264. [Google Scholar] [CrossRef]
  32. Britton, G. Functions of Intact Carotenoids. In Carotenoids. Natural Functions; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhauser Verlag: Basel, Switzerland, 2008; pp. 265–308. [Google Scholar]
  33. Wright, A.; Bubb, W.A.; Hawkins, C.L.; Davies, M.J. Singlet Oxygen–mediated Protein Oxidation: Evidence for the Formation of Reactive Side Chain Peroxides on Tyrosine Residues. Photochem. Photobiol. 2002, 76, 35–46. [Google Scholar] [CrossRef] [PubMed]
  34. Davies, M.J. Reactive species formed on proteins exposed to singlet oxygen. Photochem. Photobiol. Sci. 2004, 3, 17–25. [Google Scholar] [CrossRef] [PubMed]
  35. Foyer, C.H.; Noctor, G. Redox homeostasis and antioxidant signaling: A metabolic interface between stress perception and physiological responses. Plant Cell 2005, 17, 1866–1875. [Google Scholar] [CrossRef]
  36. Gill, S.S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010, 48, 909–930. [Google Scholar] [CrossRef]
  37. Angerhofer, A.; Bornhäuser, F.; Aust, V.; Hartwich, G.; Scheer, H. Triplet energy transfer in bacterial photosynthetic reaction centres. Biochim. Biophys. Acta (BBA)-Bioenerg. 1998, 1365, 404–420. [Google Scholar] [CrossRef]
  38. Scheer, H. An overview of chlorophylls and bacteriochlorophylls: Biochemistry, biophysics, functions and applications. In Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications; Springer: Dordrecht, The Netherlands, 2006; pp. 1–26. [Google Scholar]
  39. Makhneva, Z.K.; Ashikhmin, A.A.; Bolshakov, M.A.; Moskalenko, A.A. Carotenoids are Probably Involved in Singlet Oxygen Generation in the Membranes of Purple Photosynthetic Bacteria under Light Irradiation. Microbiology 2020, 89, 164–173. [Google Scholar] [CrossRef]
  40. Makhneva, Z.K.; Bolshakov, M.A.; Moskalenko, A.A. Carotenoids Do Not Protect Bacteriochlorophylls in Isolated Light-Harvesting LH2 Complexes of Photosynthetic Bacteria from Destructive Interactions with Singlet Oxygen. Molecules 2021, 26, 5120. [Google Scholar] [CrossRef] [PubMed]
  41. Makhneva, Z.K.; Moskalenko, A.A. Carotenoids in LH2 Complexes from Allochromatium vinosum under Illumination Are Able to Generate Singlet Oxygen Which Oxidizes BChl850. Microbiology 2022, 91, 409–416. [Google Scholar] [CrossRef]
  42. Makhneva, Z.; Bolshakov, M.; Ashikhmin, A.; Moskalenko, A. Rhodopin Incorporated into the Allochromatium vinosum LH2 Complex Is Able to Generate Singlet Oxygen under Illumination. Microbiology 2024, 93, 305–313. [Google Scholar] [CrossRef]
  43. Klenina, I.; Gryaznov, A.; Makhneva, Z.; Proskuryakov, I. Singlet-triplet Fission of Carotenoid Excitation in the Purple Phototrophic Bacteria Thermochromatium tepidum. Dokl. Biochem. Biophys. 2019, 485, 135–137. [Google Scholar] [CrossRef]
  44. Klenina, I.B.; Makhneva, Z.K.; Moskalenko, A.A.; Proskuryakov, I.I. Selective Excitation of Carotenoids of the Allochromatium vinosum Light-Harvesting LH2 Complexes Leads to Oxidation of Bacteriochlorophyll. Biochemistry 2022, 87, 1130–1137. [Google Scholar] [CrossRef] [PubMed]
  45. Ashikhmin, A.; Benditkis, A.; Moskalenko, A.; Krasnovsky, A. Phytofluene as a highly efficient UVA photosensitizer of singlet oxygen generation. Biochemistry 2020, 85, 773–780. [Google Scholar] [CrossRef] [PubMed]
  46. Ashikhmin, A.A.; Benditkis, A.S.; Moskalenko, A.A.; Krasnovsky, A.A., Jr. ζ-Carotene: Generation and quenching of singlet oxygen, comparison with phytofluene. Biochemistry 2022, 87, 1169–1178. [Google Scholar] [PubMed]
