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Development and dynamics of the photosynthetic apparatus in purple phototrophic bacteria
Development and dynamics of the photosynthetic apparatus in purple phototrophic bacteria Robert A. Niederman PII: DOI: Reference:
S0005-2728(15)00221-2 doi: 10.1016/j.bbabio.2015.10.014 BBABIO 47549
To appear in:
BBA - Bioenergetics
Received date: Revised date: Accepted date:
22 August 2015 22 October 2015 25 October 2015
Please cite this article as: Robert A. Niederman, Development and dynamics of the photosynthetic apparatus in purple phototrophic bacteria, BBA - Bioenergetics (2015), doi: 10.1016/j.bbabio.2015.10.014
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Development and Dynamics of the Photosynthetic Apparatus in Purple
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Phototrophic Bacteria
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Robert A. Niederman*
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Department of Molecular Biology and Biochemistry, Rutgers, The State University of New
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Jersey, 604 Allison Road, Piscataway, New Jersey 08854-8082, United States
For Special Issue of BBA Bioenergetics devoted to: ―Organization and dynamics of bioenergetic systems in bacteria‖
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*E-mail address: [email protected]
Keywords: Atomic force microscopy, Bacterial photosynthesis; light-harvesting; reaction center; membrane assembly; membrane dynamics
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ACCEPTED MANUSCRIPT Highlights Proteomics showed assembly factor enrichment at membrane growth initiation sites.
CCCP triggered an accumulation of factors functioning early in the assembly process.
A cooperative assembly mechanism for the RC-LH1 complex was demonstrated by AFM.
Fluorescence relaxation showed a slowing of RC electron transfer as levels of LH2 increased.
This is thought to arise mainly from an increased distance for cytochrome c2 diffusion.
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Abbreviations: AFM, atomic force microscopy; BChl, bacteriochlorophyll a; -OG, n-octyl β-D-
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glucopyranoside; CCCP, carbonyl-cyanide m-chlorophenyl-hydrazone; CM, cytoplasmic membrane; EM, electron micrograph; DOC, deoxycholate; FM, maximal fluorescence; FV,
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variable fluorescence; FV /FM, the quantum yield of the primary charge separation; ICM,
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intracytoplasmic membrane; LH, light harvesting; LH1, core light-harvesting complex; LH2, peripheral light-harvesting complex; p, light-harvesting bacteriochlorophyll connectivity, RC,
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photochemical reaction center; functional absorption cross-section; ET, the rate of RC electron transfer turnover.
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ACCEPTED MANUSCRIPT ABSTRACT The purple bacterium Rhodobacter sphaeroides provides a useful model system for studies of the
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assembly and dynamics of bacterial photosynthetic membranes. For the nascent developing
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membrane, proteomic analyses showed an ~2-fold enrichment in general membrane assembly factors, compared to chromatophores. When the protonophore carbonyl-cyanide m-
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chlorophenyl-hydrazone (CCCP) was added to an ICM inducing culture, an ~2-fold elevation in
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spectral counts vs. the control was seen for the SecA translocation ATPase, the preprotein translocase SecY, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, which
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act early in the assembly process. It is suggested that these factors accumulated with their nascent polypeptides, as putative assembly intermediates in a functionally arrested state. Since in
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Synechocystis PCC 6803, a link has been established between Chl delivery involving the highlight HilD protein and the SecY/YidC-requiring cotratranslational insertion of nascent
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polypeptides, such a connection between BChl biosynthesis and insertion and folding of nascent Rba. sphaeroides BChl binding proteins is likely to also occur. AFM imaging studies of the formation of the reaction center (RC)-light harvesting 1 (LH1) complex suggested a cooperative assembly mechanism in which, following the association between the RC template and the initial LH1 unit, addition of successive LH1 units to the RC drives the assembly process to completion. Alterations in membrane dynamics as the developing membrane becomes filled with LH2-rings was assessed by fluorescence induction/relaxation kinetics, which showed a slowing in RC electron transfer rate thought to mainly reflect alterations in donor side electron transfer. This was attributed to an increased distance for electron flow in cytochrome c2 between the RC and cytochrome bc1 complexes, as suggested in current structural models.
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ACCEPTED MANUSCRIPT 1. Introduction……………………………………………………………………………………5 2. Proteomic analyses of membrane development in Rhodobacter sphaeroides……………...6
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2.1 Analysis of changes in membrane organization during a shift from high to low light intensity
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and induction of ICM formation at low oxygen tension……………….………………………........…7 2.2. Identification of early factors involved in the assembly of proteins of the bacterial
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photosynthetic apparatus…………………………………………………………………………………...8
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3. Role of assembly factors in targeting and binding of BChl(Chl) to apoproteins followed by their oligomerization and organization into supramolecular structures……………..15
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3.1 YidC is involved in binding of Chl to assembling apoproteins in cyanobacteria……………. 15 3.2 Roles for complex-specific assembly factors in the formation of bacteriochlorophyll a-protein
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complexes………………………………………………………………………………………...16 3.3 Oligomerization of LH1 and assembly of the RC-LH1 core complex as observed in AFM
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studies…………………………………………………………………………………………………. 20 3.4. LH2 and RC-LH1play roles in establishing ICM architecture………………...……….…..22 4. Dynamics of developing ICM as examined by fluorescence emission analyses………….25 4.1 Bacteriochlorophyll a-based fluorescence induction/relaxation kinetics……………..…..…25 4.2 Alterations in membrane dynamics as assessed by fluorescence induction/relaxation analyses……………………………………………………………………………………………..……....29 4.3. Searching for a mechanism explaining the decreased reaction center electron transfer turnover rate during greening and adaptation to low light intensity……………………...…….31 References…………………………………………………………………………………...…..36
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ACCEPTED MANUSCRIPT 1. Introduction Following a period of explosive growth of research on the structure and function of the
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intracytoplasmic photosynthetic membrane (ICM) of anoxygenic purple phototrophic bacteria
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[1], largely stemming from the determination of the first atomic resolution crystal structures of the photochemical reaction center (RC) [2-5] and light-harvesting (LH) antenna [6-8] proteins, a
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new research era has emerged in which the in situ supramolecular organization and dynamics of
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photosynthetic unit components within the membrane are now undergoing detailed investigation. This epoch began just over a decade ago with the advent of atomic force microscopy (AFM)
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providing native surface views of the ICM, obtained at submolecular resolution (see 9-12 for reviews). In addition to providing the earliest in situ topological views of any multi-component
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energy transducing membrane [13], AFM has revealed a wide diversity of species-dependent topological arrangements of closely packed antenna and RC structures, capable of fulfilling the
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basic requirements for efficient collection, transmission and trapping of radiant energy. In ICM patches from Rhodobacter sphaeroides grown at low light intensity [14], a highly organized architecture was observed consisting of well-ordered, interconnected rows of dimeric RC-LH1 core complexes intercalated with areas of peripheral LH2 antenna, coexisting with extensive LH2-only domains (Fig. 1A,B). In most other species studied such as Rhodospirillum photometricum, a much less regular organization was seen [15], with mixed regions of LH2 antenna and RC-LH1 core structures intermingled with large, paracrystalline LH2 domains (Fig. 1D-F). AFM together with fluorescence induction/relaxation kinetic analyses have provided the structural and functional paradigms for functional proteomic approaches to ICM development in Rba. sphaeroides in response to alterations in light intensity and oxygen tension [20-28]. The
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ACCEPTED MANUSCRIPT fluorescence yield properties of cells adapting to both lowered light intensity and low oxygen levels that induce ICM development, have revealed a direct relationship between the slowed rate
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of RC electron transfer turnover and the increasing LH2/LH1 molar ratios and the functional
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absorption cross-section [23,24,27-29]. This has been ascribed to the imposition of constraints upon the free diffusion of redox species between the cytochrome bc1 complex and the RC as the
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ICM bilayer is expanded and becomes densely packed with LH2 rings. This possibility was
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confirmed by a comparison of ICM patches from cells grown at high and low light intensity by high-resolution AFM [14], in which the increased LH2 levels in the low-light ICM were shown
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to form the densely packed LH2-only domains that were absent in the high-light cells (Fig.1). These LH2-only domains represent the light-responsive antenna complement formed under low
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illumination. These structural and functional results have been largely confirmed by the proteomic analyses described below.
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2. Proteomic analyses of membrane development in Rhodobacter sphaeroides Rba. sphaeroides has numerous advantages that provide an ideal model system for examining the development and assembly of photosynthetic membranes. This metabolically versatile purple chlorophototrophic bacterium has an accessible genetic system together with an ICM that provides an unparalleled variety of biochemical, spectroscopic and ultrastructural probes. When grown photoheterotrophically (anaerobically in the light), the levels of LH2 relative to the RC-LH1 core complexes are related inversely to light intensity [30] and changes in illumination levels have offered a valuable paradigm for examining the differential biosynthesis of photosynthetic complexes. While under chemoheterotrophic growth conditions, ICM formation is repressed, lowering the oxygen partial pressure initiates gratuitous induction of ICM formation in the dark (greening) by invagination of the CM [31]. Under these circumstances, sequential
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ACCEPTED MANUSCRIPT assembly of the LH and RC complexes occurs in which the functionally essential RC-LH1 complex is synthesized first, followed by the peripheral LH2 antenna [29].
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2.1 Analysis of changes in membrane organization during a shift from high to low light intensity
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and induction of ICM formation at low oxygen tension
By availing ourselves of these capabilities, a proteomic analysis of the ICM development
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process was performed in which the temporal expression of membrane proteins was followed
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both during transitions from high to low light intensity [21,23,25,27] and the induction of ICM formation at low aeration [32]. Membrane protein components, which have assessable
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ultrastructural and functional correlates, are spatially localized in distinct membrane regions arising both from areas of nascent photosynthetic membrane development as well as the mature
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ICM, isolated in the respective upper pigmented and ICM vesicles (chromatophore) bands after rate-zone sedimentation of cell-free extracts in sucrose density gradients. This facilitates the
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assessment of the proteomes arising from distinct membrane regions at various stages of ICM development. For proteomic analysis, the chromatophore and nascent membrane fractions were subjected to clear native electrophoresis for isolation of bands containing the LH2 and RC–LH1 core complexes. In chromatophores, the proteomic profile of the trypsin-digested LH2 gel bands (Fig. 2), revealed increasing levels of LH2 spectral counts relative to those of the RC–LH1 complex as ICM development proceeded during a light-intensity downshift [21], essentially confirming the results of the spectral and AFM analyses (see below). A large array of other associated proteins was observed in the LH2 band, including high spectral counts for the F1FOATP synthase subunits and the cytochrome bc1 and pyridine nucleotide transhydrogenase complexes, as well as the hypothetical protein RSP6124. Significant levels of RSP6124 were previously reported [20] and although topological predictions suggested a lack of transmembrane
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ACCEPTED MANUSCRIPT domains, its spectral counts were especially elevated in the LH2 band, nearly equaling those of the LH2 polypeptides during the first 3 h following the shift from high to low light intensity
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(Fig.2). Consequently, it is possible that RSP6124 may play a transient role in the assembly or
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function of the LH2 antenna.
In contrast to chromatophores, the RC-LH1 band from the purified upper-pigmented fraction
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was enriched in cytoplasmic membrane (CM) markers (Fig. 3) including electron transfer
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proteins (2-fold) and transport proteins (3-fold), as well as general membrane protein assembly factors (2-fold)[32]. The enriched assembly factors included preprotein translocases SecY, YidC
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and YajC, together with the bacterial type 1 signal peptidase and twin arg translocation subunit TatA. These data confirm the origin of the upper pigmented band from the peripheral respiratory
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membrane and sites of active CM invagination that give rise to the ICM, in which preferential assembly of the RC-LH1 complex occurs. The complete upper pigmented fraction isolated from
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cells undergoing semiaerobic induction of ICM formation for 12 h [32] showed a much larger enrichment in CM markers (~4 fold in electron transfer components, ~14-fold in transporters), consistent with the markedly decreased levels of RC-LH1 and LH2 proteins. This fractions also showed an ~3-fold increase in general membrane protein assembly factors and detectable levels of LH complex specific assembly factors. Substantial levels of GroEL heat shock chaperonin were also found, however, reflecting the limited cell division occurring in the concentrated cell preparations used in this induction procedure. Under such circumstances, GroEL promotes the folding of nascent proteins to counteract stress-induced protein denaturation and aggregation. 2.2. Identification of early factors involved in the assembly of proteins of the bacterial photosynthetic apparatus Early factors driving the assembly of ICM proteins were identified by a proteomic approach
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ACCEPTED MANUSCRIPT in which the lipid-soluble protonophore carbonyl-cyanide m-chlorophenylhydrazone (CCCP) was utilized to block the insertion of membrane proteins during the induction of ICM formation
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under low aeration in concentrated Rba. sphaeroides cell suspensions. CCCP acts as an
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uncoupler of electron transfer from ATP synthesis [36] by inhibiting the formation of the electrochemical proton gradient generated during electron transport by the respiratory chain
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carriers [37]. Integration of CCCP into the membrane bilayer in an anionic form results in proton
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acquisition from the periplasmic space. After diffusion of the protonated CCCP form back across the membrane, protons are discharged into the cytoplasm, collapsing the electrochemical proton
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gradient and leaving CCCP in the anionic state. Multiple repetitions of this process serve to short-circuit the respiratory chain.
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In the closely-related purple bacterium, Rba. capsulatus, concentrations of CCCP blocking
polypeptide subunit into the ICM for LH2 complex assembly [38]. It
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integration of the LH2-
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cytochrome c2 export to the periplasmic space (50 M) were also shown to abolish the
was concluded that maintenance of an electrochemical proton gradient is required for translocation of the nascent pigment-binding proteins. This represents step two of the dynamic sequence of steps delineated [39] in guiding of nascent pigment binding proteins across the membrane and assembling them into functional photosynthetic complexes: (i) cotranslational chaperonin assisted folding of the nascent polypeptides into membrane insertion-competent conformations and binding to the cytoplasmic surface of the membrane; (ii) electrochemical proton gradient driven insertion and integration into the membrane; (iii) delivery and binding of the BChl and carotenoid chromophores; (iv) oligomerization of the individual protomers into oligomeric annular structures; (v) supramolecular organization into functional photosynthetic units. Here, we will concentrate largely on the second step and bring some new perspectives to
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ACCEPTED MANUSCRIPT steps three and four. With regard to the general membrane assembly factors, studies on the formation of the LH1
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complex in Rba. capsulatus using a membrane coupled cell-free translation system, demonstrated that depletion of chaperonin DnaK significantly decreased the rate of synthesis and
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membrane insertion of both the LH1-α and –β polypeptides [40,41]. Removal of the GroEL
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chaperonin affected stable LH1 insertion into the coupled membranes, and expectedly, the
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assembly process was also dependent on the presence of unidentified membrane-bound factors. The following procedures were used to assess if the CCCP-induced collapse of the
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electrochemical proton gradient resulted in the accumulation of assembly factors, in association with nascent polypeptides as predicted by step 2 of the assembly pathway [39]. ICM formation
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was gratuitously induced by shifting BChl-depleted, concentrated suspensions of aerobically grown cells to semiaerobic conditions. Over the first 8 h, a vast increase in the number of both
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the RC-LH1 core and peripheral LH2 antenna complexes occurred, where the preferential appearance of the core particles is followed by an accelerated accumulation of LH2 [29,32,42], as reflected by a >4–fold increase in the molar LH2/LH1 ratio over this time period. Since limited cell division occurred in the concentrated cell suspensions, functional changes largely reflected accelerated synthesis of pigments and their binding proteins in existing cells. Therefore, this system was ideal for examining the effects of inhibition of the electrochemical proton gradient on the active assembly of pigment-protein complexes. After the addition of CCCP at 8 h, fluorescence induction/relaxation measurements showed a marked reduction in the quantum yield of the primary charge separation, as assessed from the Fv/Fm values (see Fig. 7 legend for definitions). A >4-fold slowing of the rate of RC electron transfer turnover (2.4 to 11.5 ms) occurred within the first hour after addition of the protonophore. Since multiple cycles of CCCP
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ACCEPTED MANUSCRIPT inhibition lead to a collapse of the proton gradient and short circuit the respiratory chain, the resulting short-circuit would be expected to affect proton release phases in the Q-cycle of the bc1
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complex, limiting donor side electron transfer to the RC [43]. The physiological parameters
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assessed by these fluorescence emission measurements confirmed that 50 M CCCP should be
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more than sufficient to block the membrane-potential requirement for insertion of nascent polypeptide chains into the membrane bilayer.
