Yu. E. Erokhin

Distribution of bacteriochlorophyll between the pigment-protein complexes of the sulfur photosynthetic bacterium Allochromatium minutissimum depending on light intensity at different temperatures

Variation of the distribution of bacteriochlorophyll a (BChl a) between external antenna (LH2) and core complexes (LH1 + RC) of the photosynthetic membrane of the sulfur bacterium Allochromatium minutissimum was studied at light intensities of 5 and 90 Wt/m2 in the temperature range of 12–43°C. The increase of light intensity was shown to result in a 1.5-to 2-times increase of a photosynthetic unit (PSU). PSU sizes pass through a maximum depending on growth temperature, and the increase of light intensity (5 and 90 Wt/m2) results in a shift of the maximal PSU size to higher temperatures (15 and 20°C, respectively). In the narrow temperature interval of ∼14–17°C, the ratio of light intensity to PSU size is typical of phototrophs: lower light intensity corresponds to larger PSU size. The pattern of PSU size change depending on light intensity was shown to differ at extreme growth temperatures (12°C and over 35°C). The comparison of Alc. minutissimum PSU size with the data on Rhodobacter capsulatus and Rhodopseudomonas palustris by measuring the effective optical absorption cross-section for the reaction of photoinhibition of respiration shows a two to four times greater size of light-harvesting antenna for Alc. minutissimum, which seems to correspond to the maximum possible limit for purple bacteria.

Influence of blue light on the structure stability of antenna complexes from Allochromatium minutissimum with different content of carotenoids

Effects of photooxidation of bacteriochlorophyll (absorbtion at 850 nm) from the light-harvesting complex LH2 of Alc. minutissimum membranes on the LH2 complex structure have been studied. Photooxidation was induced by blue light that is absorbed by carotenoids. Four samples with different levels (from 100% to 3–5%) and composition of carotenoids were obtained by inhibiting the carotenoid biosynthesis in bacteria with diphenylamine. Electrophoresis in polyacrylamide gel showed that after illumination LH2 complex contained all the oxidized bacteriochlorophyll. The carotenoid composition did not change after the oxidation of the main part of bacteriochlorophyll in the LH2 complex. The results suggest that oxidation takes place in the bacteriochlorophyll part, which is essential for the molecule optical properties (the system of double conjugated bonds is changed), but does not influence the stability of the structure of the LH2 complex.

Some Spectral Characteristics of Pigment–Protein Complexes and Their Interaction in Membranes of Thiorhodospira sibirica

The structure of the photosynthetic apparatus of purple photosynthetic bacteria is much simpler than in other photosynthesizing organisms. Usually, the photosynthetic apparatus of purple bacteria consists of two light-harvesting complexes (B800-850 and B880 or LH2 and LH1, respectively) and a reaction center (RC) [1–3]. The absence of the LH2 complex in carotenoidless strains of nonsulfur purple bacteria studied thus far is a specific feature of these bacteria [1, 4, 5]. It was shown in the preceding works [2, 6] that the complete set of antenna complexes is synthesized in the sulfur photosynthesizing bacterium Chromatium minutissimum even in the presence of agents causing a 95–99% inhibition of carotenoid biosynthesis. The results of studies of antenna complexes of this bacterium were inconsistent with the existing views on the mechanisms of inhibition of carotenoid biosynthesis and the role of carotenoids in bacterial photosynthesis. The following results should be emphasized in this context: a relatively low content of phytoine in membranes treated with inhibitor, formation of LH2 complexes even against the background of a 99% inhibition of carotenoid biosynthesis, absence of the blue shift of the long-wave band of the absorption spectrum of LH1, and direct evidence for carotenoid-mediated stabilization of the structure of antenna complexes [2, 3, 6].