  47. Benditkis, A.; Ashikhmin, A.; Moskalenko, A.; Krasnovsky, A., Jr. Photogeneration and quenching of singlet molecular oxygen by bacterial C40 carotenoids with long chain of conjugated double bonds. Photosynth. Res. 2024, 159, 291–301. [Google Scholar] [CrossRef] [PubMed]
  48. Yanykin, D.; Paskhin, M.; Ashikhmin, A.A.; Bolshakov, M.A. Carotenoid-dependent singlet oxygen photogeneration in light-harvesting complex 2 of Ectothiorhodospira haloalkaliphila leads to the formation of organic hydroperoxides and damage to both pigments and protein matrix. PeerJ 2024, 12, e16615. [Google Scholar] [CrossRef] [PubMed]
  49. Imhoff, J.F.; Trüper, H.G. Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch. Microbiol. 1977, 114, 115–121. [Google Scholar] [CrossRef]
  50. Moskalenko, A.; Toropygina, O.; Makhneva, Z. Behavior of carotenoids in Rhodospirillum rubrum cells under cultivation with diphenylamine. Dokl Akad Nauk 1997, 355, 259–261. [Google Scholar]
  51. Bol’shakov, M.; Ashikhmin, A.; Makhneva, Z.; Moskalenko, A. Effect of illumination intensity and inhibition of carotenoid biosynthesis on assembly of peripheral light-harvesting complexes in purple sulfur bacteria Allochromatium vinosum ATCC 17899. Microbiology 2016, 85, 420–429. [Google Scholar] [CrossRef]
  52. Makhneva, Z.K.; Bol’shakov, M.A.; Ashikhmin, A.A.; Erokhin, Y.E.; Moskalenko, A.A. Influence of blue light on the structure stability of antenna complexes from Allochromatium minutissimum with different content of carotenoids. Biochem. Suppl. Ser. A Membr. Cell Biol. 2009, 3, 123–127. [Google Scholar] [CrossRef]
  53. Khorobrykh, S.A.; Khorobrykh, A.A.; Yanykin, D.V.; Ivanov, B.N.; Klimov, V.V.; Mano, J.i. Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: Evidence with a specific fluorescent probe. Biochemistry 2011, 50, 10658–10665. [Google Scholar] [CrossRef] [PubMed]
  54. Yanykin, D.; Khorobrykh, A.; Terentyev, V.; Klimov, V. Two pathways of photoproduction of organic hydroperoxides on the donor side of photosystem 2 in subchloroplast membrane fragments. Photosynth. Res. 2017, 133, 129–138. [Google Scholar] [CrossRef] [PubMed]
  55. Moskalenko, A.; Makhneva, Z. Light-harvesting complexes from purple sulfur bacteria Allochromatium minutissimum assembled without carotenoids. J. Photochem. Photobiol. B Biol. 2012, 108, 1–7. [Google Scholar] [CrossRef] [PubMed]
  56. Ashikhmin, A.; Makhneva, Z.; Moskalenko, A. The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis. Photosynth. Res. 2014, 119, 291–303. [Google Scholar] [CrossRef]
  57. Bystranowska, D.; Szewczuk, Z.; Lisowski, M.; Sitkiewicz, E.; Dobryszycki, P.; Ożyhar, A.; Kochman, M. Intramolecular cross-linking in the native JHBP molecule. Arch. Biochem. Biophys. 2012, 517, 12–19. [Google Scholar] [CrossRef]
  58. Correia, M.; Neves-Petersen, M.T.; Jeppesen, P.B.; Gregersen, S.; Petersen, S.B. UV-Light Exposure of Insulin: Pharmaceutical Implications upon Covalent Insulin Dityrosine Dimerization and Disulphide Bond Photolysis. PLoS ONE 2012, 7, e50733. [Google Scholar] [CrossRef]
  59. Correia, M.; Neves-Petersen, M.T.; Parracino, A.; di Gennaro, A.K.; Petersen, S.B. Photophysics, Photochemistry and Energetics of UV Light Induced Disulphide Bridge Disruption in apo-α-Lactalbumin. J. Fluoresc. 2012, 22, 323–337. [Google Scholar] [CrossRef]
  60. Correia, M.; Thiagarajan, V.; Coutinho, I.; Gajula, G.P.; Petersen, S.B.; Neves-Petersen, M.T. Modulating the structure of EGFR with UV light: New possibilities in cancer therapy. PLoS ONE 2014, 9, e111617. [Google Scholar] [CrossRef] [PubMed]