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To test the effects of CCCP on protein insertion into the ICM, chromatophore fractions isolated from the inducing cells at 4 h following the onset of CCCP treatment, and from the
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untreated 12-h control cells were subjected to clear native electrophoresis for LC-MS/MS analysis of trypsinized gel bands [32]. The effects of CCCP treatment on the LH, RC and related
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energy transduction complexes from major gel bands are shown in Fig. 4. CCCP markedly blocked the membrane insertion of polypeptides of the LH2, RC-LH1 and F1FO-ATPase
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complexes, especially as sampled in the respective gel bands in which they were highly enriched. A 2.7-fold reduction in spectral counts was observed for the LH2 complex from the bottom (LH2) band, consistent with the accelerated rate of LH2 synthesis and assembly seen at this stage of the induction process. In contrast, 1.7–1.9-fold reductions were found for the RC-LH1 complex in the top (RC-LH1) band, and in the upper intermediate band for the F1-ATP and FO-ATP synthases. Likewise, the pyridine nucleotide transhydrogenase, an additional energy transducing ICM-associated protein complex, showed a spectral count reduction of 1.7-fold, averaged over all-four gel bands. Accordingly, this complex, along with the RC-LH1 core and F1FO-ATPase complexes, with spectral count decreases in the of 1.7-1.9-fold range, reflect blockage by CCCP of the membrane insertion of nascent polypeptides of complexes with rates of assembly at a basal level during this stage of ICM induction, rather than at the accelerated rate
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ACCEPTED MANUSCRIPT seen for the LH2 complex. Moreover, these results provide novel in vivo evidence that confirms the necessity of the maintenance of an electrochemical proton gradient as an essential element in
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membrane translocation of the integral LH2 apoproteins [38] and extends these findings to the
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RC-LH1, transhydrogenase and F1FO-ATPase complexes. A CCCP-induced reduction in spectral counts was also observed for enzymes of BChl and carotenoid synthesis (1.6- and 1.7-fold,
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respectively). Although the majority of these enzymes represent soluble proteins, they may be
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degraded in the absence of appropriate associations with membrane components required for coordinating their respective pathways of pigment biosynthesis with concomitant formation of
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pigment binding apoproteins of the LH and RC complexes. It still seemed possible that the decline in spectral counts in the energy transducing
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membrane proteins reflected a slowing of protein synthesis arising from decreases in ATP levels caused by the effects of electrochemical proton gradient dissipation on oxidative
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phosphorylation. This was shown not to be the case by the similar levels of spectral counts between the CCCP-treated and -untreated chromatophores for 35 selected soluble proteins, representing enzymes of carbohydrate and amino acid metabolism (Fig. 4). This indicated that the rate of protein biosynthesis was unaffected during the 4-h protonophore treatment. Therefore, the CCCP-induced major decline in levels of the energy-transducing membrane protein complexes can be attributed to protonophore-induced blockage in the electrochemical protein gradient necessary for their membrane insertion rather than a general arrest in protein biosynthesis. As expected, CCCP treatment resulted in the accumulation of general membrane assembly factors in the unfractionated upper-pigmented fraction, as shown in the proteomic profile seen in Fig. 5. An ~2-fold elevation in the spectral counts vs. control levels was observed for the SecA
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ACCEPTED MANUSCRIPT translocation ATPase, the preprotein translocase SecY, SecD and SecF insertion components, and chaperonins DnaJ and DnaK. Since each of these components functions in early stages of the
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membrane insertion process, they may have accumulated in association with nascent
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polypeptides in a functionally arrested state. The resulting transient assembly intermediates, formed as a consequence of CCCP-induced blockage of the electrochemical gradient, apparently
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exist in a state that is not readily dissociated or degraded. In contrast, YidC and YagC, which
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play roles in later stages of the Sec translocase dependent prokaryotic membrane assembly process [44], showed a CCCP-induced decline in their levels and appear to have undergone
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degradation. A varied level of elevation of CCCP-induced spectral counts was observed for the highly conserved SPFH domain protein [45], the FtsH metalloproteinase/chaperonin [46], and
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HflC, a member of the SPFH group which functions along with HflK as a modulator of FtsH activity. FtsH has been shown to cross-link with YidC and HflK/C, suggesting that these proteins
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are linked to play a role in quality control of the membrane protein integration process [47]. Other components involved in early aspects of membrane protein assembly that were slightly elevated by CCCP treatment included the signal recognition particle homologue Ffh/SRP54 [48] and signal peptidase l [49]. Assembly factors that failed to accumulate, and showed essentially no changes in levels in the upper-pigmented fraction after CCCP treatment, included complex specific assembly factors (not shown) functioning in cofactor binding and assembly steps, following the initial insertion of nascent polypeptides into the membrane. Among these components with the highest spectral counts were the cytochrome c-type biogenesis protein, cycH [50] iron-sulfur cluster-binding protein [51] and LH1 assembly factors [52]. Overall, these data suggest that nascent polypeptides destined for BChl binding and with roles in energy
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ACCEPTED MANUSCRIPT transducing complexes, are inserted cotranslationally into the membrane in Sec-dependent manner.
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The observation in AFM topographs of ―captive‖ proteins in association with LH1 rings
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formed in an LH-only Rba. sphaeroides strain (Fig. 6)[53], provides in vivo structural support for the existence of membrane protein-assembly factor intermediates as suggested from the
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proteomic analysis of CCCP-treated cells [32]. The presence of complete LH1 rings still
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associated with their assembly factors is thought to reflect the absence of an RC template, leading to LH1 formation around other proteins of appropriate dimensions. Integral membrane
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proteins of sufficient size to serve in this capacity include BChl synthase (BchG), catalyzing bacteriochlorophyllide esterification and LhaA, a RC-LH1 core assembly factor with a possible
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role in delivery of BChl to apoproteins (see below). Not only have proteomic approaches applied to the upper-pigmented fraction facilitated
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elucidation of relative levels of associated general membrane assembly factors and their LH complex-specific counterparts [32], but also it was possible to assess the dynamics of the ICM assembly process in which general assembly factors involved in early assembly stages accumulated as putative transient intermediates as a result of CCCP-induced blockage of the membrane insertion of nascent polypeptides. Distinct transient assembly intermediates have been shown to accumulate upon disruption of photosystem assembly in the cyanobacterium Synechocystis PCC 6803 [54], leading to the identification of Psb28, a factor involved in Chl biosynthesis as well as formation of apoprotein subunits of Chl-binding proteins (the PSII antenna complex CP47, PSI RC core proteins PsaA and PsaB)[55]. Subsequently, the multifunctional assembly factor Psb27 was found to associate with the unassembled PSII antenna complex CP43, larger CP43 containing complexes, as well as the PsaB [56]. The associations
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ACCEPTED MANUSCRIPT forming these transient assembly intermediates were demonstrated through comigration in native gel electrophoresis, copurification with His-tagged PSI and a yeast two-hybrid assay.
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3. Role of assembly factors in targeting BChl(Chl) to apoproteins followed by their
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oligomerization and organization into supramolecular structures
3.1 YidC is involved in binding of Chl to assembling apoproteins in cyanobacteria
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In an investigation of mechanisms involved in the delivery of Chl to nascent photosystem
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apoproteins, a strain of Synechocystis PCC 6803 was constructed for pull down assays to detect proteins associated with Chl synthase (ChlG), the terminal enzyme of Chl biosynthesis [57,58].
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Among the associated proteins was HliD, a tightly bound protein induced at high light intensity, also associated with the putative PSII assembly factor Ycf39 and the YidC insertase. In addition,
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YidC was shown by immunoblotting to be associated with ribosomal subunits. Deletion of hilD resulted in elevated levels of the Chl G substrate, chlorophyllide, suggesting a role for HilD in
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the formation of ChlG complexes containing Ycf39 and other assembly factors. These studies established a link between Chl delivery by HilD and the SecY/YidC-requiring insertion of nascent photosystem polypeptides into cyanobacterial photosynthetic membranes [57]. Evidence for such a possible BChl-protein assembly supercomplex is discussed below. The polypeptide foldase activity of Yid C may be a crucial player in both insertion of Chl into nascent pigment binding proteins and establishment of suitable Chl binding sites. The identification of YidC together with ribosomal subunits as part of the ChlG)/pigment apoprotein assembly supercomplex is important, since ribosomal pausing has been shown to occur at distinct sites during elongation of the D1 subunit of PSII [59] and may be coordinated with Chl binding. It was suggested [57] that Yid C arranges the growing nascent polypeptide chains into a series of programmed configurations amenable to Chl insertion and that the concerted pausing by
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ACCEPTED MANUSCRIPT the ribosome provides the stops along the growing nascent polypeptide chain for correct attachment of Chl molecules, as stipulated by the adjacent ChlG, ultimately forming the correctly
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folded and occupied binding pockets of active Chl containing proteins.
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Among the earliest functions established for YidC in Escherichia coli is its role as a membrane chaperonin, facilitating Sec translocase-dependent and -independent lateral bilayer
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movement of nascent polypeptides [60]. In the case of the SecYEG translocon-dependent
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pathway, YidC occupies the lateral gate of SecYEG and becomes displaced in a sequential manner by nascent polypeptides, thereby facilitating the lipid bilayer transfer of transmembrane
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segments [61]. YidC also functions in the folding of nascent polypeptides and in promoting helix-helix interactions in conjunction with the SecYEG machinery [62]. The Sec translocase-
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independent YidC pathway drives the membrane integration of the subunits a and c of the FO sector of the F1FO ATPase [63] and Subunit II (CyoA) of the cytochrome o complex [64], a
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conserved function in respiratory chain biogenesis. YidC binds directly to the SecDFYajC in forming a heterotetrameric accessory complex [65] linking the Sec translocase complex to YidC, while SecD and SecF also participates in SecY independent insertion of proteins into the bilayer. Moreover, a holo-translocon consisting of SecYEG-SecDF-YajC-YidC along with an electrochemical proton gradient drives both the co-translational membrane protein insertion and posttranslational secretion processes [66]. Thus, YidC acts as a multifunctional assembly factor and is capable of catalyzing a multitude of activities to assist in the targeting and binding of cofactors during the insertion of nascent apoproteins into the membrane in a fully folded and functionally active state. 3.2 Roles for complex-specific assembly factors in the formation of bacteriochlorophyll a-protein complexes
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ACCEPTED MANUSCRIPT A supercomplex of protein assembly factors can also be envisioned for the implementation of a concerted mechanism for BChl synthase (BchG)/BChl delivery/apoprotein assembly, thus
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minimizing free pigment exposure by rapid transfer between the synthase and a nearby
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translocon channel, thus avoiding the threat of photooxidative damage to the developing photosynthetic apparatus. Among the complex-specific assembly factors encoded by genes
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located in the photosynthetic gene cluster of Rba. sphaeroides are LhaA, an LH1-specific
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assembly factor, and the related LH2-specific factor PucC [52]. These proteins are members the major facilitator superfamily of transport proteins, forming part of the putative ―chlorophyll
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delivery branch.‖ A role for LhaA in BChl cofactor assembly was suggested from immunoblotting analyses, indicating an enrichment of both LhaA and the BChl synthase protein
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in the Rba. sphaeroides upper-pigmented fraction [67,68] that was shown to contain nascent
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photosynthetic membranes that serve as a ―hotspot‖ for both BChl [67] and pigment binding
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protein biosynthesis [See 27,28 for reviews]. It is essential that the targeting process deliver the nascent apoproteins to the membrane at the precise location and time of BChl availability through coordination of protein integration with pigment synthesis. Thus, a supercomplex can be envisioned that consists in part of BChl synthase (BChG) and such putative BChl delivery proteins as LhaA and PucC, together with the translocase component YidC. This would establish the appropriate protein architecture for pigment binding and lead to fully folded and assembled photosynthetic complexes. Since apoproteins of the RC-LH1 and LH2 complexes were the major components undergoing synthesis in the oxygen-depleted, concentrated cell suspensions during CCCP treatment [32], it is possible that the Sec translocase participates in their membrane insertion, insofar as putative early assembly intermediate were found to accumulate in the absence of an electrochemical proton gradient. These apparent intermediates contained the SecA
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ACCEPTED MANUSCRIPT translocation ATPase, the preprotein translocase SecY, and the SecD and SecF insertion components.
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Genes for additional factors with photosynthetic complex assembly roles [52] have been
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identified within the photosynthesis gene clusters of Rba sphaeroides and Rba. capsulatus. Included are the genes encoding PufQ, a putative BChl carrier protein [69-71], PuhB, an
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apparent dimeric RC assembly factor which secondarily affects LH1 assembly [72], PuhC,
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required for optimal RC and LH1 levels, and thought to affect RC/LH1/PufX reorganization [73], and PuhE, a negative modulator of BChl synthesis [74]. In Rba sphaeroides, transcripts of
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pufQ, puhB and puhE were detected in a DNA microarray analysis of the oxygen and lightregulation of transcriptome expression [75]. Evidence of a role for PufQ in BChl
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biosynthesis/delivery comes from genetic studies in Rba. capsulatus, which showed a linear relation between pufQ expression levels and the formation of BChl, as well as conserved BChl
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binding sequences [69]. PufQ was identified in Rba. capsulatus membranes by immunoblotting, where detectable levels were correlated with BChl synthesis after a transfer from aerobic to semiaerobic growth conditions [70]. Moreover, PufQ was also shown to have an affinity for the BChl precursor protochlorophyllide, when reconstituted into liposomes [71]. Further studies are necessary before a definitive role can be established for any of these putative assembly factors. It is important to note that the pufQ gene is at the 5’ end of the PufQBALMX operon encoding PufQ, the LH1- and -polypeptides, respectively, the RC-L and RC-M subunits and PufX. The PufX protein promotes the dimerization of the RC-LH1 core complex [76,77], in which it is interspersed within the LH1 ring a 1:1 stoichiometry with the RC [77]. The dimerization process prevents complete encirclement of LH1 around the RC, forming an LH1-based portal for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome
18
ACCEPTED MANUSCRIPT bc1 complex [78]. PufX is also implicated in the supramolecular organization of the Rba. sphaeroides ICM through alleged promotion of a uniform long-range order to the RC-LH1
PT
complex, as suggested by linear dichroism (LD) spectroscopy [79,80]. LD interrogates the
RI
difference in absorption between horizontally and vertically polarized light, relative to the
SC
orientation of membrane preparations that are ordered macroscopically in polyacrylamide gels. This facilitates determination of the transition-dipole moment orientations of relevant
NU
chromophores, viz. the accessory RC BChl, the BChl special pair and LH1 and LH2 BChls. In both LD and dark-minus-light difference LD spectra of oriented membranes containing the
MA
native RC-LH1-PufX complex, the RC was distinguished by distinctive accessory BChl and BChl special pair spectral features, not found with RC-LH1 complexes lacking PufX [79].
D
-
TE
As first seen in EMs of freeze fracture replicas of elongated tubular membranes in the LH2
Rba. sphaeroides strain M21, dimeric RC–LH1 complexes form highly ordered arrays that are
-
AC CE P
arranged helically along the longitudinal axes of the tubules [82]. LD measurements of oriented LH2 membranes showed that the RC BChls are locked in a unique, uniform orientation within individual RCs over the entire intact membrane. [79]. Oriented wild-type chromatophore preparations also exhibited a remarkable degree of structural orientation in which all RC-LH1 core structures were shown to align in regular arrays with all RC BChl transition moments oriented in the same direction, despite the presence of LH2 as the predominant antenna complex in these native membranes. Moreover, the relative contributions of the LH1 and LH2 complexes to the overall LD spectrum in chromatophore vesicles in which the LD of LH2 was reduced relative to that in the LH1 absorption peak, showed that LH2 preferentially resides in highly curved parts of ICM membrane vesicles, since the LD and absorption peaks were equalized in flattened membrane patches [80].
19
ACCEPTED MANUSCRIPT While considerable information on how the supramolecular structure and organization of photosynthetic unit cores is established has arisen from these studies, it has recently been
PT
suggested from variations in the level of supramolecular organization of the Rba. sphaeroides
RI
ICM induced by alterations in PufX, that PufX may play an indirect role in the ordering of RC-LH1 dimers over the membrane [83]. Instead long-range order may stem from the role of
SC
PufX in promoting an appropriate dimer configuration with the creation of ordered RC–LH1
NU
domains depending not only on the presence of RC–LH1 dimers in their appropriate bent conformation, but also on the structure of LH1 on the outside of the complex. Thus, the sole
MA
influence that PufX exerts on membrane ordering may be via the facilitation of the dimerization process in which the dimer has one long axis of symmetry and is bent along the dimer interface
TE
D
[84].