Effect of low pH values on chromatophores, core complexes, and peripheral light harvesting complexes isolated from the sulfur bacterium Allochromatium minutissimum

Spectral methods have been used to trace pheophytinization of bacteriochlorophyll (BChl) in the membranes of chromatophores isolated from normal and carotenoidless cells of the purple bacterium Allochromatium minutissimum as well as in the core complexes and peripheral light harvesting complexes in the media with different detergents at low pH values. The well-marked staging of damage of native BChl forms with the absorption band of 885 nm has been revealed: (1) the formation and increase of the absorption band of monomeric BChl (785 nm); (2) pheophytinization of resultant monomeric BChl, and (3) aggregation of bacteriopheophytin (BPheo). Compared to the initial carotenoid complexes, carotenoidless pigment protein complexes were less resistant to the effect of low pH values, especially at the stages of BChl monomerization and pheophytinization. However, BPheo aggregation in them was slower. The electrophoresis in PAAG has shown that BChl pheophytinization in peripheral light harvesting complexes is accompanied by disruption of the ring-shaped structures of the complexes, with appearance of typical fragments consisting of α- and β- peptides and carrying monomeric BPheo, and by formation of α-peptide aggregates carrying BPheo aggregates.

Role of bacteriochlorophyll in stabilization of the structure of the core and peripheral light-harvesting complexes from purple photosynthetic bacteria

Pheophytinization of bacteriochlorophyll (BChl) at low pH was investigated in the core (LH1) and peripheral (LH2) light-harvesting complexes, as well as in the ensemble of the reaction center (RC) with the LH1 complex. The stages in disintegration of the native BChl forms in the LH1 complex and in its ensemble with RC were revealed. They were observed as emergence of the absorption band of monomeric BChl and an increase in its intensity, followed by its transformation into the band of monomeric bacteriopheophytin (BPh) and then into the band of aggregated BPh. Unlike the LH1 complex, in the case of the LH2 complex, monomeric BChl was never detected as an intermediate product. While the spectra revealed formation of monomeric BPh, its accumulation did not occur, since its aggregation is very rapid compared to that in the LH1 complex and in the RC-LH1 ensemble. PAAG electrophoresis revealed that pheophytinization of BChl in the LH2 complex was accompanied by disruption of the stable cylindrical structure of this complex with emergence of characteristic fragments consisting of α and β peptides and bearing monomeric BPh, as well as of the α peptide aggregates bearing BPh aggregates. Unlike the LH2 complex, BChl pheophytinization in the LH1 complex did not result in its fragmentation. This is an indication of different types of structural stabilization in the LH1 and LH2 complexes. In the LH2 complex, coordination of bacteriochlorophyll Mg2+ by conservative histidine residues of the α and β polypeptides is the main factor responsible for the maintenance of its cylindrical structure. Stability of the LH1 complex is probably based primarily on the highly specific hydrophobic interactions between the surfaces of individual polypeptide chains, since the presence of hydrogen bonds results in autonomy of each αβBChl2 subunit, rather than in stabilization of the LH1 complex as a whole.

Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting the B800-850 complex and its dependence on the spectral composition of the light is discussed.

Formation of 55-kDa fragments under impaired coordination bonds and hydrophobic interactions in peripheral light-harvesting complexes isolated from photosynthetic purple bacteria

Size exclusion chromatography was used to assess the relative size of intact and diphenylamine-treated (DPA, with suppressed carotenoid synthesis) peripheral light-harvesting complexes (LH2 complexes) of the sulfur bacterium Allochromatium minutissimum. Both LH2 complexes were nonamers and had the same elution volume Ve, coinciding with that for the LH2 complex of Rhodoblastus acidophilus (strain 10050). Their molecular weight was 150 kDa. Both pheophytinization of bacteriochlorophyll (BChl) at low pH and treatment with the detergent LDAO, which affects the hydrophobic interactions between the neighboring protomers, result in the fragmentation of the ring of the isolated LH2 complexes and formation of 55-kDa fragments with molecular weights corresponding to one-third of the initial value. Fragmentation caused by both pheophytinization and detergent treatment was much more rapid in DPA LH2 complexes than in the intact ones. The 55-kDa fragments formed at low pH values contained monomeric bacteriopheophytin, while the fragments of a similar molecular weight formed at pH 8.0 in the presence of the detergent contained monomeric BChl. The observed fragmentation was hypothesized to reflect the inherent C3 symmetry of the LH2 complexes, with the preliminarily assembled trimers used as building blocks.