  61. Correia, M.; Snabe, T.; Thiagarajan, V.; Petersen, S.B.; Campos, S.R.R.; Baptista, A.M.; Neves-Petersen, M.T. Photonic Activation of Plasminogen Induced by Low Dose UVB. PLoS ONE 2015, 10, e0116737. [Google Scholar] [CrossRef] [PubMed]
  62. Neves-Petersen, M.T.; Gryczynski, Z.; Lakowicz, J.; Fojan, P.; Pedersen, S.; Petersen, E.; Bjørn Petersen, S. High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue. Protein Sci. 2002, 11, 588–600. [Google Scholar] [CrossRef] [PubMed]
  63. Makhneva, Z.; Ashikhmin, A.; Bolshakov, M.; Moskalenko, A. Bacteriochlorophyll interaction with singlet oxygen in membranes of purple photosynthetic bacteria: Does the protective function of carotenoids exist? Dokl. Biochem. Biophys. 2019, 486, 216–219. [Google Scholar] [CrossRef]
  64. Lindahl, M.; Yang, D.H.; Andersson, B. Regulatory proteolysis of the major light-harvesting chlorophyll a/b protein of photosystem II by a light-induced membrane-associated enzymic system. Eur. J. Biochem. 1995, 231, 503–509. [Google Scholar] [PubMed]
  65. Zolla, L.; Rinalducci, S. Involvement of active oxygen species in degradation of light-harvesting proteins under light stresses. Biochemistry 2002, 41, 14391–14402. [Google Scholar] [CrossRef] [PubMed]
  66. Nakagawa, M.; Okajima, H.; Hino, T. Photosensitized oxygenation of Nb-methoxycarbonyltryptophan methyl ester and Nb-methoxycarbonyltryptamine. Isolation and novel transformations of a 3a-hydroxyperoxypyrroloindole. J. Am. Chem. Soc. 1976, 98, 635–637. [Google Scholar] [CrossRef]
  67. Saito, I.; Matsuura, T.; Nakagawa, M.; Hino, T. Peroxidic intermediates in photosensitized oxygenation of tryptophan derivatives. Acc. Chem. Res. 1977, 10, 346–352. [Google Scholar] [CrossRef]
  68. Tomita, M.; Irie, M.; Ukita, T. Sensitized photooxidation of histidine and its derivatives. Prod. Mech. React. Biochem. 1969, 8, 5149–5160. [Google Scholar]
  69. Kang, P.; Foote, C.S. Synthesis of a 13C, 15N labeled imidazole and characterization of the 2, 5-endoperoxide and its decomposition. Tetrahedron Lett. 2000, 41, 9623–9626. [Google Scholar] [CrossRef]
  70. Giles, S. A Computational Investigation of a Biologically Relevant Rearrangement: Significance of Molecular Structure and Orbital Interactions as Related to the Transformation of a Model Endoperoxide into a Hydroperoxide Product. J. Young Investig. 2007, 16. [Google Scholar]
  71. Liu, F.; Lu, W.; Fang, Y.; Liu, J. Evolution of oxidation dynamics of histidine: Non-reactivity in the gas phase, peroxides in hydrated clusters, and pH dependence in solution. Phys. Chem. Chem. Phys. 2014, 16, 22179–22191. [Google Scholar] [CrossRef] [PubMed]
  72. Wei, C.; Song, B.; Yuan, J.; Feng, Z.; Jia, G.; Li, C. Luminescence and Raman spectroscopic studies on the damage of tryptophan, histidine and carnosine by singlet oxygen. J. Photochem. Photobiol. A Chem. 2007, 189, 39–45. [Google Scholar] [CrossRef]
  73. Nakane, K.; Sato, S.; Niwa, T.; Tsushima, M.; Tomoshige, S.; Taguchi, H.; Ishikawa, M.; Nakamura, H. Proximity histidine labeling by umpolung strategy using singlet oxygen. J. Am. Chem. Soc. 2021, 143, 7726–7731. [Google Scholar] [CrossRef] [PubMed]
  74. Nakane, K.; Nagasawa, H.; Fujimura, C.; Koyanagi, E.; Tomoshige, S.; Ishikawa, M.; Sato, S. Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling. Int. J. Mol. Sci. 2022, 23, 11622. [Google Scholar] [CrossRef]