3.3 Oligomerization of LH1 and assembly of the RC-LH1 core complex as observed in AFM
AC CE P
studies
In an AFM imaging study of the assembly of single RC-LH1 molecules using membranes from an LH1 only mutant [53], a series of aberrant forms of the LH1 complex in which the basic repeating unit (LH1-11(BChl)2) was seen to assume ring structures of variable size, elliptical structures, spirals and arcs that appear to represent assembly products (Fig. 6). The spiral complexes occur as a result of a failure in the closing of the ring, while the short arcs, first observed in lithium dodecyl sulfate/polyacrylamide gel electrophoresis [82], arise from premature termination of the LH1 complex assembly process. Occasionally, captive proteins were observed that were enclosed within the LH1 ring (Fig. 6) and as noted above, they could correspond on the basis of their size to assembly intermediates in which BChl synthase or complex specific assembly factors such as LhaA are bound and still associated. The presence of
20
ACCEPTED MANUSCRIPT such intermediates may reflect LH1 formation around proteins other than the RC template, which is absent in this strain [53]. A mixture of fully formed RC-LH1 complexes, emptied LH1 rings
PT
and isolated RCs was imaged (Fig. 6C) when formation of LH1 was restricted by a lowering of the cognate pufBA transcripts levels through removal of the stem-loop structure between the pufA
SC
RI
and pufL genes and progressive truncation at the C-terminus of the LH1- polypeptide. Overall, the resulting assortment of structures suggested a cooperative mechanism for assembly of the
NU
RC-LH1 complex in which once an association occurs between the RC and the first LH1 repeating unit, RC associations with successive LH1 subunits drive the LH1 ring assembly
MA
process to completion. Partial LH1 rings were not observed in the LH1-limited constructs (Fig. 6C), establishing that the necessity of an RC template for LH1 ring assembly. These AFM
TE
D
studies essentially confirm previous results (85) from an examination of ICM induction under low aeration in which the RC-H subunit was detected first, followed by PufX and thereafter by a
AC CE P
low level of LH1- and -subunits, which successively increased. The assembly process was proposed to reflect the initial formation of a RC-(LH1-11(Bchl)2)-PufX complex, with progressive addition of LH1-11(BChl)2 subunits, that eventually encircle the RC. Forces guiding the assembly of the LH2 complex, which unlike LH1 consists of an ―empty‖ ring structure, have been studied within the native ICM of Rsp. photometricum by AFM imaging of single molecules, combined with force measurements [86]. The binding of LH2 protomers was found to be fairly weak, indicating the importance of LH2 ring architecture in stabilizing the structure of the LH2 complex. In addition, intermolecular forces were shown to influence LH2 structural and functional integrity, as demonstrated from AFM unfolding kinetics in which the G for unfolding of an LH2 subunit consisting of an LH2-11(BChl)3 structure was estimated to be 222 kJ mol-1. Since a similar G might be anticipated for unfolding of an LH1-11(BChl)2
21
ACCEPTED MANUSCRIPT subunit, the oligomerization of an LH1-only complex would be expected to be driven by small cumulative inter-subunit forces that facilitate ring formation (53). In the case of a spiral complex,
PT
crowding pressure from adjacent rings might be expected to misalign the nascent complex so that
RI
additional subunits are assembled to form a spiral. Overall, the studies on forces driving the assembly of the LH2 complex [86] showed that: (i) weakly bound subunits are capable of self-
SC
assembling to form rings; (ii) the stability of the complex is guarantied by the ring-shape alone;
NU
(iii) the individual subunits are essentially solid and their stability further depends on the intramembrane molecular environment.
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3.4. LH2 and RC-LH1 play roles in establishing ICM architecture The discovery of a vesicular ICM similar to that of the wild-type in the Rba. sphaeoroides
TE
D
NF57 LH2-only mutant (i.e. lacking the RC and LH1 complexes) suggested that the LH2 oligomeric ring structure possesses the membrane bending properties responsible for the ICM
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vesiculation process [87,88]. On the other hand, a tubular ICM system was found in the M21 mutant lacking LH2 that was sometimes seen running between two cells appearing to be arrested in cell division (not shown). The observed ultrastructural differences suggested that in the absence of LH2, ICM morphogenesis is incomplete and is arrested at a tubular stage. The presence of vesicular ICM in the NF57 strain indicated that LH2 alone is both necessary and sufficient for vesicular ICM formation and that membrane morphogenesis can be completed in the absence of RC-LH1 core particles. As noted in section 3.2, the RC-LH1 complex exists in Rba. sphaeroides mainly as an Sshaped dimeric structure. The dimerization process is facilitated by the PufX protein, which serves as an integral component of the core complex (RC2LH1- pair28PufX2)[77]. In an effort to determine the extent to which the dimeric RC-LH1 complex controls the morphology of the
22
ACCEPTED MANUSCRIPT ICM, a 3-D RC-LH1-PufX model was constructed from single particle EM analysis of negatively stained complexes [84]. The two halves of the dimer were inclined toward one
PT
another on the periplasmic aspect of the complex, creating an uncommon V-shaped structure
RI
measuring ~200 A° along the long dimer axis and 100 A° along the short axis. The strikingly bent modeled structure has revealed the basis for the imposition of membrane curvature by the
SC
core complex and the ability to drive the formation of budded, tubular membrane structure,
NU
together with the arrangement of dimeric RC-LH1 complexes in highly ordered helical arrays along the tubules and the ordered packing of the dimers in 2D crystals. It can also be envisioned
MA
that RC-LH1 dimers utilize their distinctive curvature properties to facilitate their aggregation into the rows of RC-LH1 complexes observed uniquely in AFM topographs of Rba. sphaeroides
TE
D
[13] to facilitate excitation energy transfer from LH1 to the RC. In an investigation of the membrane bending effects of the highly bent RC-LH1 complex,
AC CE P
molecular dynamics simulations were employed for constructing an all-atom structural model within the EM density map [89]. A membrane of high local curvature was simulated which agreed well with the size of RC-LH1 tubules and further demonstrated how the intrinsic geometric shape of RC-LH1 dimers, consisting of a structure bent at the dimer interface, propagates the extended organization of a tubular ICM. In a similar approach, using the atomic resolution X-ray structure of the Rhodoblastus acidophilus LH2 and a homology-modeled Rba. sphaeroides LH2 structure [90], membrane embedded arrays of LH2 rings structures were found to assume a dynamic curvature, consistent with the putative LH2 membrane bending properties. An array of seven LH2 rings, as observed in AFM for LH2 complexes of Rsp. photometricum membranes (Fig. 1E), gave simulations in which these structures were seen to tilt away from neighboring complexes on the cytoplasmic side which created a curvature for the LH2
23
ACCEPTED MANUSCRIPT aggregates, that consequently curving the membrane. Thus, the aggregation dependent properties of individual LH2 rings were proposed to account for the membrane curving properties.
PT
Although a collections of seven rings grouped together is often encountered in Rsp.
RI
photometricum membranes, the ICM of this organism consists of regular bundles of flattened thylakoid-like discs rather than vesicular ICM. Moreover, while EMs of high-light grown Rba.
SC
sphaeroides in which LH2 concentration is low, clearly show a vesicular ICM [14], the LH2
NU
rings therein do not not form large aggregates, but instead are present mainly as individual structures in dimeric association or short single-file rows (Fig. 1C, 14). Also, simulations based
MA
on the atomic resolution LH2 structure of Rbl. acidolphilus showed the same arrangement of seven grouped LH2 rings as for Rba. sphaeroides LH2 and no substantial differences in
TE
D
membrane curvature were simulated, despite the existence of a lamellar ICM in Rbl. acidolphilus. Thus, while LH2 is clearly a membrane-bending protein, the overall mechanism of
AC CE P
membrane bending and vesiculation remain elusive. In this connection, the possibility of a role for protein-lipid interactions in establishing ICM architecture in Rba. sphaeroides has has been suggested from the finding of a putative LH2-lipid binding site and enrichment of the non-bilayer phospholipid phosphatidylethanolamine in the lipids associated at the boundary with LH2 [91]. The lipid-binding site included the keto carbonyl group of spheroidenone and a Glu residue located two residues from the N-terminus of the -subunit. When changed to Ala by site-directed mutagenesis, a reduction of the phosphatidylethanolamine level in the lipid boundary phase was observed together with its replacement by phosphatidylcholine. ICM from the strain harboring the mutated protein retained a normal spherical vesicular morphology, but by exchanging the carotenoid from spheroidenone to neurosporene resulted in tubular membrane invagination in this strain. It was suggested that
24
ACCEPTED MANUSCRIPT this specific Glu residue as well as the nature of the carotenoid polar head group govern interactions with the surrounding phospholipid that together with LH2 participate in the ICM
PT
vesiculation process. This led to the conclusion that the preferential binding and packing of
RI
phospatidylethanolamine at the LH2-lipid interface is essential for maintaining ICM morphology.
SC
4. Dynamics of the developing ICM as examined by fluorescence emission analyses
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4.1 Bacteriochlorophyll a-based fluorescence induction/relaxation kinetics Fluorescence induction/relaxation measurements have served as a valuable method for
MA
assessment of the functional absorption cross-section, the charge separation and electron transport capabilities of anoxygenic phototropic bacteria [92-97]. While light-induced absorption
TE
D
changes for monitoring electron transfer reactions have largely prevailed for the evaluation of the physiological status of the purple anoxygenic phototrophs, several additional functional
AC CE P
parameters can be conveniently assessed from the analysis of the fluorescence emitted when RC phototraps are photooxidized and effectively become ―closed‖ during the fluorescence induction phase, and open again during the fluorescence relaxation phase. These include: (i) the quantum yield of the primary charge separation; (ii) the rates of RC electron transfer turnover; (iii) the functional absorption cross-section and connectivity of the photosynthetic apparatus (Fig. 7). Moreover, fluorescence spectroscopy is a highly sensitive technique for examining membrane dynamics in a component specific manner with both intrinsic and extrinsic fluorophores. The pioneering discovery of the relation between the fluorescence yield and the fraction of closed ―RC‖ phototraps by Duysens [96] has served as the basis for the current understanding of how excitation energy transfer, via inductive resonance, serves to couple the large LH antenna complement to multiple RC. When a RC is closed and, providing that little energy is lost via
25
ACCEPTED MANUSCRIPT non-radiative dissipative pathways, excitations can visit neighboring open RCs and ultimately undergo trapping. A structural basis for this concept has recently been borne out by AFM
PT
topographs of native Rba. sphaeroides membranes [13,14](Fig. 1A), in which many RC-LH1 core complexes are arranged in multiple rows of dimeric structures, facilitating the migration of
RI
an LH1 excitation along a series of dimers until an open RC is found. Analysis of the induction
SC
and relaxation characteristics of the fluorescence emitted during these trapping events is the basis
NU
for determining the various physiological parameters that interrogate the functional status of the photosynthetic apparatus.
MA
The variable fluorescence signal arising from the RC phototraps reflects the redox status of the RC, where a low fluorescence yield indicates an open RC (uncharged, P870 capable of
TE
+
D
performing photochemistry) and a high fluorescence yield indicates a fully closed RC (P870 photooxidized to P870 , charged RC transiently nonfunctional). The difference between the
AC CE P
minimal fluorescence (F0) and the maximal fluorescence (FM) yields upon closing of the RC, provides an estimate of the quantum yield of the primary charge separation, obtained from the FV/FM ratio, where variable fluorescence FV = FM-F0. In addition, analysis of the induction kinetics also provides an estimate of the functional absorption cross-section (σ) of the LH complexes, as well as their connectivity (p), the latter reflected by the sigmoidicity of the fluorescence induction curve. The fluorescence relaxation kinetics arise from the reopening of the RC as governed by the RC electron transfer turnover rate (τET). Importantly, the kinetics and energetics of the RC donor and acceptor side electron transfer reactions determine the nature of the fluorescence relaxation phase [43]. As noted by van Grondelle [97], three different RC redox states are known to cause substantially different fluorescence yields during the induction phase: (i) fully active ―open‖ RCs (oxidized P870, QA, QB) having a low fluorescence yield; (ii) closed
26
ACCEPTED MANUSCRIPT -
RCs which have twice the yield of open RCs if QA is reduced to the semiquinone anion (QA. ); (iii) closed RCs which have ~3-times the open RC yield when the primary donor is fully +
PT
oxidized to the P870 .
RI
Kato et al. [98] have recently noted that while Chl fluorescence techniques can serve as a
SC
highly accessible alternative method for monitoring the charge separation and O2 evolution activities of PSII in oxygenic phototrophs, fluorescence only indirectly assesses such parameters
NU
as RC charge, and is dependent on excitation light intensity and wavelength1 and whether the
MA
sample is dispersed in solution or in the solid state. Moreover, imprecise spectroscopic interpretations can arise from proteins that remain spectroscopically active after losing their
D
functional activities. Although these caveats may also apply to BChl fluorescence emission of
TE
purple anoxygenic phototrophs as assessed in induction/relaxation measurements, the results of our fluorescence kinetic analyses are largely corroborated by related structural and functional
AC CE P
correlates: (i) FV/FM ratios are in the range of 0.7-0.8, close to the ~0.65 obtained in Rbl. acidophilus in the presence of the QB-site inhibitor terbutryn [95]; (ii) fluorescence induction curves display essentially exponential kinetics consistent with single turnover behavior (Fig. 7); (iii) FM levels obtained by single turnover flashes were essentially matched by those arising from multiple turnover pulses on a tens of millisecond timescale; (iv) values obtained for functional absorption cross-sections ranged from 28-130 Å2 [23,29], corresponding closely to the range of BChl molecules/RC (30-120) which make up the photosynthetic unit. The lower value, 1Koblízek
et al. (29) observed a dependence of excitation wavelength on functional absorption cross-section values, which for steady-state Rba. sphaeroides cells excited at 470 and 795 nm were ~55 Å2 for 470 nm and ~65 Å2 for 795 nm. This can be accounted for by differences in energy transfer efficiency from carotenoids to LH1 BChl, which in the LH1 complex isolated by lithium dodecyl native electrophoresis [99] yielded a value of 61 % [100], accounting for the lower values for 470 nm, while a higher efficiency of B800 B850 energy transfer accounts for the higher 795 nm value. 27
ACCEPTED MANUSCRIPT representing the number of LH1 BChl molecules per RC, is close to that of 32 obtained in Rba. sphaeroides by spectral deconvolution coupled with RC photooxidation measurements [101],
PT
and from fluorescence induction in Rbl. acidophilus containing both the B800-850 and B800-820
RI
LH2 antenna complexes [95]. We believe that together, these data attest to the validity of near-IR fluorescence induction/relaxation analyses for monitoring selected photosynthetic activities of
SC
the purple anoxygenic phototrophs.
NU
It has been widely proposed that in the PSII RC of oxygenic phototrophs, variable fluorescence mainly reflects the redox status of primary stable electron acceptor QA (RC acceptor
MA
side)[102]. As noted above, recent reports [42,43] on the nature of the fluorescence relaxation phase in Rba. sphaeroides suggest a major role for the primary P870 electron donor (c-type
TE
D
cytochrome), depending upon the type of RC association of the donor cytochrome in the organism under examination. Accordingly, Asztalos et al. [43], using intact cells of Rba.
AC CE P
sphaeroides, Rhodospirillum rubrum and Rubriviax gelatinosus, showed that laser diode excitation at 808 nm yielded complex, multi-expontential fluorescence decay kinetics. Longer excitations were correlated with the slowing of the relaxation phase, which was determined by the redox status, size and accessibility of the reduced cytochrome c2 and quinone pools for bacterial strains limited on either the donor or acceptor side of the RC, respectively. In Rba. sphaeroides and Rsp. rubrum, in which mobile cytochromes c2 serves as the immediate P870 electron donor, relaxation is preferentially controlled by the re-reduction rate of the oxidized RC by the reduced cytochrome c2, and the acceptor side has only a minor role. For Rba. sphaeroides, the multiphasic P+ reduction kinetics have been explained by a proximal–distal cytochrome c2 binding model [103-105] where the fastest component (~1 s) reflects tight binding at the proximal site. The origin of the second phase (20-200s) is less clear and may result from
28
ACCEPTED MANUSCRIPT cytochrome c2 binding at the distal site or the cytochrome may move from other binding sites. The third phase (100s-10 ms) is thought to arise from the shuttling of cytochrome c2 between
PT
the RC and cytochrome bc1 complexes. The close correlation between the equilibrium redox +
RI
titration on the RC donor side and the fluorescence relaxation (43) further suggested that P
SC
reduction acts as the rate-limiting step the Rba. sphaeroides RC re-opening process. On the other hand, in Rvx. gelatinosus where the cytochrome subunit is physically attached to
NU
the RC, the acceptor side determines the fluorescence relaxation kinetics with the rate constant of -
MA
350 s—1 for QA interquinone electron transfer as the determining factor. Importantly, an analysis of the nature of the relaxation phase as a means of assessing membrane dynamics has
D
recently been applied to Rba. sphaeroides cells undergoing different developmental regimens
TE
(23,24,42), as will be described below.