Interdependence of carotenoid biosynthesis and assembly of pigment-protein complexes in Ectothiorhodospira haloalkaliphila cells

In the majority of species of photosynthetic bacteria, the lightharvesting antenna consists of two typesof pigment–protein complexes—peripheral LH2 andnearcentral LH1—which are located in the innercytoplasmic membrane [1]. The LH1 complex surrounds the reaction center (RC), forming a “core”complex with it (or the LH1–RC complex). Bothlightharvesting complexes were obtained in the pureform and are well studied [2–6]. Depending on thespecies of bacteria, the LH1 complex has the majorabsorption maximum at ~865–890 nm in the nearinfrared region of the spectrum, whereas the LH2 complex has two maxima (at ~800 and ~850 nm) [2, 3]. Inthe purple bacteria, these complexes are constructedaccording to a similar modular principle and consist oftwo types of polypeptides (α and β), bacteriochlorophyll (BChl), and carotenoids [4–6]. Carotenoids areadditional pigments. They perform several importantfunctions in vivo (lightharvesting, protecting, andstructural functions) [7, 8]. It was stated that the LH2complexes are not assembled in the cells of carotenoidless mutants of purple nonsulfur bacteria [9].There is another approach to obtaining carotenoidless LH2 complexes—the use of an inhibitor ofcarotenogenesis during cell culturing. Using theinhibitor diphenylamine (DPA), we obtained carotenoidless cells (DPA cells) of the purple sulfur bacterium

Effect of acid pH on the core and peripheral light-harvesting complexes of purple bacteria

75 Photosynthesis in the absence of lighttharvesting complexes would be poorly effective because of a low photon absorption crossssection of pigments in reacc tion centers (RCs). All species of purple bacteria conn tain core lighttharvesting complexes LH1, which directly transfer the excitation energy to RCs, and most species also contain peripheral lighttharvesting complexes LH2, which transfer the excitation energy to RC via LH1 complexes. Both LH1 and LH2 have been fairly well studied in terms of their structure, function, and spectroscopic parameters. However, the complete spatial structure has been resolved for LH2, including the mutual arrangement of bacteriochloroo phyll (BChl), carotenoid, and peptide molecules [1, 2], while similar highhresolution data on LH1 are as yet absent. Both complexes consist of protomers, i.e., hett erodimers of two types of polypeptides (α and β chains), which bind BChl and carotenoid molecules [3, 4] and are arranged in two concentric cylinders spanning the membrane. This structure is stabilized by pigment–pigment interaction between BChl porphyy rin rings, coordination bonding between Mg 2+ ions and peptides, and numerous weak bonds (hydrogen, van der Waals, and hydrophobic) between the latter and carotenoid polyene chains and BChl phytol chains. The α polypeptide chains form the inner cyll inder, where their helices are located close to and interact with each other, while the β chains are posii tioned in the outer cylinder, where the distances between them prevent direct β–β interactions. The transmembrane segments of α and β chains can interr act only indirectly, via the molecules of pigments and bound water, with a direct interaction being possible only at their NN and CCterminals. The overlapping porphyrin rings of BChl dimers within the membrane lie perpendicular to its surface, closer to the periplasm, between the cylinders formed by α and β polypeptides and compose an exciton ensemble with the absorption peak at 850 nm for LH2 and 870 nm for LH1 comm plexes. The central Mg 2+ ions of these BChl molecules are ligated by conserved histidine residues of α and β polypeptides. In addition, LH2 complexes contain BChl molecules absorbing at 800 nm that are arranged between β chains so that their porphyrin rings are parr allel to the membrane surface. The LH2 complex from Rhodopseudomonas acidophila consists of nine proo tomers [1], while that from Phaeospirillum moliss chianum, of eight protomers. The cylindrical structure of LH1 complex from Rhodospirillum (Rsp.) rubrum is formed by 16 protomers …

Effect of carotenoids on the interaction between pigment-protein complexes in membranes of the sulfur photosynthetic bacterium Chromatium minutissimum.