  75. Krieger-Liszkay, A. Singlet oxygen production in photosynthesis. J. Exp. Bot. 2004, 56, 337–346. [Google Scholar] [CrossRef]
  76. Kramer, H.; Mathis, P. Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures. Biochim. Biophys. Acta (BBA)-Bioenerg. 1980, 593, 319–329. [Google Scholar] [CrossRef]
  77. Santabarbara, S.; Bordignon, E.; Jennings, R.C.; Carbonera, D. Chlorophyll triplet states associated with photosystem II of thylakoids. Biochemistry 2002, 41, 8184–8194. [Google Scholar] [CrossRef]
  78. Rinalducci, S.; Pedersen, J.Z.; Zolla, L. Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II. Biochim. Biophys. Acta (BBA)-Bioenerg. 2004, 1608, 63–73. [Google Scholar] [CrossRef]
  79. Egorov, S.Y.; Krasnovsky, A.A., Jr. Laser-induced luminescence of singlet molecular oxygen: Generation by drugs and pigments of biological importance. Laser Appl. Life Sci. 1991, 1403, 611–621. [Google Scholar]
  80. Krasnovsky Jr, A.; Cheng, P.; Blankenship, R.; Moore, T.; Gust, D. The photophysics of monomeric bacteriochlorophylls c and d and their derivatives: Properties of triplet state and singlet oxygen photogeneration and quenching. Photochem. Photobiol. 1993, 57, 324–330. [Google Scholar] [CrossRef] [PubMed]
  81. Borland, C.; McGarvey, D.; Truscott, T.; Codgell, R.; Land, E.J. Photophysical studies of bacteriochlorophyll a and bacteriopheophytin a—Singlet oxygen generation. J. Photochem. Photobiol. B Biol. 1987, 1, 93–101. [Google Scholar] [CrossRef]
  82. Hoebeke, M.; Damoiseau, X. Determination of the singlet oxygen quantum yield of bacteriochlorin a: A comparative study in phosphate buffer and aqueous dispersion of dimiristoyl-l-α-phosphatidylcholine liposomes. Photochem. Photobiol. Sci. 2002, 1, 283–287. [Google Scholar] [CrossRef] [PubMed]
  83. Foote, C. Singlet Oxygen; Wasserman, H.H., Murray, R.W., Eds.; Academic: Cambridge, MA, USA, 1979. [Google Scholar]
  84. Smith, K.A.; Ardelt, B.K.; Huner, N.P.A.; Krol, M.; Myscich, E.; Low, P.S. Identification and Partial Characterization of the Denaturation Transition of the Light Harvesting Complex II of Spinach Chloroplast Membranes 1. Plant Physiol. 1989, 90, 492–499. [Google Scholar] [CrossRef] [PubMed]
  85. Smith, H.; Whitelam, G.C. The shade avoidance syndrome: Multiple responses mediated by multiple phytochromes. Plant Cell Environ. 1997, 20, 840–844. [Google Scholar] [CrossRef]
  86. Gryaznov, A.A.; Klenina, I.B.; Makhneva, Z.K.; Moskalenko, A.A.; Proskuryakov, I.I. Singlet-triplet division of excitation of carotenoids of light harvesting complexes of Thermochromatium tepidum. Biophysics 2019, 64, 1045–1051. (In Russian) [Google Scholar] [CrossRef]
  87. Gradinaru, C.C.; Kennis, J.T.; Papagiannakis, E.; Van Stokkum, I.H.; Cogdell, R.J.; Fleming, G.R.; Niederman, R.A.; Van Grondelle, R. An unusual pathway of excitation energy deactivation in carotenoids: Singlet-to-triplet conversion on an ultrafast timescale in a photosynthetic antenna. Proc. Natl. Acad. Sci. USA 2001, 98, 2364–2369. [Google Scholar] [CrossRef] [PubMed]
  88. Makhneva, Z.; Bolshakov, M.; Moskalenko, A. Heterogeneity of carotenoid content and composition in LH2 of the purple sulphur bacterium Allochromatium minutissimum grown under carotenoid-biosynthesis inhibition. Photosynth. Res. 2008, 98, 633–641. [Google Scholar] [CrossRef]
  89. Hu, H.; Li, C.; Lyu, C.; Meng, X.; Li, B.; Shu, C. Effect of protein oxidation on the structural characteristics of hazelnut protein isolate. Food Sci. Technol. 2023, 43, e103922. [Google Scholar] [CrossRef]