4.2 Alterations in membrane dynamics as assessed by fluorescence induction/relaxation analyses
AC CE P
A near-IR fast-repetition rate fluorescence analysis2 by Koblízek et al. [23] in steady-state Rba. sphaeroides cells undergoing gratuitous induction of ICM formation at reduced oxygen tension in the dark was the first to suggest that ICM development was accompanied by major changes in membrane dynamics. A linear relation was observed between the rise in the 795
nm
and the elevations in the LH2/RC; this was expected since both parameters reflect the preferential insertion of LH2 into the membrane, while the LH1/RC remained constant, making up the core of the photosynthetic unit. Moreover, a correlation between the growth of the LH2 antenna and the gradual slowing of RC electron transfer turnover was observed. The
ET
was
relatively fast (0.8-1.6 ms) following aerobic growth and during the first 3 h at low aeration, but 2
Although for these initial studies, a near-IR fast repetition rate fluorometer [106] was employed in which the fluorescence signal was excited by a sequence of submicrosecond flashes rather
29
ACCEPTED MANUSCRIPT than a single 144 s single turnover flash as in the subsequent in the subsequent FIRe analyses, the results from both systems have proved to be essentially the same.
PT
a decrease to 7 ms was observed after 13 h, occurring in parallel with the rise in functional absorption cross-section. This was attributed to an imposition of constraints on electron transfer
RI
as the membrane bilayer becomes densely packed with accumulating LH2 rings. This possibility
SC
is supported further by fluorescence induction/relaxation measurements in cells undergoing adaptation from high to low light intensity. The rates of RC electron transfer turnover and
NU
reoxidation of UQ pool were decreased by ~4- and 3-fold, respectively, over 4 days [23].
MA
Moreover, the slowing of ET was directly related to the increase in the functional absorption cross-section (450 nm ranged from ~70 Å2 at outset to 130 Å2 after 4 days) [23,24]. Again, an
D
essentially linear relation was observed between the size of the absorption cross-section and the
TE
LH2:LH1 molar ratios. Thus, when applied to the monitoring of changes in photosynthetic
AC CE P
activities during ICM development, taken together with AFM surface views of the alterations in membrane structure (13,14), fluorescence induction/relaxation measurements have served as a valuable probe of the membrane dynamics accompanying the membrane remodeling process (23,27).
While we initially attributed the slowing of the ET as a function of increasing LH2 levels to the imposition of constraints upon free diffusion of ubiquinone redox species between the RC and cytochrome bc1 complex (RC acceptor side kinetics)[23,24] as discussed above, a somewhat more complicated picture has emerged from the studies on Rba. sphaeroides by Kis et al. [42]. In cells adapting from aerobic to phototrophic growth conditions under constant illumination, donor electron transfer reactions dominated the relaxation kinetics after a single-turnover flash, while contributions on the acceptor side were enhanced only after multiple turnover flashes. It was
30
ACCEPTED MANUSCRIPT concluded that the slowing of electron transfer turnover results from changes on the RC donor side in which an increased distance for electron flow occurs in the cytochrome c2 periplasmic
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diffusion pathway between the RC and cytochrome bc1 complexes. This accounts for the initial
RI
relaxation kinetics when the developed (mature) membrane becomes more densely packed with LH2 rings and/or the distal RC position [103-105] becomes less accessible to cytochrome c2.
SC
Moreover, Kis et al. [42] showed that the slowed rate of electron transfer during ICM
NU
development did not result from changes in cytochrome c2 pool size. On the other hand, slow reoxidation of the UQ pool reflected contributions of the acceptor side, which is enhanced after
MA
long excitation (multiple turnovers). This is also compatible with our results [23,24], which were interpreted to show slowing of UQ pool oxidation with increasing LH2 levels after multiple
TE
D
turnover flashes. However, AFM images of Rba. sphaeroides LH2-only membranes have formed the basis for a functional model of highly curved native membranes, which holds that these
AC CE P
structures would permit the free diffusion of electron carriers through the LH2 domains, while still maintaining efficient excitation energy transfer [107]. Clearly, more work needs to be done on the question of whether quinone redox species flow is impeded by the presence of hexagonally packed LH2-only domains. 4.3. Searching for a mechanism explaining the decreases in reaction center electron transfer turnover rate during greening and adaptation to low light intensity A second question arises concerning the growth of the Rba. sphaeroides ICM during adaptation to reduced oxygen tension or low light intensity and the distance traveled by cytochrome c2 as predicted from the fluorescence induction studies of Kis et al. [42]. The intracelleular status of the Rba. sphaeroides ICM is now a matter of intense debate [16,108,109]. The cryo-electron tomography images of Tucker et al. [16] revealed a complex scenario for the
31
ACCEPTED MANUSCRIPT ultrastructure assumed by the ICM. While some ICM vesicles were observed without connections to other structures, others were located nearer to the CM, frequently forming
PT
interconnections with physical association to the CM, providing apparent access to the
RI
periplasm. Some nearly spherical single invaginations, with ―necks‖ attaching them to the CM were also seen, along with small CM indentations, representing the ICM precursor membrane
SC
isolated in the upper pigmented band in sucrose density gradients of cell-free extracts. Thus,
NU
fully detached ICM vesicles, possessing all the machinery for converting light energy into ATP, regarded as bacterial membrane organelles, only represent a part of the picture, for which the
MA
tomography methodology does not permit quantification of their relative content. A functional approach to the question of whether an ICM continuum exists with free access to
TE
D
the periplasmic space has been taken recently [108], where the ionophore gramicidin was employed to investigate the relative size of the electroosmotic units in intact cells, isolated
AC CE P
chromatophores and spheroplasts, lacking an outer membrane. In intact cells and in spheroplasts, the half-time of the decay induced by a single channel was ~6 ms and ~23 ms, respectively, as measured from the carotenoid electrochromic shift, which was three to four orders of magnitude slower that in isolated chromatophores. This was interpreted to mean that that the area of functionally active membrane in cells or spheroplasts is at least three orders of magnitude greater than that in individual chromatophore vesicles, which at most could contribute 10% of the active photosynthetic membrane in the cell. This is to be compared with the results of Prince et al. [110] who showed that ≤85 % of the cytochrome c2 was released upon spheroplast formation and thus an upper limit for free chromatophores would be ~15%; however, this is likely to amount to more at low light intensities. It was emphasized [108] that a single internal membrane system with connections to the periplasmic space may provide significant advantages in renewing the
32
ACCEPTED MANUSCRIPT photosynthetic apparatus and in the reallocation of the components shared with additional bioenergetic pathways.
PT
A more recent report on the cryo-electron tomography of the ICM from Rba. sphaeroides has
RI
appeared [109] in which the results were interpreted to show that ICM vesicles form a continuous reticulum rather than existing as discrete vesicular structures. The possibility must be
SC
considered that the different 3-D ICM arrangements are due to strain difference, since the Ga
NU
strain used in this study was derived from the 2.4.1 strain in which the yield of ICM vesicles is much lower than in strain NCIB8253 [27]. Most of the photopigment is retained in an upper-
MA
pigmented band in sucrose gradients, arising from CM invagination sites. This means that the internal membrane invagination and budding process may be limited in the 2.4.1 and Ga strains.
TE
D
In this study [109], the cytochrome bc1 complex was localized in the ICM, through immunoelectron microscopy using immuno-gold labeling, with antibodies raised against peptides
AC CE P
representing putative, well conserved cytoplasmic loops of cytochrome b from Rsp. rubrum. Labeling was mostly found on small membrane fragments and in the ―neck‖ regions protruding from the ICM vesicles suggesting that that the complexes localize to the fragile membrane regions that interconnect the chromatophores in the network. These fragile regions were thought to arise from tubular structures reconstructed from tomographic data, seen lying between ICM vesicles. Further, it was proposed that the bc1 complexes within these regions are surrounded by RC-LH1 complexes, bordered by arrays of the peripheral LH2 antenna complex. The localization of the cytochrome bc1 complex in the native ICM has also been examined recently by Cartron et al. [111]. EMs of negatively-stained Rba. sphaeroides chromatophores in which the C-terminus of cytochrome c1 contained a His10-tag that was labeled with gold nanobeads, showed that the majority of the labeled complexes were dimeric (Fig. 8A,B). The bc1
33
ACCEPTED MANUSCRIPT complexes appeared to be adjacent to RC-LH1 complexes, as seen in AFM topographs of goldlabeled chromatophores (Fig. 8C). While this provides evidence that restricted electron transfer
PT
domains might exist, these were not seen as fixed RC-LH1-PufX-cytochrome bc1
RI
supercomplexes. Instead, cytochrome bc1 complexes sit adjacent to the core complexes in disordered areas, not necessarily in direct contact with the majority of the RC-LH1-PufX
SC
complexes (Fig. 8). These findings were combined with a quantitative MS analysis in which the
NU
levels of the RC, cytochrome bc1 complex, ATP synthase, and cytochrome aa3 and cbb3 complexes were determined as a basis for constructing an atomic resolution model of a 50-nm
MA
chromatophore vesicle. The modeled vesicle contained 67 LH2 complexes, 11 LH1–RC dimers, 2 RC–LH1 monomers, 4 cytochrome bc1 dimers and 2 ATP synthases. Through simulation of the
TE
D
energy, electron and proton transfer processes, the calculated half-maximum ATP turnover rate suggested that this photosynthetic unit architecture is optimized for accommodating to low light
AC CE P
intensities.
Since the current atomic-level structural model for a 50-nm low-light chromatophore has four dimeric cytochrome bc1 complexes in close arrangement with the RC-LH1 complex, with seven RC-LH1 at some distance, the average distances over the internal vesicle surface for diffusion of cytochrome c2 between cytochrome bc1 and the RC is 10–30 nm. This may account for the observed slowing of the RC donor pathway, but localization of the bc1 complex in high-light chromatophores in which the LH2 content is reduced, will be essential for confirming this possibility. Acknowledgements We thank Prof. Peter Lobel and Dr. Haiyan Zheng for conducting the proteomics analyses and Dr. Kamil Woronowicz, Dr. Raoul Frese, Oluwatobi B. Olubanjo and Daniel Sha for
34
ACCEPTED MANUSCRIPT participating in the studies performed in my laboratory. Work in the author’s laboratory was supported by grants from the U. S. Department of Energy, Grant No. DE-FG02-08ER15957
PT
from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy
AC CE P
TE
D
MA
NU
SC
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Sciences and the National Science Foundation (Subaward No. 12-764).
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(2000) 14822–14830.
106] Z.S. Kolber, C.L. van Dover, R.A. Niederman, P.G. Falkowski, Bacterial photosynthesis in
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surface waters of the open ocean, Nature 407 (2000) 177– 179. [107] J.D. Olsen, J.D. Tucker, J.A. Timney, P. Qian, C. Vassilev, C.N. Hunter, The organization
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of LH2 complexes in membranes from Rhodobacter sphaeroides, J. Biol. Chem. 283 (2008) 30772–30779.
[108] A.Verméglio, J. Lavergne, F. Rappaport, Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach, Photosynth. Res. (2014) PMID: 25512104.
[109] S. Scheuring, R. Nevo, L.-N. Liu, S. Mangenot, D. Charuvi, T. Boudier V. Prima, P. Hubert, P, J.N. Sturgis, Z.Reich, The architecture of Rhodobacter sphaeroides chromatophores, Biochim Biophys Acta. 1837 (2014) 1263-1270. [110] R.C. Prince, A. Baccarini-Melandri, G.A. Hauska, B.A. Melandri, A.R Crofts, Asymmetry of an energy transducing membrane. The location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, Biochim Biophys Acta 387 (1975) 212-227.
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ACCEPTED MANUSCRIPT [111] M.L. Cartron, J.D Olsen, M. Sener, P.J. Jackson, A.A. Brindley, P. Qian, M.J. Dickman, G.J., Leggett, K. Schulten, C.N. Hunter, Integration of energy and electron transfer
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processes in the photosynthetic membrane of Rhodobacter sphaeroides, Biochim. Biophys.
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Acta 1837 (2014) 1769-1780.
[112] L. Esser, M. Elberry, F. Zhou, C.-A. Yu, L. Yu, D. Xia,Inhibitor-complexed structures of
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the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides. J. Biol.
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Chem. 283 (2008) 2846–2857.
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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. High-resolution AFM topographs of the cytoplasmic surface of native ICM represented
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by Rba sphaeroides membrane patches (A-C) [14] and flattened thylakoid-like discs from Rps. photometricum (D-F) [15]. The Rba. sphaeroides membrane patches were prepared with 0.03%
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-dodecyl maltoside, which is at a sub-critical micelle concentration, in order to open up ICM vesicles so that the resulting ICM patches are located closer to the mica surface, thereby
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maximizing the area available to the AFM tip during tapping mode acquisition of topographs. This represents a subsolubilizing level of detergent without apparent effects on membrane
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structure [10]. ICM preparations in panels (A) and (B) are from cells grown at low light intensity (4 W/m2). (C) Cells grown at high light intensity (220 W/m2). LH2 complexes are seen as
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separate nonameric ring structures (∼6–7 nm in diameter), assuming a zigzag appearance especially in LH2-only domains (B), as a result of vertical displacement when the natively
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curved membranes adsorb onto the planar mica surface, leading to lines of raised LH2 complexes. This is also seen in panel (A) for a membrane patch also containing a row of RCLH1 dimers (∼8.5 nm RC-RC separation within dimer) with the RC-H subunit projecting from the membrane surface. Panel C shows a vast predominance of dimeric RC-LH1 complexes in patch form high light cells with only a few LH2 and monomeric RC-LH1 complexes. This predominance of RC-LH1 dimers was seen previously [16] in non-detergent treated membrane preparations representing nascent ICM growth initiation sites isolated from the upper-pigmented band. For the high- and low-light chromatophores, the LH2/LH1 molar ratios, determined as described by Sturgis et al. [17] were 0.53 and 1.23 respectively. (D-F) High-resolution AFM topographs of native Rsp. photometricum ICM preparations obtained by contact mode AFM. Here the RC-H subunit has been removed by nanodissection with the AFM tip leaving
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ACCEPTED MANUSCRIPT cytoplasmic surfaces of the L and M subunits of the RC exposed. (D) High-light-adapted (~100 W/m2) ICM [15]. M, closed monomeric RC-LH1 core complex; LH2, nonameric LH2 complex.
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The core complexes and LH2 rings are arranged randomly and are found in an ∼1:3.5 ratio. (E,F)
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High-resolution topographs of low-light-adapted (10 to 20 W/m2) ICM. In panel E, an area of
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mixed core complexes and LH2 rings is shown, with an overall core complex:LH2 ratio of ∼1:7; areas of paracrystalline hexagonal arrays of LH2 are also seen that are devoid of core complexes.
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In panel F, an area is seen with substantially more core complexes than the average distribution. In these regions, as in panel D, several core complexes are found in contact, and the overall
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∼1:3.5 core complex:LH2 ratio is retained. The arrow in panel (D) delineates a free lipid bilayer space between LH2 complexes, which is thought to be functionally important in facilitating
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membrane mobility for quinone-quinol exchange to and from the core complex [18], just as the openings in the dimeric RC-LH1 complexes at the ends of linear core chains are thought to serve
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in this function in Rba. sphaeroides [19].
Fig. 2. Proteomic analysis of chromatophore LH2 gel band derived from clear-native electrophoresis, showing the distribution of spectral counts [25]. Chromatophores were isolated after 3 and 24 h from cells undergoing acclimation from high to low light intensity. For clearnative electrophoresis, chromatophores were solubilized with n-octyl β-D-glucopyranoside (-OG) and deoxycholate (DOC), both at concentrations of 15 mM, and subjected to clear-native electrophoresis essentially as described by Wittig et al. [33]. Resolved gel bands were excised and subjected to in-gel trypsin digestion, followed by LC-MS/MS at sub-femtomol sensitivity. Tandem mass spectrometry data files were searched against the NCBI assembly of the annotated Rba. sphaeroides 2.4.1 genome [34]. Proteomic data were quantified by spectral counting, which
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ACCEPTED MANUSCRIPT measures protein abundance on the basis of the number of tandem mass spectral observation for all constituent peptides. Accordingly, spectral counts mainly reflect the ability of trypsin to act at
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potential cleavage sites and are therefore only semiquantitative in nature. Proteins that are not
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highly abundant with a large number of accessible sites can give rise to higher counts than more abundant proteins with far fewer sites; in cases where no sites are available, no counts are found,
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e. g., PufB (LH1-β polypeptide) and PucA (LH2-α), although the latter polypeptide was detected
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after chymotrypsin cleavage [25]. Since the gel lanes were loaded with equal amounts of total protein, valid comparisons can be made between the same protein components in the
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chromatophore and upper-pigmented band preparations. Since spectral count sampling statistics have high technical reproducibility, they reflect precise assessments of relative protein
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abundance for differential protein expression studies such as those reported here. The spectral count distributions shown are for clusters of orthologous groups [35], in which the usual energy
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production and conversion categories have been divided into subgroups, which account for the distinct metabolic capabilities unique to Rba. sphaeroides.