Pigment–protein complexes in the photosynthetic membranes of purple bacteria fall into three major types: antenna light-harvesting complexes LH1 (B880), antenna light-harvesting complexes LH2 (B800–850), and photosynthetic reaction centers (RC) [1]. Antenna complexes are composed of two lowmolecular-weight subunits ( α and β ). The polypeptide chain of each subunit contains one transmembrane hydrophobic segment and two hydrophilic domains exposed to the membrane surface. The polypeptides are arranged in two rings (external and internal). In the LH1 and LH2 complexes, these rings are composed of 16 and 8–9 polypeptides, respectively [2, 3]. A bacteriochlorophyll (BChl) dimer is sandwiched between the polypeptide rings in the LH1 complexes, whereas in the LH2 complexes, the sandwich structure is composed of the polypeptide rings, a BChl dimer, and a BChl monomer. The BChl forms incorporated in the light-harvesting antenna complexes can be easily identified by specific absorption spectra. In the near-infrared spectral range, the LH1 complexes contain only one absorption band of the BChl dimer at 880 nm, whereas the LH2 complexes are characterized by two absorption bands of BChl: at 800 nm (the BChl monomer) and at 850 nm (the BChl dimer) [1, 4]. Light-harvesting antenna complexes also contain carotenoids. The carotenoid molecules are embedded between the hydrophobic polypeptide chains, one end of the carotenoid molecule being in contact with the BChl dimer and the other end being exposed to the cytoplasm membrane surface. The carotenoid molecule interacts with a number of amino acid residues of the two polypeptides. The LH1 and LH2 complexes contain 16 and 13–14 carotenoid molecules, respectively [1–3]. In some cases, five or six carotenoid molecules are located at the outer side of the complex [1–3]. According to the presently accepted views [5, 6], carotenoids are capable of stabilizing the structure of the LH1 and LH2 complexes [5, 6]. Carotenoids can be removed from the LH1 and LH2 complexes using two methodological approaches: mutagenesis or bacterial growth in the presence of carotenoid biosynthesis inhibitors [4, 7]. Carotenoidless mutants contain only one light-harvesting antenna complex (LH1 or LH2), the structure of the remaining complex being modified. For example, point substitutions of amino acids of one of the polypeptides were found in the carotenoidless mutant LH1 complexes. The binding site of the monomer BChl is absent in the pseudo-LH2 complexes. In addition, the BChl dimer absorption band in the spectra of these complexes is shifted toward a shorter wavelength, thereby indicating structural modification of mutant carotenoidless complexes [1, 7]. The second approach provides milder conditions of carotenoid removal. Although this approach allows as much as 95 − 99% of carotenoids to be removed, the whole set of intact antenna complexes is conserved in an unmodified state. Using this approach, we have managed, for the first time, to isolate almost purely carotenoidless membranes of Chromatium minutissimum containing the whole set of antenna complexes [4]. Given that the positions of the polypeptide chain ends of antenna complexes depend on carotenoids [8] and taking into account that carotenoidless membranes are enriched with RC (i.e., the LH1 complex is an open ring), it was reasonable to study interaction between pigment–protein complexes in carotenoidless and control membranes of the purple sulfur bacterium Chr. minutissimum. This was the goal of our study. In this work, we describe the results of analysis of cross-linked associates of the pigment–protein complexes isolated from carotenoidless and carotenoid-containing membranes pretreated with a bifunctional cross-linking agent, dithiobis -succinimidyl propionate (DSP).