  90. Xu, W.; Bao, Y.; Zhou, Y.; Hong, H.; Gao, R. Effect of protein oxidation on the structure and emulsifying properties of fish gelatin. Food Res. Int. 2024, 195, 114963. [Google Scholar] [CrossRef]
  91. Wang, X.; Wu, Q.; Mao, X.; Zhang, J. Effect of Alkyl Peroxyl Radical Oxidation on the Oxidative Stability of Walnut Protein Emulsions and Their Adsorbed Proteins. Foods 2024, 13, 1513. [Google Scholar] [CrossRef]
  92. Li, S.; Huang, Y.; An, F.; Huang, Q.; Geng, F.; Ma, M. Hydroxyl radical-induced early stage oxidation improves the foaming and emulsifying properties of ovalbumin. Poult. Sci. 2019, 98, 1047–1054. [Google Scholar] [CrossRef] [PubMed]
  93. Yu, Q.; Shi, T.; Xiong, Z.; Yuan, L.; Hong, H.; Gao, R.; Bao, Y. Oxidation affects dye binding of myofibrillar proteins via alteration in net charges mediated by a reduction in isoelectric point. Food Res. Int. 2023, 163, 112204. [Google Scholar] [CrossRef] [PubMed]
Microbiolres 16 00036 g001
Figure 1. HPLC analysis of pigment composition of the LH2 complexes isolated from cells’ purple sulfur bacterium E. haloalkaliphila; (1) LH2-control preparations, (2) LH2-DPA preparations, (3) LH2-Neu preparations, (4) LH2-Sph preparations, or (5) LH2-Rho preparations. Peak identification: (a) bacteriochlorophyll, (b) didehydrorhodopin, (c) rhodopin, (d) spirilloxanthin, (e) anhydrorhodovibrin, (f) lycopene, (g) spheroidene, and (h) neurosporene. The chromatograms were normalized according to bacteriochlorophyll peaks. Detection was performed using DAD detector at 190–800 nm.
Figure 1. HPLC analysis of pigment composition of the LH2 complexes isolated from cells’ purple sulfur bacterium E. haloalkaliphila; (1) LH2-control preparations, (2) LH2-DPA preparations, (3) LH2-Neu preparations, (4) LH2-Sph preparations, or (5) LH2-Rho preparations. Peak identification: (a) bacteriochlorophyll, (b) didehydrorhodopin, (c) rhodopin, (d) spirilloxanthin, (e) anhydrorhodovibrin, (f) lycopene, (g) spheroidene, and (h) neurosporene. The chromatograms were normalized according to bacteriochlorophyll peaks. Detection was performed using DAD detector at 190–800 nm.
Microbiolres 16 00036 g001
Microbiolres 16 00036 g002
Figure 2. Room-temperature absorption spectra (A) and difference (light minus dark) absorption spectra (B) of LH2 preparations. (1) LH2-control preparations, (2) LH2-DPA preparations, (3) LH2-Neu preparations, (4) LH2-Sph preparations, or (5) LH2-Rho preparations. Illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements of the absorption spectra were done immediately after illumination or dark incubation without dilution of the sample solution.
Figure 2. Room-temperature absorption spectra (A) and difference (light minus dark) absorption spectra (B) of LH2 preparations. (1) LH2-control preparations, (2) LH2-DPA preparations, (3) LH2-Neu preparations, (4) LH2-Sph preparations, or (5) LH2-Rho preparations. Illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements of the absorption spectra were done immediately after illumination or dark incubation without dilution of the sample solution.
Microbiolres 16 00036 g002
Microbiolres 16 00036 g003
Figure 3. Fluorescence spectra of LH2-control preparations before (A) and after (B) illumination. Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements of the fluorescence spectra were done immediately after pre-illumination or dark incubation without dilution of the sample solution. All measurements were performed at least three times and the typical spectra are shown.