Fig. 3. Proteomic analysis of upper-pigmented (A) and chromatophore fractions (B) in the clear native electrophoresis RC-LH1 enriched gel band. Membrane fractions were isolated from cells after a 24-h adaptation to a light intensity of 100 W/m2 [32]; the respective membrane fractions were solubilized with digitonin and
, and the resolved gel bands were subjected to in-
gel trypsin digestion and LC-MS/MS. The distributions shown are for clusters of orthologous groups [35], modified as described in the legend of Fig. 2.
Fig. 4. Effects of CCCP on the LH, RC and related energy transduction complexes of the ICM as
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gel bands were subjected to in-gel trypsin digestion and LC-MS/MS [32].
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Fig. 5. Proteomic analysis of upper-pigmented fraction isolated from CCCP-treated cell suspensions undergoing ICM induction showing effect of CCCP on general membrane assembly
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factor levels (A,B). Following removal from sucrose gradients, the upper-pigmented band was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and after entering, gel
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bands were excised for in-gel trypsin digestion followed by LC-MS/MS in which a 135-min LC
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gradient was used for optimal peptide resolution.
Fig. 6. (A, B) LH1-only membrane patches prepared using sub-critical micelle concentrations of
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-dodecylmaltoglucoside, showing aberrant complexes as depicted in 3-D [53]. (A) LH-1 only membrane patch showing an incomplete LH1 ring (blue arrow), LH1 arcs (cyan arrows) and captive proteins (red arrows)(see also E, F). (B) LH-1 only membrane showing aberrant complexes consisting of a spiral structure and an open ring enclosed within the white and blue boxes, respectively. The noise levels were reduced in these topographs by using a low-pass filter. (C) Membrane patch from a LH1-limited mutant [53], showing a 3-D representation of an intact RC-LH1 complex (magenta arrow), an isolated RC (white arrow), an RC-LH1 complex without the H-subunit (blue arrow), and an empty LH1 ring (red arrow). (D-F) AFM topographs of LH1 complexes reconstituted in 2D LH1 crystals adsorbed onto mica, prepared from an LH1-only Rba. sphaeroides strain (Topographs kindly provided by Prof. C.N. Hunter). While the majority of complexes consisted of empty ()16 LH1 rings of ~115 Å diameter (D), the complexes
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ACCEPTED MANUSCRIPT shown in E. and F. were among several that resemble putative assembly intermediates with a membrane protein entrapped in their interior, tentatively identified as LhaA, an LH1 assembly
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factor, which like the Lac permease belongs to the major facilitator superfamily, forming part of
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its putative ―Chl delivery branch‖ 52]. The space filling image (G) of the Lac permease [81], closely resembles the large transmembrane protein enclosed within the LH1 annulus (E, F),
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apparently representing the assembly factor still in association with its LH1 substrate. It is
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possible that entrapment of LhaA within the LH1 ring arose from the lack of RCs in this strain, which may normally displace this complex specific assembly factor during the core assembly
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process in which the RC is believed to act as a template [53].
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Fig. 7. Fluorescence kinetic transient elicited upon 450-nm excitation of Rba. sphaeroides cells undergoing induction of ICM development at low aeration [24] measured in a Satlantic
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Fluorescence Induction/Relaxation system (Satlantic Inc., Halifax, Nova Scotia). Measurements were made on whole cells diluted typically to ~ 30 nM BChl in growth medium to produce transients in the linear range. A blue LED (450 nm, 30-nm bandwidth) served as the excitation source. Fluorescence emission is passed through an 880-nm interference filter (50-nm bandwidth) and detected by a sensitive avalanche photodiode module. The digitized fluorescence kinetic transients obtained at 880 nm are processed by computer-assisted analysis, which translates measured signals to programmed physiological parameters, following a charge separation elicited between the RC-BChl special pair and QA. Four phases can be distinguished in the plotted transients: phase I, induction phase (strong pulse of 144 μs duration), eliciting a single-turnover flash (STF), cumulatively saturating the photosystem to permit measurement of fluorescence induction from F0 to FM; phase II, relaxation phase in which indirect modulated
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ACCEPTED MANUSCRIPT light is applied to assess the relaxation kinetics of the fluorescence yield on a 500 ms time scale, reflecting reopening of the RC; phase III, in which a 20 ms flash induces multiple turnovers
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saturating the photosystem and UQ pool (MTF, multiple turnover flash); and phase IV, applying
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indirect modulated light that records the kinetics of UQ pool reoxidation on a 1 s time scale. The transients represent signal-averaged sets of 20 traces per sample, which minimized noise levels
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of individual traces. Note the difference in the rate of fluorescence induction between the 0 and
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48 h samples, reflecting marked differences in the functional absorption cross-section. The registered variable fluorescence signal reflects the RC redox state in which a low fluorescence
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yield indicates an open RC (no charge, RC capable of performing photochemistry), while a high fluorescence yield indicates a closed RC (positively charged RC, transiently non-functional
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photochemistry). Parameters derived from the induction kinetics include: the quantum yield of the primary charge separation estimated from FV/FM where FV, the variable fluorescence, is the
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difference between the minimal fluorescence (F0) and the maximal fluorescence (FM); the functional absorption cross section (), calculated from the slope of the single turnover saturation curve, related to the rate of increase in fluorescence yield. Since the extent to which the relaxation kinetics reflected RC-BChl special pair re-reduction and/or QA re-oxidation was previously unknown, the multiphasic relaxation profile is expressed as the RC electron transfer turnover rate, designated as ET. Analysis of the induction phase (I) gave functional absorption cross-section (450 nm) values of 40.6, 71.5 and 101.5 Å2 in cells sampled at 0, 3 and 48 h, respectively, while the respective values for the RC electron transfer turnover rate (ET) of 3.1, 1.7 and 6.0 ms were obtained from the relaxation phase (II). ET represents the average of the first and second phases of the three phases derived from the exponential decay kinetics.
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ACCEPTED MANUSCRIPT Fig. 8. EM and AFM analyses of chromatophore membrane showing location of cytochrome bc1 complex labeled with gold NTA-Nanogold® [111]. (A) A negatively stained patch consisting of
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a single membrane bilayer with the gold-labeled cytochrome bc1 complexes mainly clustering
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near one edge. (B) The negatively stained features in A. were identified as the LH2 complex (green), RC–LH1 complex (blue/red), and cytochrome bc1 dimers (purple), identified from gold
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labeling. (C) Upper panel: close-up of false color 3-D depiction of AFM topograph obtained
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under liquid, showing intact, uncollapsed chromatophores labeled with nanogold. Image has been filtered with a low pass filter to reduce noise. A cluster of four putative nanogold beads
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indicating possible positions of dimeric bc1 complexes are contained within the red outline. The recessed area extending from the cluster shows a protein-free region. The pairs of colored
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asterisks denote the RC-H subunits of RC–LH1 core complexes; the 9-nm separation determined for the peaks indicated by the paired blue, red or magenta asterisks is consistent with the peak-to-
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peak separation of H-subunits in a RC–LH1 dimer complex. Bottom panel: interpretation of AFM data in which putative nanogold beads are indicated by the orange circles denoting the possible positions of the cytochrome bc1 complexes contained within the red outline in the upper panel. The dimeric RC-LH1 complexes are also shown (blue/red). The edge-to-edge separation of the pairs of gold beads was found to be 2.4 ± 0.5 nm; compatible with the structure of the dimeric Rba. sphaeroides cytochrome bc1 complex [112].
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Robert A. Niederman, Development and Dynamics of the Photosynthetic Apparatus in Purple Phototrophic Bacteria
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The author has no conflicts of interest associated with this study.
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S0005-2728(15)00221-2 doi: 10.1016/j.bbabio.2015.10.014 BBABIO 47549
To appear in:
BBA - Bioenergetics
Received date: Revised date: Accepted date:
22 August 2015 22 October 2015 25 October 2015
Please cite this article as: Robert A. Niederman, Development and dynamics of the photosynthetic apparatus in purple phototrophic bacteria, BBA - Bioenergetics (2015), doi: 10.1016/j.bbabio.2015.10.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development and Dynamics of the Photosynthetic Apparatus in Purple
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Phototrophic Bacteria
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Robert A. Niederman*
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Department of Molecular Biology and Biochemistry, Rutgers, The State University of New
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Jersey, 604 Allison Road, Piscataway, New Jersey 08854-8082, United States
For Special Issue of BBA Bioenergetics devoted to: ―Organization and dynamics of bioenergetic systems in bacteria‖
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*E-mail address: [email protected]
Keywords: Atomic force microscopy, Bacterial photosynthesis; light-harvesting; reaction center; membrane assembly; membrane dynamics
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ACCEPTED MANUSCRIPT Highlights Proteomics showed assembly factor enrichment at membrane growth initiation sites.
CCCP triggered an accumulation of factors functioning early in the assembly process.
A cooperative assembly mechanism for the RC-LH1 complex was demonstrated by AFM.
Fluorescence relaxation showed a slowing of RC electron transfer as levels of LH2 increased.
This is thought to arise mainly from an increased distance for cytochrome c2 diffusion.
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Abbreviations: AFM, atomic force microscopy; BChl, bacteriochlorophyll a; -OG, n-octyl β-D-
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glucopyranoside; CCCP, carbonyl-cyanide m-chlorophenyl-hydrazone; CM, cytoplasmic membrane; EM, electron micrograph; DOC, deoxycholate; FM, maximal fluorescence; FV,
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variable fluorescence; FV /FM, the quantum yield of the primary charge separation; ICM,
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intracytoplasmic membrane; LH, light harvesting; LH1, core light-harvesting complex; LH2, peripheral light-harvesting complex; p, light-harvesting bacteriochlorophyll connectivity, RC,
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photochemical reaction center; functional absorption cross-section; ET, the rate of RC electron transfer turnover.
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ACCEPTED MANUSCRIPT ABSTRACT The purple bacterium Rhodobacter sphaeroides provides a useful model system for studies of the
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assembly and dynamics of bacterial photosynthetic membranes. For the nascent developing
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membrane, proteomic analyses showed an ~2-fold enrichment in general membrane assembly factors, compared to chromatophores. When the protonophore carbonyl-cyanide m-
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chlorophenyl-hydrazone (CCCP) was added to an ICM inducing culture, an ~2-fold elevation in
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spectral counts vs. the control was seen for the SecA translocation ATPase, the preprotein translocase SecY, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, which
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act early in the assembly process. It is suggested that these factors accumulated with their nascent polypeptides, as putative assembly intermediates in a functionally arrested state. Since in
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Synechocystis PCC 6803, a link has been established between Chl delivery involving the highlight HilD protein and the SecY/YidC-requiring cotratranslational insertion of nascent
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polypeptides, such a connection between BChl biosynthesis and insertion and folding of nascent Rba. sphaeroides BChl binding proteins is likely to also occur. AFM imaging studies of the formation of the reaction center (RC)-light harvesting 1 (LH1) complex suggested a cooperative assembly mechanism in which, following the association between the RC template and the initial LH1 unit, addition of successive LH1 units to the RC drives the assembly process to completion. Alterations in membrane dynamics as the developing membrane becomes filled with LH2-rings was assessed by fluorescence induction/relaxation kinetics, which showed a slowing in RC electron transfer rate thought to mainly reflect alterations in donor side electron transfer. This was attributed to an increased distance for electron flow in cytochrome c2 between the RC and cytochrome bc1 complexes, as suggested in current structural models.
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ACCEPTED MANUSCRIPT 1. Introduction……………………………………………………………………………………5 2. Proteomic analyses of membrane development in Rhodobacter sphaeroides……………...6
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2.1 Analysis of changes in membrane organization during a shift from high to low light intensity
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and induction of ICM formation at low oxygen tension……………….………………………........…7 2.2. Identification of early factors involved in the assembly of proteins of the bacterial
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photosynthetic apparatus…………………………………………………………………………………...8
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3. Role of assembly factors in targeting and binding of BChl(Chl) to apoproteins followed by their oligomerization and organization into supramolecular structures……………..15
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3.1 YidC is involved in binding of Chl to assembling apoproteins in cyanobacteria……………. 15 3.2 Roles for complex-specific assembly factors in the formation of bacteriochlorophyll a-protein
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complexes………………………………………………………………………………………...16 3.3 Oligomerization of LH1 and assembly of the RC-LH1 core complex as observed in AFM
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studies…………………………………………………………………………………………………. 20 3.4. LH2 and RC-LH1play roles in establishing ICM architecture………………...……….…..22 4. Dynamics of developing ICM as examined by fluorescence emission analyses………….25 4.1 Bacteriochlorophyll a-based fluorescence induction/relaxation kinetics……………..…..…25 4.2 Alterations in membrane dynamics as assessed by fluorescence induction/relaxation analyses……………………………………………………………………………………………..……....29 4.3. Searching for a mechanism explaining the decreased reaction center electron transfer turnover rate during greening and adaptation to low light intensity……………………...…….31 References…………………………………………………………………………………...…..36
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ACCEPTED MANUSCRIPT 1. Introduction Following a period of explosive growth of research on the structure and function of the
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intracytoplasmic photosynthetic membrane (ICM) of anoxygenic purple phototrophic bacteria
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[1], largely stemming from the determination of the first atomic resolution crystal structures of the photochemical reaction center (RC) [2-5] and light-harvesting (LH) antenna [6-8] proteins, a
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new research era has emerged in which the in situ supramolecular organization and dynamics of
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photosynthetic unit components within the membrane are now undergoing detailed investigation. This epoch began just over a decade ago with the advent of atomic force microscopy (AFM)
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providing native surface views of the ICM, obtained at submolecular resolution (see 9-12 for reviews). In addition to providing the earliest in situ topological views of any multi-component
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energy transducing membrane [13], AFM has revealed a wide diversity of species-dependent topological arrangements of closely packed antenna and RC structures, capable of fulfilling the
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basic requirements for efficient collection, transmission and trapping of radiant energy. In ICM patches from Rhodobacter sphaeroides grown at low light intensity [14], a highly organized architecture was observed consisting of well-ordered, interconnected rows of dimeric RC-LH1 core complexes intercalated with areas of peripheral LH2 antenna, coexisting with extensive LH2-only domains (Fig. 1A,B). In most other species studied such as Rhodospirillum photometricum, a much less regular organization was seen [15], with mixed regions of LH2 antenna and RC-LH1 core structures intermingled with large, paracrystalline LH2 domains (Fig. 1D-F). AFM together with fluorescence induction/relaxation kinetic analyses have provided the structural and functional paradigms for functional proteomic approaches to ICM development in Rba. sphaeroides in response to alterations in light intensity and oxygen tension [20-28]. The
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ACCEPTED MANUSCRIPT fluorescence yield properties of cells adapting to both lowered light intensity and low oxygen levels that induce ICM development, have revealed a direct relationship between the slowed rate
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of RC electron transfer turnover and the increasing LH2/LH1 molar ratios and the functional
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absorption cross-section [23,24,27-29]. This has been ascribed to the imposition of constraints upon the free diffusion of redox species between the cytochrome bc1 complex and the RC as the
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ICM bilayer is expanded and becomes densely packed with LH2 rings. This possibility was
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confirmed by a comparison of ICM patches from cells grown at high and low light intensity by high-resolution AFM [14], in which the increased LH2 levels in the low-light ICM were shown
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to form the densely packed LH2-only domains that were absent in the high-light cells (Fig.1). These LH2-only domains represent the light-responsive antenna complement formed under low
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illumination. These structural and functional results have been largely confirmed by the proteomic analyses described below.
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2. Proteomic analyses of membrane development in Rhodobacter sphaeroides Rba. sphaeroides has numerous advantages that provide an ideal model system for examining the development and assembly of photosynthetic membranes. This metabolically versatile purple chlorophototrophic bacterium has an accessible genetic system together with an ICM that provides an unparalleled variety of biochemical, spectroscopic and ultrastructural probes. When grown photoheterotrophically (anaerobically in the light), the levels of LH2 relative to the RC-LH1 core complexes are related inversely to light intensity [30] and changes in illumination levels have offered a valuable paradigm for examining the differential biosynthesis of photosynthetic complexes. While under chemoheterotrophic growth conditions, ICM formation is repressed, lowering the oxygen partial pressure initiates gratuitous induction of ICM formation in the dark (greening) by invagination of the CM [31]. Under these circumstances, sequential
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ACCEPTED MANUSCRIPT assembly of the LH and RC complexes occurs in which the functionally essential RC-LH1 complex is synthesized first, followed by the peripheral LH2 antenna [29].
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2.1 Analysis of changes in membrane organization during a shift from high to low light intensity
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and induction of ICM formation at low oxygen tension
By availing ourselves of these capabilities, a proteomic analysis of the ICM development
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process was performed in which the temporal expression of membrane proteins was followed
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both during transitions from high to low light intensity [21,23,25,27] and the induction of ICM formation at low aeration [32]. Membrane protein components, which have assessable
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ultrastructural and functional correlates, are spatially localized in distinct membrane regions arising both from areas of nascent photosynthetic membrane development as well as the mature
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ICM, isolated in the respective upper pigmented and ICM vesicles (chromatophore) bands after rate-zone sedimentation of cell-free extracts in sucrose density gradients. This facilitates the
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assessment of the proteomes arising from distinct membrane regions at various stages of ICM development. For proteomic analysis, the chromatophore and nascent membrane fractions were subjected to clear native electrophoresis for isolation of bands containing the LH2 and RC–LH1 core complexes. In chromatophores, the proteomic profile of the trypsin-digested LH2 gel bands (Fig. 2), revealed increasing levels of LH2 spectral counts relative to those of the RC–LH1 complex as ICM development proceeded during a light-intensity downshift [21], essentially confirming the results of the spectral and AFM analyses (see below). A large array of other associated proteins was observed in the LH2 band, including high spectral counts for the F1FOATP synthase subunits and the cytochrome bc1 and pyridine nucleotide transhydrogenase complexes, as well as the hypothetical protein RSP6124. Significant levels of RSP6124 were previously reported [20] and although topological predictions suggested a lack of transmembrane
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(Fig.2). Consequently, it is possible that RSP6124 may play a transient role in the assembly or
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function of the LH2 antenna.
In contrast to chromatophores, the RC-LH1 band from the purified upper-pigmented fraction
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was enriched in cytoplasmic membrane (CM) markers (Fig. 3) including electron transfer
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proteins (2-fold) and transport proteins (3-fold), as well as general membrane protein assembly factors (2-fold)[32]. The enriched assembly factors included preprotein translocases SecY, YidC
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and YajC, together with the bacterial type 1 signal peptidase and twin arg translocation subunit TatA. These data confirm the origin of the upper pigmented band from the peripheral respiratory
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membrane and sites of active CM invagination that give rise to the ICM, in which preferential assembly of the RC-LH1 complex occurs. The complete upper pigmented fraction isolated from
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cells undergoing semiaerobic induction of ICM formation for 12 h [32] showed a much larger enrichment in CM markers (~4 fold in electron transfer components, ~14-fold in transporters), consistent with the markedly decreased levels of RC-LH1 and LH2 proteins. This fractions also showed an ~3-fold increase in general membrane protein assembly factors and detectable levels of LH complex specific assembly factors. Substantial levels of GroEL heat shock chaperonin were also found, however, reflecting the limited cell division occurring in the concentrated cell preparations used in this induction procedure. Under such circumstances, GroEL promotes the folding of nascent proteins to counteract stress-induced protein denaturation and aggregation. 2.2. Identification of early factors involved in the assembly of proteins of the bacterial photosynthetic apparatus Early factors driving the assembly of ICM proteins were identified by a proteomic approach
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under low aeration in concentrated Rba. sphaeroides cell suspensions. CCCP acts as an
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uncoupler of electron transfer from ATP synthesis [36] by inhibiting the formation of the electrochemical proton gradient generated during electron transport by the respiratory chain
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carriers [37]. Integration of CCCP into the membrane bilayer in an anionic form results in proton
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acquisition from the periplasmic space. After diffusion of the protonated CCCP form back across the membrane, protons are discharged into the cytoplasm, collapsing the electrochemical proton
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gradient and leaving CCCP in the anionic state. Multiple repetitions of this process serve to short-circuit the respiratory chain.
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polypeptide subunit into the ICM for LH2 complex assembly [38]. It
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integration of the LH2-
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cytochrome c2 export to the periplasmic space (50 M) were also shown to abolish the
was concluded that maintenance of an electrochemical proton gradient is required for translocation of the nascent pigment-binding proteins. This represents step two of the dynamic sequence of steps delineated [39] in guiding of nascent pigment binding proteins across the membrane and assembling them into functional photosynthetic complexes: (i) cotranslational chaperonin assisted folding of the nascent polypeptides into membrane insertion-competent conformations and binding to the cytoplasmic surface of the membrane; (ii) electrochemical proton gradient driven insertion and integration into the membrane; (iii) delivery and binding of the BChl and carotenoid chromophores; (iv) oligomerization of the individual protomers into oligomeric annular structures; (v) supramolecular organization into functional photosynthetic units. Here, we will concentrate largely on the second step and bring some new perspectives to
9
ACCEPTED MANUSCRIPT steps three and four. With regard to the general membrane assembly factors, studies on the formation of the LH1
PT
complex in Rba. capsulatus using a membrane coupled cell-free translation system, demonstrated that depletion of chaperonin DnaK significantly decreased the rate of synthesis and
RI
membrane insertion of both the LH1-α and –β polypeptides [40,41]. Removal of the GroEL
SC
chaperonin affected stable LH1 insertion into the coupled membranes, and expectedly, the
NU
assembly process was also dependent on the presence of unidentified membrane-bound factors. The following procedures were used to assess if the CCCP-induced collapse of the
MA
electrochemical proton gradient resulted in the accumulation of assembly factors, in association with nascent polypeptides as predicted by step 2 of the assembly pathway [39]. ICM formation
TE
D
was gratuitously induced by shifting BChl-depleted, concentrated suspensions of aerobically grown cells to semiaerobic conditions. Over the first 8 h, a vast increase in the number of both
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the RC-LH1 core and peripheral LH2 antenna complexes occurred, where the preferential appearance of the core particles is followed by an accelerated accumulation of LH2 [29,32,42], as reflected by a >4–fold increase in the molar LH2/LH1 ratio over this time period. Since limited cell division occurred in the concentrated cell suspensions, functional changes largely reflected accelerated synthesis of pigments and their binding proteins in existing cells. Therefore, this system was ideal for examining the effects of inhibition of the electrochemical proton gradient on the active assembly of pigment-protein complexes. After the addition of CCCP at 8 h, fluorescence induction/relaxation measurements showed a marked reduction in the quantum yield of the primary charge separation, as assessed from the Fv/Fm values (see Fig. 7 legend for definitions). A >4-fold slowing of the rate of RC electron transfer turnover (2.4 to 11.5 ms) occurred within the first hour after addition of the protonophore. Since multiple cycles of CCCP
10
ACCEPTED MANUSCRIPT inhibition lead to a collapse of the proton gradient and short circuit the respiratory chain, the resulting short-circuit would be expected to affect proton release phases in the Q-cycle of the bc1
PT
complex, limiting donor side electron transfer to the RC [43]. The physiological parameters
RI
assessed by these fluorescence emission measurements confirmed that 50 M CCCP should be
SC
more than sufficient to block the membrane-potential requirement for insertion of nascent polypeptide chains into the membrane bilayer.
NU
To test the effects of CCCP on protein insertion into the ICM, chromatophore fractions isolated from the inducing cells at 4 h following the onset of CCCP treatment, and from the
MA
untreated 12-h control cells were subjected to clear native electrophoresis for LC-MS/MS analysis of trypsinized gel bands [32]. The effects of CCCP treatment on the LH, RC and related
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D
energy transduction complexes from major gel bands are shown in Fig. 4. CCCP markedly blocked the membrane insertion of polypeptides of the LH2, RC-LH1 and F1FO-ATPase
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complexes, especially as sampled in the respective gel bands in which they were highly enriched. A 2.7-fold reduction in spectral counts was observed for the LH2 complex from the bottom (LH2) band, consistent with the accelerated rate of LH2 synthesis and assembly seen at this stage of the induction process. In contrast, 1.7–1.9-fold reductions were found for the RC-LH1 complex in the top (RC-LH1) band, and in the upper intermediate band for the F1-ATP and FO-ATP synthases. Likewise, the pyridine nucleotide transhydrogenase, an additional energy transducing ICM-associated protein complex, showed a spectral count reduction of 1.7-fold, averaged over all-four gel bands. Accordingly, this complex, along with the RC-LH1 core and F1FO-ATPase complexes, with spectral count decreases in the of 1.7-1.9-fold range, reflect blockage by CCCP of the membrane insertion of nascent polypeptides of complexes with rates of assembly at a basal level during this stage of ICM induction, rather than at the accelerated rate
11
ACCEPTED MANUSCRIPT seen for the LH2 complex. Moreover, these results provide novel in vivo evidence that confirms the necessity of the maintenance of an electrochemical proton gradient as an essential element in
PT
membrane translocation of the integral LH2 apoproteins [38] and extends these findings to the
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RC-LH1, transhydrogenase and F1FO-ATPase complexes. A CCCP-induced reduction in spectral counts was also observed for enzymes of BChl and carotenoid synthesis (1.6- and 1.7-fold,
SC
respectively). Although the majority of these enzymes represent soluble proteins, they may be
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degraded in the absence of appropriate associations with membrane components required for coordinating their respective pathways of pigment biosynthesis with concomitant formation of
MA
pigment binding apoproteins of the LH and RC complexes. It still seemed possible that the decline in spectral counts in the energy transducing
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D
membrane proteins reflected a slowing of protein synthesis arising from decreases in ATP levels caused by the effects of electrochemical proton gradient dissipation on oxidative
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phosphorylation. This was shown not to be the case by the similar levels of spectral counts between the CCCP-treated and -untreated chromatophores for 35 selected soluble proteins, representing enzymes of carbohydrate and amino acid metabolism (Fig. 4). This indicated that the rate of protein biosynthesis was unaffected during the 4-h protonophore treatment. Therefore, the CCCP-induced major decline in levels of the energy-transducing membrane protein complexes can be attributed to protonophore-induced blockage in the electrochemical protein gradient necessary for their membrane insertion rather than a general arrest in protein biosynthesis. As expected, CCCP treatment resulted in the accumulation of general membrane assembly factors in the unfractionated upper-pigmented fraction, as shown in the proteomic profile seen in Fig. 5. An ~2-fold elevation in the spectral counts vs. control levels was observed for the SecA
12
ACCEPTED MANUSCRIPT translocation ATPase, the preprotein translocase SecY, SecD and SecF insertion components, and chaperonins DnaJ and DnaK. Since each of these components functions in early stages of the
PT
membrane insertion process, they may have accumulated in association with nascent
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polypeptides in a functionally arrested state. The resulting transient assembly intermediates, formed as a consequence of CCCP-induced blockage of the electrochemical gradient, apparently
SC
exist in a state that is not readily dissociated or degraded. In contrast, YidC and YagC, which
NU
play roles in later stages of the Sec translocase dependent prokaryotic membrane assembly process [44], showed a CCCP-induced decline in their levels and appear to have undergone
MA
degradation. A varied level of elevation of CCCP-induced spectral counts was observed for the highly conserved SPFH domain protein [45], the FtsH metalloproteinase/chaperonin [46], and
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D
HflC, a member of the SPFH group which functions along with HflK as a modulator of FtsH activity. FtsH has been shown to cross-link with YidC and HflK/C, suggesting that these proteins
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are linked to play a role in quality control of the membrane protein integration process [47]. Other components involved in early aspects of membrane protein assembly that were slightly elevated by CCCP treatment included the signal recognition particle homologue Ffh/SRP54 [48] and signal peptidase l [49]. Assembly factors that failed to accumulate, and showed essentially no changes in levels in the upper-pigmented fraction after CCCP treatment, included complex specific assembly factors (not shown) functioning in cofactor binding and assembly steps, following the initial insertion of nascent polypeptides into the membrane. Among these components with the highest spectral counts were the cytochrome c-type biogenesis protein, cycH [50] iron-sulfur cluster-binding protein [51] and LH1 assembly factors [52]. Overall, these data suggest that nascent polypeptides destined for BChl binding and with roles in energy
13
ACCEPTED MANUSCRIPT transducing complexes, are inserted cotranslationally into the membrane in Sec-dependent manner.
PT
The observation in AFM topographs of ―captive‖ proteins in association with LH1 rings
RI
formed in an LH-only Rba. sphaeroides strain (Fig. 6)[53], provides in vivo structural support for the existence of membrane protein-assembly factor intermediates as suggested from the
SC
proteomic analysis of CCCP-treated cells [32]. The presence of complete LH1 rings still
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associated with their assembly factors is thought to reflect the absence of an RC template, leading to LH1 formation around other proteins of appropriate dimensions. Integral membrane
MA
proteins of sufficient size to serve in this capacity include BChl synthase (BchG), catalyzing bacteriochlorophyllide esterification and LhaA, a RC-LH1 core assembly factor with a possible
TE
D
role in delivery of BChl to apoproteins (see below). Not only have proteomic approaches applied to the upper-pigmented fraction facilitated
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elucidation of relative levels of associated general membrane assembly factors and their LH complex-specific counterparts [32], but also it was possible to assess the dynamics of the ICM assembly process in which general assembly factors involved in early assembly stages accumulated as putative transient intermediates as a result of CCCP-induced blockage of the membrane insertion of nascent polypeptides. Distinct transient assembly intermediates have been shown to accumulate upon disruption of photosystem assembly in the cyanobacterium Synechocystis PCC 6803 [54], leading to the identification of Psb28, a factor involved in Chl biosynthesis as well as formation of apoprotein subunits of Chl-binding proteins (the PSII antenna complex CP47, PSI RC core proteins PsaA and PsaB)[55]. Subsequently, the multifunctional assembly factor Psb27 was found to associate with the unassembled PSII antenna complex CP43, larger CP43 containing complexes, as well as the PsaB [56]. The associations
14
ACCEPTED MANUSCRIPT forming these transient assembly intermediates were demonstrated through comigration in native gel electrophoresis, copurification with His-tagged PSI and a yeast two-hybrid assay.
PT
3. Role of assembly factors in targeting BChl(Chl) to apoproteins followed by their
RI
oligomerization and organization into supramolecular structures
3.1 YidC is involved in binding of Chl to assembling apoproteins in cyanobacteria
SC
In an investigation of mechanisms involved in the delivery of Chl to nascent photosystem
NU
apoproteins, a strain of Synechocystis PCC 6803 was constructed for pull down assays to detect proteins associated with Chl synthase (ChlG), the terminal enzyme of Chl biosynthesis [57,58].
MA
Among the associated proteins was HliD, a tightly bound protein induced at high light intensity, also associated with the putative PSII assembly factor Ycf39 and the YidC insertase. In addition,
TE
D
YidC was shown by immunoblotting to be associated with ribosomal subunits. Deletion of hilD resulted in elevated levels of the Chl G substrate, chlorophyllide, suggesting a role for HilD in
AC CE P
the formation of ChlG complexes containing Ycf39 and other assembly factors. These studies established a link between Chl delivery by HilD and the SecY/YidC-requiring insertion of nascent photosystem polypeptides into cyanobacterial photosynthetic membranes [57]. Evidence for such a possible BChl-protein assembly supercomplex is discussed below. The polypeptide foldase activity of Yid C may be a crucial player in both insertion of Chl into nascent pigment binding proteins and establishment of suitable Chl binding sites. The identification of YidC together with ribosomal subunits as part of the ChlG)/pigment apoprotein assembly supercomplex is important, since ribosomal pausing has been shown to occur at distinct sites during elongation of the D1 subunit of PSII [59] and may be coordinated with Chl binding. It was suggested [57] that Yid C arranges the growing nascent polypeptide chains into a series of programmed configurations amenable to Chl insertion and that the concerted pausing by
15
ACCEPTED MANUSCRIPT the ribosome provides the stops along the growing nascent polypeptide chain for correct attachment of Chl molecules, as stipulated by the adjacent ChlG, ultimately forming the correctly
PT
folded and occupied binding pockets of active Chl containing proteins.