Figure 3. Fluorescence spectra of LH2-control preparations before (A) and after (B) illumination. Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements of the fluorescence spectra were done immediately after pre-illumination or dark incubation without dilution of the sample solution. All measurements were performed at least three times and the typical spectra are shown.
Microbiolres 16 00036 g003
Microbiolres 16 00036 g004
Figure 4. Difference (light minus dark) fluorescence spectra of LH2-control preparations (A), LH2-DPA preparations (B), LH2-Neu preparations (C), LH2-Sph preparations (D), or LH2-Rho preparations (E). Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements of the fluorescence spectra were performed immediately after pre-illumination or dark incubation without dilution of the sample solution. All measurements were performed at least three times and the typical spectra are shown.
Figure 4. Difference (light minus dark) fluorescence spectra of LH2-control preparations (A), LH2-DPA preparations (B), LH2-Neu preparations (C), LH2-Sph preparations (D), or LH2-Rho preparations (E). Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements of the fluorescence spectra were performed immediately after pre-illumination or dark incubation without dilution of the sample solution. All measurements were performed at least three times and the typical spectra are shown.
Microbiolres 16 00036 g004
Microbiolres 16 00036 g005
Figure 5. Dependence of viscosity (A) of LH2 solution on temperature and its derivative (B). Measurements were performed before (1, 3, 5) and after (2, 4, 6) illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) of LH2-control preparations (1, 2), LH2-DPA preparations (3, 4), or LH2-Rho preparations (5, 6) with blue-green light. Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements were performed immediately after pre-illumination or dark incubation with a 3.8-fold dilution of the sample by medium containing 50 mM Tris-HCl. All measurements were performed at least three times and the typical kinetics are shown.
Figure 5. Dependence of viscosity (A) of LH2 solution on temperature and its derivative (B). Measurements were performed before (1, 3, 5) and after (2, 4, 6) illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) of LH2-control preparations (1, 2), LH2-DPA preparations (3, 4), or LH2-Rho preparations (5, 6) with blue-green light. Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. Measurements were performed immediately after pre-illumination or dark incubation with a 3.8-fold dilution of the sample by medium containing 50 mM Tris-HCl. All measurements were performed at least three times and the typical kinetics are shown.
Microbiolres 16 00036 g005
Microbiolres 16 00036 g006
Figure 6. Kinetics of the Spy-LHP fluorescence related to its oxidation by hydroperoxides formed by a 20 min illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) of LH2-control preparations (1), LH2-DPA preparations (2), LH2-Neu preparations (3), LH2-Sph preparations (4), or LH2-Rho preparations (5). Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. “0” on the timescale indicates the moment of addition of the sample containing LH2 complexes to SPY-LHP solution. Measurements were performed immediately after pre-illumination or dark incubation. For other details, see Section 2. All measurements were performed at least three times and the typical kinetics are shown.
Figure 6. Kinetics of the Spy-LHP fluorescence related to its oxidation by hydroperoxides formed by a 20 min illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) of LH2-control preparations (1), LH2-DPA preparations (2), LH2-Neu preparations (3), LH2-Sph preparations (4), or LH2-Rho preparations (5). Illumination or dark incubation of the LH2 preparations was carried out in the medium containing 50 mM Tris-HCl, at LH2 concentration of 16.7 nmol at 25 °C. “0” on the timescale indicates the moment of addition of the sample containing LH2 complexes to SPY-LHP solution. Measurements were performed immediately after pre-illumination or dark incubation. For other details, see Section 2. All measurements were performed at least three times and the typical kinetics are shown.
Microbiolres 16 00036 g006
Microbiolres 16 00036 g007
Figure 7. Effect of Rose Bengal on the light-induced (20 min, 650 mmol photons s−1 m−2, 375 > λ > 600 nm) hydroperoxides formation in LH2-control preparations (A), LH2-DPA preparations (B), LH2-Neu preparations (C), LH2-Sph preparations (D), or LH2-Rho preparations (E). Illumination or dark incubation of the LH2 preparations was carried out in the absence (1) or in the presence (2) of Rose Bengal. For other details, see caption to Figure 4 and Section 2. All measurements were performed at least three times and the typical kinetics are shown.