RI
Among the earliest functions established for YidC in Escherichia coli is its role as a membrane chaperonin, facilitating Sec translocase-dependent and -independent lateral bilayer
SC
movement of nascent polypeptides [60]. In the case of the SecYEG translocon-dependent
NU
pathway, YidC occupies the lateral gate of SecYEG and becomes displaced in a sequential manner by nascent polypeptides, thereby facilitating the lipid bilayer transfer of transmembrane
MA
segments [61]. YidC also functions in the folding of nascent polypeptides and in promoting helix-helix interactions in conjunction with the SecYEG machinery [62]. The Sec translocase-
TE
D
independent YidC pathway drives the membrane integration of the subunits a and c of the FO sector of the F1FO ATPase [63] and Subunit II (CyoA) of the cytochrome o complex [64], a
AC CE P
conserved function in respiratory chain biogenesis. YidC binds directly to the SecDFYajC in forming a heterotetrameric accessory complex [65] linking the Sec translocase complex to YidC, while SecD and SecF also participates in SecY independent insertion of proteins into the bilayer. Moreover, a holo-translocon consisting of SecYEG-SecDF-YajC-YidC along with an electrochemical proton gradient drives both the co-translational membrane protein insertion and posttranslational secretion processes [66]. Thus, YidC acts as a multifunctional assembly factor and is capable of catalyzing a multitude of activities to assist in the targeting and binding of cofactors during the insertion of nascent apoproteins into the membrane in a fully folded and functionally active state. 3.2 Roles for complex-specific assembly factors in the formation of bacteriochlorophyll a-protein complexes
16
ACCEPTED MANUSCRIPT A supercomplex of protein assembly factors can also be envisioned for the implementation of a concerted mechanism for BChl synthase (BchG)/BChl delivery/apoprotein assembly, thus
PT
minimizing free pigment exposure by rapid transfer between the synthase and a nearby
RI
translocon channel, thus avoiding the threat of photooxidative damage to the developing photosynthetic apparatus. Among the complex-specific assembly factors encoded by genes
SC
located in the photosynthetic gene cluster of Rba. sphaeroides are LhaA, an LH1-specific
NU
assembly factor, and the related LH2-specific factor PucC [52]. These proteins are members the major facilitator superfamily of transport proteins, forming part of the putative ―chlorophyll
MA
delivery branch.‖ A role for LhaA in BChl cofactor assembly was suggested from immunoblotting analyses, indicating an enrichment of both LhaA and the BChl synthase protein
D
in the Rba. sphaeroides upper-pigmented fraction [67,68] that was shown to contain nascent
TE
photosynthetic membranes that serve as a ―hotspot‖ for both BChl [67] and pigment binding
AC CE P
protein biosynthesis [See 27,28 for reviews]. It is essential that the targeting process deliver the nascent apoproteins to the membrane at the precise location and time of BChl availability through coordination of protein integration with pigment synthesis. Thus, a supercomplex can be envisioned that consists in part of BChl synthase (BChG) and such putative BChl delivery proteins as LhaA and PucC, together with the translocase component YidC. This would establish the appropriate protein architecture for pigment binding and lead to fully folded and assembled photosynthetic complexes. Since apoproteins of the RC-LH1 and LH2 complexes were the major components undergoing synthesis in the oxygen-depleted, concentrated cell suspensions during CCCP treatment [32], it is possible that the Sec translocase participates in their membrane insertion, insofar as putative early assembly intermediate were found to accumulate in the absence of an electrochemical proton gradient. These apparent intermediates contained the SecA
17
ACCEPTED MANUSCRIPT translocation ATPase, the preprotein translocase SecY, and the SecD and SecF insertion components.
PT
Genes for additional factors with photosynthetic complex assembly roles [52] have been
RI
identified within the photosynthesis gene clusters of Rba sphaeroides and Rba. capsulatus. Included are the genes encoding PufQ, a putative BChl carrier protein [69-71], PuhB, an
SC
apparent dimeric RC assembly factor which secondarily affects LH1 assembly [72], PuhC,
NU
required for optimal RC and LH1 levels, and thought to affect RC/LH1/PufX reorganization [73], and PuhE, a negative modulator of BChl synthesis [74]. In Rba sphaeroides, transcripts of
MA
pufQ, puhB and puhE were detected in a DNA microarray analysis of the oxygen and lightregulation of transcriptome expression [75]. Evidence of a role for PufQ in BChl
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D
biosynthesis/delivery comes from genetic studies in Rba. capsulatus, which showed a linear relation between pufQ expression levels and the formation of BChl, as well as conserved BChl
AC CE P
binding sequences [69]. PufQ was identified in Rba. capsulatus membranes by immunoblotting, where detectable levels were correlated with BChl synthesis after a transfer from aerobic to semiaerobic growth conditions [70]. Moreover, PufQ was also shown to have an affinity for the BChl precursor protochlorophyllide, when reconstituted into liposomes [71]. Further studies are necessary before a definitive role can be established for any of these putative assembly factors. It is important to note that the pufQ gene is at the 5’ end of the PufQBALMX operon encoding PufQ, the LH1- and -polypeptides, respectively, the RC-L and RC-M subunits and PufX. The PufX protein promotes the dimerization of the RC-LH1 core complex [76,77], in which it is interspersed within the LH1 ring a 1:1 stoichiometry with the RC [77]. The dimerization process prevents complete encirclement of LH1 around the RC, forming an LH1-based portal for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome
18
ACCEPTED MANUSCRIPT bc1 complex [78]. PufX is also implicated in the supramolecular organization of the Rba. sphaeroides ICM through alleged promotion of a uniform long-range order to the RC-LH1
PT
complex, as suggested by linear dichroism (LD) spectroscopy [79,80]. LD interrogates the
RI
difference in absorption between horizontally and vertically polarized light, relative to the
SC
orientation of membrane preparations that are ordered macroscopically in polyacrylamide gels. This facilitates determination of the transition-dipole moment orientations of relevant
NU
chromophores, viz. the accessory RC BChl, the BChl special pair and LH1 and LH2 BChls. In both LD and dark-minus-light difference LD spectra of oriented membranes containing the
MA
native RC-LH1-PufX complex, the RC was distinguished by distinctive accessory BChl and BChl special pair spectral features, not found with RC-LH1 complexes lacking PufX [79].
D
-
TE
As first seen in EMs of freeze fracture replicas of elongated tubular membranes in the LH2
Rba. sphaeroides strain M21, dimeric RC–LH1 complexes form highly ordered arrays that are
-
AC CE P
arranged helically along the longitudinal axes of the tubules [82]. LD measurements of oriented LH2 membranes showed that the RC BChls are locked in a unique, uniform orientation within individual RCs over the entire intact membrane. [79]. Oriented wild-type chromatophore preparations also exhibited a remarkable degree of structural orientation in which all RC-LH1 core structures were shown to align in regular arrays with all RC BChl transition moments oriented in the same direction, despite the presence of LH2 as the predominant antenna complex in these native membranes. Moreover, the relative contributions of the LH1 and LH2 complexes to the overall LD spectrum in chromatophore vesicles in which the LD of LH2 was reduced relative to that in the LH1 absorption peak, showed that LH2 preferentially resides in highly curved parts of ICM membrane vesicles, since the LD and absorption peaks were equalized in flattened membrane patches [80].
19
ACCEPTED MANUSCRIPT While considerable information on how the supramolecular structure and organization of photosynthetic unit cores is established has arisen from these studies, it has recently been
PT
suggested from variations in the level of supramolecular organization of the Rba. sphaeroides
RI
ICM induced by alterations in PufX, that PufX may play an indirect role in the ordering of RC-LH1 dimers over the membrane [83]. Instead long-range order may stem from the role of
SC
PufX in promoting an appropriate dimer configuration with the creation of ordered RC–LH1
NU
domains depending not only on the presence of RC–LH1 dimers in their appropriate bent conformation, but also on the structure of LH1 on the outside of the complex. Thus, the sole
MA
influence that PufX exerts on membrane ordering may be via the facilitation of the dimerization process in which the dimer has one long axis of symmetry and is bent along the dimer interface
TE
D
[84].
3.3 Oligomerization of LH1 and assembly of the RC-LH1 core complex as observed in AFM
AC CE P
studies
In an AFM imaging study of the assembly of single RC-LH1 molecules using membranes from an LH1 only mutant [53], a series of aberrant forms of the LH1 complex in which the basic repeating unit (LH1-11(BChl)2) was seen to assume ring structures of variable size, elliptical structures, spirals and arcs that appear to represent assembly products (Fig. 6). The spiral complexes occur as a result of a failure in the closing of the ring, while the short arcs, first observed in lithium dodecyl sulfate/polyacrylamide gel electrophoresis [82], arise from premature termination of the LH1 complex assembly process. Occasionally, captive proteins were observed that were enclosed within the LH1 ring (Fig. 6) and as noted above, they could correspond on the basis of their size to assembly intermediates in which BChl synthase or complex specific assembly factors such as LhaA are bound and still associated. The presence of
20
ACCEPTED MANUSCRIPT such intermediates may reflect LH1 formation around proteins other than the RC template, which is absent in this strain [53]. A mixture of fully formed RC-LH1 complexes, emptied LH1 rings
PT
and isolated RCs was imaged (Fig. 6C) when formation of LH1 was restricted by a lowering of the cognate pufBA transcripts levels through removal of the stem-loop structure between the pufA
SC
RI
and pufL genes and progressive truncation at the C-terminus of the LH1- polypeptide. Overall, the resulting assortment of structures suggested a cooperative mechanism for assembly of the
NU
RC-LH1 complex in which once an association occurs between the RC and the first LH1 repeating unit, RC associations with successive LH1 subunits drive the LH1 ring assembly
MA
process to completion. Partial LH1 rings were not observed in the LH1-limited constructs (Fig. 6C), establishing that the necessity of an RC template for LH1 ring assembly. These AFM
TE
D
studies essentially confirm previous results (85) from an examination of ICM induction under low aeration in which the RC-H subunit was detected first, followed by PufX and thereafter by a
AC CE P
low level of LH1- and -subunits, which successively increased. The assembly process was proposed to reflect the initial formation of a RC-(LH1-11(Bchl)2)-PufX complex, with progressive addition of LH1-11(BChl)2 subunits, that eventually encircle the RC. Forces guiding the assembly of the LH2 complex, which unlike LH1 consists of an ―empty‖ ring structure, have been studied within the native ICM of Rsp. photometricum by AFM imaging of single molecules, combined with force measurements [86]. The binding of LH2 protomers was found to be fairly weak, indicating the importance of LH2 ring architecture in stabilizing the structure of the LH2 complex. In addition, intermolecular forces were shown to influence LH2 structural and functional integrity, as demonstrated from AFM unfolding kinetics in which the G for unfolding of an LH2 subunit consisting of an LH2-11(BChl)3 structure was estimated to be 222 kJ mol-1. Since a similar G might be anticipated for unfolding of an LH1-11(BChl)2
21
ACCEPTED MANUSCRIPT subunit, the oligomerization of an LH1-only complex would be expected to be driven by small cumulative inter-subunit forces that facilitate ring formation (53). In the case of a spiral complex,
PT
crowding pressure from adjacent rings might be expected to misalign the nascent complex so that
RI
additional subunits are assembled to form a spiral. Overall, the studies on forces driving the assembly of the LH2 complex [86] showed that: (i) weakly bound subunits are capable of self-
SC
assembling to form rings; (ii) the stability of the complex is guarantied by the ring-shape alone;
NU
(iii) the individual subunits are essentially solid and their stability further depends on the intramembrane molecular environment.
MA
3.4. LH2 and RC-LH1 play roles in establishing ICM architecture The discovery of a vesicular ICM similar to that of the wild-type in the Rba. sphaeoroides
TE
D
NF57 LH2-only mutant (i.e. lacking the RC and LH1 complexes) suggested that the LH2 oligomeric ring structure possesses the membrane bending properties responsible for the ICM
AC CE P
vesiculation process [87,88]. On the other hand, a tubular ICM system was found in the M21 mutant lacking LH2 that was sometimes seen running between two cells appearing to be arrested in cell division (not shown). The observed ultrastructural differences suggested that in the absence of LH2, ICM morphogenesis is incomplete and is arrested at a tubular stage. The presence of vesicular ICM in the NF57 strain indicated that LH2 alone is both necessary and sufficient for vesicular ICM formation and that membrane morphogenesis can be completed in the absence of RC-LH1 core particles. As noted in section 3.2, the RC-LH1 complex exists in Rba. sphaeroides mainly as an Sshaped dimeric structure. The dimerization process is facilitated by the PufX protein, which serves as an integral component of the core complex (RC2LH1- pair28PufX2)[77]. In an effort to determine the extent to which the dimeric RC-LH1 complex controls the morphology of the
22
ACCEPTED MANUSCRIPT ICM, a 3-D RC-LH1-PufX model was constructed from single particle EM analysis of negatively stained complexes [84]. The two halves of the dimer were inclined toward one
PT
another on the periplasmic aspect of the complex, creating an uncommon V-shaped structure
RI
measuring ~200 A° along the long dimer axis and 100 A° along the short axis. The strikingly bent modeled structure has revealed the basis for the imposition of membrane curvature by the
SC
core complex and the ability to drive the formation of budded, tubular membrane structure,
NU
together with the arrangement of dimeric RC-LH1 complexes in highly ordered helical arrays along the tubules and the ordered packing of the dimers in 2D crystals. It can also be envisioned
MA
that RC-LH1 dimers utilize their distinctive curvature properties to facilitate their aggregation into the rows of RC-LH1 complexes observed uniquely in AFM topographs of Rba. sphaeroides
TE
D
[13] to facilitate excitation energy transfer from LH1 to the RC. In an investigation of the membrane bending effects of the highly bent RC-LH1 complex,
AC CE P
molecular dynamics simulations were employed for constructing an all-atom structural model within the EM density map [89]. A membrane of high local curvature was simulated which agreed well with the size of RC-LH1 tubules and further demonstrated how the intrinsic geometric shape of RC-LH1 dimers, consisting of a structure bent at the dimer interface, propagates the extended organization of a tubular ICM. In a similar approach, using the atomic resolution X-ray structure of the Rhodoblastus acidophilus LH2 and a homology-modeled Rba. sphaeroides LH2 structure [90], membrane embedded arrays of LH2 rings structures were found to assume a dynamic curvature, consistent with the putative LH2 membrane bending properties. An array of seven LH2 rings, as observed in AFM for LH2 complexes of Rsp. photometricum membranes (Fig. 1E), gave simulations in which these structures were seen to tilt away from neighboring complexes on the cytoplasmic side which created a curvature for the LH2
23
ACCEPTED MANUSCRIPT aggregates, that consequently curving the membrane. Thus, the aggregation dependent properties of individual LH2 rings were proposed to account for the membrane curving properties.
PT
Although a collections of seven rings grouped together is often encountered in Rsp.
RI
photometricum membranes, the ICM of this organism consists of regular bundles of flattened thylakoid-like discs rather than vesicular ICM. Moreover, while EMs of high-light grown Rba.
SC
sphaeroides in which LH2 concentration is low, clearly show a vesicular ICM [14], the LH2
NU
rings therein do not not form large aggregates, but instead are present mainly as individual structures in dimeric association or short single-file rows (Fig. 1C, 14). Also, simulations based
MA
on the atomic resolution LH2 structure of Rbl. acidolphilus showed the same arrangement of seven grouped LH2 rings as for Rba. sphaeroides LH2 and no substantial differences in
TE
D
membrane curvature were simulated, despite the existence of a lamellar ICM in Rbl. acidolphilus. Thus, while LH2 is clearly a membrane-bending protein, the overall mechanism of
AC CE P
membrane bending and vesiculation remain elusive. In this connection, the possibility of a role for protein-lipid interactions in establishing ICM architecture in Rba. sphaeroides has has been suggested from the finding of a putative LH2-lipid binding site and enrichment of the non-bilayer phospholipid phosphatidylethanolamine in the lipids associated at the boundary with LH2 [91]. The lipid-binding site included the keto carbonyl group of spheroidenone and a Glu residue located two residues from the N-terminus of the -subunit. When changed to Ala by site-directed mutagenesis, a reduction of the phosphatidylethanolamine level in the lipid boundary phase was observed together with its replacement by phosphatidylcholine. ICM from the strain harboring the mutated protein retained a normal spherical vesicular morphology, but by exchanging the carotenoid from spheroidenone to neurosporene resulted in tubular membrane invagination in this strain. It was suggested that
24
ACCEPTED MANUSCRIPT this specific Glu residue as well as the nature of the carotenoid polar head group govern interactions with the surrounding phospholipid that together with LH2 participate in the ICM
PT
vesiculation process. This led to the conclusion that the preferential binding and packing of
RI
phospatidylethanolamine at the LH2-lipid interface is essential for maintaining ICM morphology.
SC
4. Dynamics of the developing ICM as examined by fluorescence emission analyses
NU
4.1 Bacteriochlorophyll a-based fluorescence induction/relaxation kinetics Fluorescence induction/relaxation measurements have served as a valuable method for
MA
assessment of the functional absorption cross-section, the charge separation and electron transport capabilities of anoxygenic phototropic bacteria [92-97]. While light-induced absorption
TE
D
changes for monitoring electron transfer reactions have largely prevailed for the evaluation of the physiological status of the purple anoxygenic phototrophs, several additional functional
AC CE P
parameters can be conveniently assessed from the analysis of the fluorescence emitted when RC phototraps are photooxidized and effectively become ―closed‖ during the fluorescence induction phase, and open again during the fluorescence relaxation phase. These include: (i) the quantum yield of the primary charge separation; (ii) the rates of RC electron transfer turnover; (iii) the functional absorption cross-section and connectivity of the photosynthetic apparatus (Fig. 7). Moreover, fluorescence spectroscopy is a highly sensitive technique for examining membrane dynamics in a component specific manner with both intrinsic and extrinsic fluorophores. The pioneering discovery of the relation between the fluorescence yield and the fraction of closed ―RC‖ phototraps by Duysens [96] has served as the basis for the current understanding of how excitation energy transfer, via inductive resonance, serves to couple the large LH antenna complement to multiple RC. When a RC is closed and, providing that little energy is lost via
25
ACCEPTED MANUSCRIPT non-radiative dissipative pathways, excitations can visit neighboring open RCs and ultimately undergo trapping. A structural basis for this concept has recently been borne out by AFM
PT
topographs of native Rba. sphaeroides membranes [13,14](Fig. 1A), in which many RC-LH1 core complexes are arranged in multiple rows of dimeric structures, facilitating the migration of
RI
an LH1 excitation along a series of dimers until an open RC is found. Analysis of the induction
SC
and relaxation characteristics of the fluorescence emitted during these trapping events is the basis
NU
for determining the various physiological parameters that interrogate the functional status of the photosynthetic apparatus.