Figure 7. Effect of Rose Bengal on the light-induced (20 min, 650 mmol photons s−1 m−2, 375 > λ > 600 nm) hydroperoxides formation in LH2-control preparations (A), LH2-DPA preparations (B), LH2-Neu preparations (C), LH2-Sph preparations (D), or LH2-Rho preparations (E). Illumination or dark incubation of the LH2 preparations was carried out in the absence (1) or in the presence (2) of Rose Bengal. For other details, see caption to Figure 4 and Section 2. All measurements were performed at least three times and the typical kinetics are shown.
Microbiolres 16 00036 g007
Table 1. Pigment composition of the LH2 complexes isolated from cells’ purple sulfur bacterium E. haloalkaliphila. The measurements were performed at least in triplicate. The standard deviation did not exceed five percent.
Table 1. Pigment composition of the LH2 complexes isolated from cells’ purple sulfur bacterium E. haloalkaliphila. The measurements were performed at least in triplicate. The standard deviation did not exceed five percent.
Detected CarotenoidContent of Carotenoid in LH2 Preparations, Molecules per One LH2
LH2-ControlLH2-DPALH2-NeuLH2-SphLH2-Rho
Didehydrorhodopin0.81———0.22
Rhodopin0.81———5.46
Spirilloxanthin2.36———0.07
Anhydrorhodovibrin2.64————
Lycopene1.38—0.07——
Spheroidene———4.16—
Neurosporene—<0.016.78——
ζ—carotene—0.220.02——
Phytoene—<0.01———
Phytofluene—0.17———
Table 2. Effect of the illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) of colloidal solutions containing LH2 complexes with different carotenoid composition on the ζ-potential. For details, see caption to Figure 1 and Section 2. The data are the means of 10 replications with the standard deviation of the mean.
Table 2. Effect of the illumination (650 mmol photons s−1 m−2, 375 > λ > 600 nm) of colloidal solutions containing LH2 complexes with different carotenoid composition on the ζ-potential. For details, see caption to Figure 1 and Section 2. The data are the means of 10 replications with the standard deviation of the mean.
ζ–Potential, mV
LH2–ControlLH2–DPALH2–NeuLH2–SphLH2–Rho
Before illumination−28.8 a ± 1.4−12.7 d± 0.8−23.8 b ± 1.3−23.2 b ± 1.4−15.8 c ± 0.9
After illumination−25.7 b ± 1.6−11.9 d± 0.8−24.2 b ± 0.7−23.3 b ± 0.8−15.5 c ± 1.3
Letters indicate statistically significant differences between different seed groups (p ≤ 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yanykin, D.V.; Paskhin, M.O.; Shumeyko, S.A.; Ashikhmin, A.A.; Bolshakov, M.A. Effect of Carotenoid Composition on Stability and Light-Induced Oxidative Damage of the LH2 Complexes Isolated from Ectothiorhodospira haloalkaliphila. Microbiol. Res. 2025, 16, 36. https://doi.org/10.3390/microbiolres16020036

AMA Style

Yanykin DV, Paskhin MO, Shumeyko SA, Ashikhmin AA, Bolshakov MA. Effect of Carotenoid Composition on Stability and Light-Induced Oxidative Damage of the LH2 Complexes Isolated from Ectothiorhodospira haloalkaliphila. Microbiology Research. 2025; 16(2):36. https://doi.org/10.3390/microbiolres16020036

Chicago/Turabian Style

Yanykin, Denis V., Mark O. Paskhin, Sergey A. Shumeyko, Aleksandr A. Ashikhmin, and Maxim A. Bolshakov. 2025. "Effect of Carotenoid Composition on Stability and Light-Induced Oxidative Damage of the LH2 Complexes Isolated from Ectothiorhodospira haloalkaliphila" Microbiology Research 16, no. 2: 36. https://doi.org/10.3390/microbiolres16020036

APA Style

Yanykin, D. V., Paskhin, M. O., Shumeyko, S. A., Ashikhmin, A. A., & Bolshakov, M. A. (2025). Effect of Carotenoid Composition on Stability and Light-Induced Oxidative Damage of the LH2 Complexes Isolated from Ectothiorhodospira haloalkaliphila. Microbiology Research, 16(2), 36. https://doi.org/10.3390/microbiolres16020036

Article Metrics

Back to TopTop