MA
The variable fluorescence signal arising from the RC phototraps reflects the redox status of the RC, where a low fluorescence yield indicates an open RC (uncharged, P870 capable of
TE
+
D
performing photochemistry) and a high fluorescence yield indicates a fully closed RC (P870 photooxidized to P870 , charged RC transiently nonfunctional). The difference between the
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minimal fluorescence (F0) and the maximal fluorescence (FM) yields upon closing of the RC, provides an estimate of the quantum yield of the primary charge separation, obtained from the FV/FM ratio, where variable fluorescence FV = FM-F0. In addition, analysis of the induction kinetics also provides an estimate of the functional absorption cross-section (σ) of the LH complexes, as well as their connectivity (p), the latter reflected by the sigmoidicity of the fluorescence induction curve. The fluorescence relaxation kinetics arise from the reopening of the RC as governed by the RC electron transfer turnover rate (τET). Importantly, the kinetics and energetics of the RC donor and acceptor side electron transfer reactions determine the nature of the fluorescence relaxation phase [43]. As noted by van Grondelle [97], three different RC redox states are known to cause substantially different fluorescence yields during the induction phase: (i) fully active ―open‖ RCs (oxidized P870, QA, QB) having a low fluorescence yield; (ii) closed
26
ACCEPTED MANUSCRIPT -
RCs which have twice the yield of open RCs if QA is reduced to the semiquinone anion (QA. ); (iii) closed RCs which have ~3-times the open RC yield when the primary donor is fully +
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oxidized to the P870 .
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Kato et al. [98] have recently noted that while Chl fluorescence techniques can serve as a
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highly accessible alternative method for monitoring the charge separation and O2 evolution activities of PSII in oxygenic phototrophs, fluorescence only indirectly assesses such parameters
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as RC charge, and is dependent on excitation light intensity and wavelength1 and whether the
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sample is dispersed in solution or in the solid state. Moreover, imprecise spectroscopic interpretations can arise from proteins that remain spectroscopically active after losing their
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functional activities. Although these caveats may also apply to BChl fluorescence emission of
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purple anoxygenic phototrophs as assessed in induction/relaxation measurements, the results of our fluorescence kinetic analyses are largely corroborated by related structural and functional
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correlates: (i) FV/FM ratios are in the range of 0.7-0.8, close to the ~0.65 obtained in Rbl. acidophilus in the presence of the QB-site inhibitor terbutryn [95]; (ii) fluorescence induction curves display essentially exponential kinetics consistent with single turnover behavior (Fig. 7); (iii) FM levels obtained by single turnover flashes were essentially matched by those arising from multiple turnover pulses on a tens of millisecond timescale; (iv) values obtained for functional absorption cross-sections ranged from 28-130 Å2 [23,29], corresponding closely to the range of BChl molecules/RC (30-120) which make up the photosynthetic unit. The lower value, 1Koblízek
et al. (29) observed a dependence of excitation wavelength on functional absorption cross-section values, which for steady-state Rba. sphaeroides cells excited at 470 and 795 nm were ~55 Å2 for 470 nm and ~65 Å2 for 795 nm. This can be accounted for by differences in energy transfer efficiency from carotenoids to LH1 BChl, which in the LH1 complex isolated by lithium dodecyl native electrophoresis [99] yielded a value of 61 % [100], accounting for the lower values for 470 nm, while a higher efficiency of B800 B850 energy transfer accounts for the higher 795 nm value. 27
ACCEPTED MANUSCRIPT representing the number of LH1 BChl molecules per RC, is close to that of 32 obtained in Rba. sphaeroides by spectral deconvolution coupled with RC photooxidation measurements [101],
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and from fluorescence induction in Rbl. acidophilus containing both the B800-850 and B800-820
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LH2 antenna complexes [95]. We believe that together, these data attest to the validity of near-IR fluorescence induction/relaxation analyses for monitoring selected photosynthetic activities of
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the purple anoxygenic phototrophs.
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It has been widely proposed that in the PSII RC of oxygenic phototrophs, variable fluorescence mainly reflects the redox status of primary stable electron acceptor QA (RC acceptor
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side)[102]. As noted above, recent reports [42,43] on the nature of the fluorescence relaxation phase in Rba. sphaeroides suggest a major role for the primary P870 electron donor (c-type
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cytochrome), depending upon the type of RC association of the donor cytochrome in the organism under examination. Accordingly, Asztalos et al. [43], using intact cells of Rba.
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sphaeroides, Rhodospirillum rubrum and Rubriviax gelatinosus, showed that laser diode excitation at 808 nm yielded complex, multi-expontential fluorescence decay kinetics. Longer excitations were correlated with the slowing of the relaxation phase, which was determined by the redox status, size and accessibility of the reduced cytochrome c2 and quinone pools for bacterial strains limited on either the donor or acceptor side of the RC, respectively. In Rba. sphaeroides and Rsp. rubrum, in which mobile cytochromes c2 serves as the immediate P870 electron donor, relaxation is preferentially controlled by the re-reduction rate of the oxidized RC by the reduced cytochrome c2, and the acceptor side has only a minor role. For Rba. sphaeroides, the multiphasic P+ reduction kinetics have been explained by a proximal–distal cytochrome c2 binding model [103-105] where the fastest component (~1 s) reflects tight binding at the proximal site. The origin of the second phase (20-200s) is less clear and may result from
28
ACCEPTED MANUSCRIPT cytochrome c2 binding at the distal site or the cytochrome may move from other binding sites. The third phase (100s-10 ms) is thought to arise from the shuttling of cytochrome c2 between
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the RC and cytochrome bc1 complexes. The close correlation between the equilibrium redox +
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titration on the RC donor side and the fluorescence relaxation (43) further suggested that P
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reduction acts as the rate-limiting step the Rba. sphaeroides RC re-opening process. On the other hand, in Rvx. gelatinosus where the cytochrome subunit is physically attached to
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the RC, the acceptor side determines the fluorescence relaxation kinetics with the rate constant of -
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350 s—1 for QA interquinone electron transfer as the determining factor. Importantly, an analysis of the nature of the relaxation phase as a means of assessing membrane dynamics has
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recently been applied to Rba. sphaeroides cells undergoing different developmental regimens
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(23,24,42), as will be described below.
4.2 Alterations in membrane dynamics as assessed by fluorescence induction/relaxation analyses
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A near-IR fast-repetition rate fluorescence analysis2 by Koblízek et al. [23] in steady-state Rba. sphaeroides cells undergoing gratuitous induction of ICM formation at reduced oxygen tension in the dark was the first to suggest that ICM development was accompanied by major changes in membrane dynamics. A linear relation was observed between the rise in the 795
nm
and the elevations in the LH2/RC; this was expected since both parameters reflect the preferential insertion of LH2 into the membrane, while the LH1/RC remained constant, making up the core of the photosynthetic unit. Moreover, a correlation between the growth of the LH2 antenna and the gradual slowing of RC electron transfer turnover was observed. The
ET
was
relatively fast (0.8-1.6 ms) following aerobic growth and during the first 3 h at low aeration, but 2
Although for these initial studies, a near-IR fast repetition rate fluorometer [106] was employed in which the fluorescence signal was excited by a sequence of submicrosecond flashes rather
29
ACCEPTED MANUSCRIPT than a single 144 s single turnover flash as in the subsequent in the subsequent FIRe analyses, the results from both systems have proved to be essentially the same.
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a decrease to 7 ms was observed after 13 h, occurring in parallel with the rise in functional absorption cross-section. This was attributed to an imposition of constraints on electron transfer
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as the membrane bilayer becomes densely packed with accumulating LH2 rings. This possibility
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is supported further by fluorescence induction/relaxation measurements in cells undergoing adaptation from high to low light intensity. The rates of RC electron transfer turnover and
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reoxidation of UQ pool were decreased by ~4- and 3-fold, respectively, over 4 days [23].
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Moreover, the slowing of ET was directly related to the increase in the functional absorption cross-section (450 nm ranged from ~70 Å2 at outset to 130 Å2 after 4 days) [23,24]. Again, an
D
essentially linear relation was observed between the size of the absorption cross-section and the
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LH2:LH1 molar ratios. Thus, when applied to the monitoring of changes in photosynthetic
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activities during ICM development, taken together with AFM surface views of the alterations in membrane structure (13,14), fluorescence induction/relaxation measurements have served as a valuable probe of the membrane dynamics accompanying the membrane remodeling process (23,27).
While we initially attributed the slowing of the ET as a function of increasing LH2 levels to the imposition of constraints upon free diffusion of ubiquinone redox species between the RC and cytochrome bc1 complex (RC acceptor side kinetics)[23,24] as discussed above, a somewhat more complicated picture has emerged from the studies on Rba. sphaeroides by Kis et al. [42]. In cells adapting from aerobic to phototrophic growth conditions under constant illumination, donor electron transfer reactions dominated the relaxation kinetics after a single-turnover flash, while contributions on the acceptor side were enhanced only after multiple turnover flashes. It was
30
ACCEPTED MANUSCRIPT concluded that the slowing of electron transfer turnover results from changes on the RC donor side in which an increased distance for electron flow occurs in the cytochrome c2 periplasmic
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diffusion pathway between the RC and cytochrome bc1 complexes. This accounts for the initial
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relaxation kinetics when the developed (mature) membrane becomes more densely packed with LH2 rings and/or the distal RC position [103-105] becomes less accessible to cytochrome c2.
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Moreover, Kis et al. [42] showed that the slowed rate of electron transfer during ICM
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development did not result from changes in cytochrome c2 pool size. On the other hand, slow reoxidation of the UQ pool reflected contributions of the acceptor side, which is enhanced after
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long excitation (multiple turnovers). This is also compatible with our results [23,24], which were interpreted to show slowing of UQ pool oxidation with increasing LH2 levels after multiple
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D
turnover flashes. However, AFM images of Rba. sphaeroides LH2-only membranes have formed the basis for a functional model of highly curved native membranes, which holds that these
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structures would permit the free diffusion of electron carriers through the LH2 domains, while still maintaining efficient excitation energy transfer [107]. Clearly, more work needs to be done on the question of whether quinone redox species flow is impeded by the presence of hexagonally packed LH2-only domains. 4.3. Searching for a mechanism explaining the decreases in reaction center electron transfer turnover rate during greening and adaptation to low light intensity A second question arises concerning the growth of the Rba. sphaeroides ICM during adaptation to reduced oxygen tension or low light intensity and the distance traveled by cytochrome c2 as predicted from the fluorescence induction studies of Kis et al. [42]. The intracelleular status of the Rba. sphaeroides ICM is now a matter of intense debate [16,108,109]. The cryo-electron tomography images of Tucker et al. [16] revealed a complex scenario for the
31
ACCEPTED MANUSCRIPT ultrastructure assumed by the ICM. While some ICM vesicles were observed without connections to other structures, others were located nearer to the CM, frequently forming
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interconnections with physical association to the CM, providing apparent access to the
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periplasm. Some nearly spherical single invaginations, with ―necks‖ attaching them to the CM were also seen, along with small CM indentations, representing the ICM precursor membrane
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isolated in the upper pigmented band in sucrose density gradients of cell-free extracts. Thus,
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fully detached ICM vesicles, possessing all the machinery for converting light energy into ATP, regarded as bacterial membrane organelles, only represent a part of the picture, for which the
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tomography methodology does not permit quantification of their relative content. A functional approach to the question of whether an ICM continuum exists with free access to
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D
the periplasmic space has been taken recently [108], where the ionophore gramicidin was employed to investigate the relative size of the electroosmotic units in intact cells, isolated
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chromatophores and spheroplasts, lacking an outer membrane. In intact cells and in spheroplasts, the half-time of the decay induced by a single channel was ~6 ms and ~23 ms, respectively, as measured from the carotenoid electrochromic shift, which was three to four orders of magnitude slower that in isolated chromatophores. This was interpreted to mean that that the area of functionally active membrane in cells or spheroplasts is at least three orders of magnitude greater than that in individual chromatophore vesicles, which at most could contribute 10% of the active photosynthetic membrane in the cell. This is to be compared with the results of Prince et al. [110] who showed that ≤85 % of the cytochrome c2 was released upon spheroplast formation and thus an upper limit for free chromatophores would be ~15%; however, this is likely to amount to more at low light intensities. It was emphasized [108] that a single internal membrane system with connections to the periplasmic space may provide significant advantages in renewing the
32
ACCEPTED MANUSCRIPT photosynthetic apparatus and in the reallocation of the components shared with additional bioenergetic pathways.
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A more recent report on the cryo-electron tomography of the ICM from Rba. sphaeroides has
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appeared [109] in which the results were interpreted to show that ICM vesicles form a continuous reticulum rather than existing as discrete vesicular structures. The possibility must be
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considered that the different 3-D ICM arrangements are due to strain difference, since the Ga
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strain used in this study was derived from the 2.4.1 strain in which the yield of ICM vesicles is much lower than in strain NCIB8253 [27]. Most of the photopigment is retained in an upper-
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pigmented band in sucrose gradients, arising from CM invagination sites. This means that the internal membrane invagination and budding process may be limited in the 2.4.1 and Ga strains.
TE
D
In this study [109], the cytochrome bc1 complex was localized in the ICM, through immunoelectron microscopy using immuno-gold labeling, with antibodies raised against peptides
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representing putative, well conserved cytoplasmic loops of cytochrome b from Rsp. rubrum. Labeling was mostly found on small membrane fragments and in the ―neck‖ regions protruding from the ICM vesicles suggesting that that the complexes localize to the fragile membrane regions that interconnect the chromatophores in the network. These fragile regions were thought to arise from tubular structures reconstructed from tomographic data, seen lying between ICM vesicles. Further, it was proposed that the bc1 complexes within these regions are surrounded by RC-LH1 complexes, bordered by arrays of the peripheral LH2 antenna complex. The localization of the cytochrome bc1 complex in the native ICM has also been examined recently by Cartron et al. [111]. EMs of negatively-stained Rba. sphaeroides chromatophores in which the C-terminus of cytochrome c1 contained a His10-tag that was labeled with gold nanobeads, showed that the majority of the labeled complexes were dimeric (Fig. 8A,B). The bc1
33
ACCEPTED MANUSCRIPT complexes appeared to be adjacent to RC-LH1 complexes, as seen in AFM topographs of goldlabeled chromatophores (Fig. 8C). While this provides evidence that restricted electron transfer
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domains might exist, these were not seen as fixed RC-LH1-PufX-cytochrome bc1
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supercomplexes. Instead, cytochrome bc1 complexes sit adjacent to the core complexes in disordered areas, not necessarily in direct contact with the majority of the RC-LH1-PufX
SC
complexes (Fig. 8). These findings were combined with a quantitative MS analysis in which the
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levels of the RC, cytochrome bc1 complex, ATP synthase, and cytochrome aa3 and cbb3 complexes were determined as a basis for constructing an atomic resolution model of a 50-nm
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chromatophore vesicle. The modeled vesicle contained 67 LH2 complexes, 11 LH1–RC dimers, 2 RC–LH1 monomers, 4 cytochrome bc1 dimers and 2 ATP synthases. Through simulation of the
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D
energy, electron and proton transfer processes, the calculated half-maximum ATP turnover rate suggested that this photosynthetic unit architecture is optimized for accommodating to low light
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intensities.
Since the current atomic-level structural model for a 50-nm low-light chromatophore has four dimeric cytochrome bc1 complexes in close arrangement with the RC-LH1 complex, with seven RC-LH1 at some distance, the average distances over the internal vesicle surface for diffusion of cytochrome c2 between cytochrome bc1 and the RC is 10–30 nm. This may account for the observed slowing of the RC donor pathway, but localization of the bc1 complex in high-light chromatophores in which the LH2 content is reduced, will be essential for confirming this possibility. Acknowledgements We thank Prof. Peter Lobel and Dr. Haiyan Zheng for conducting the proteomics analyses and Dr. Kamil Woronowicz, Dr. Raoul Frese, Oluwatobi B. Olubanjo and Daniel Sha for
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ACCEPTED MANUSCRIPT participating in the studies performed in my laboratory. Work in the author’s laboratory was supported by grants from the U. S. Department of Energy, Grant No. DE-FG02-08ER15957
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from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy
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D
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Sciences and the National Science Foundation (Subaward No. 12-764).
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ACCEPTED MANUSCRIPT [111] M.L. Cartron, J.D Olsen, M. Sener, P.J. Jackson, A.A. Brindley, P. Qian, M.J. Dickman, G.J., Leggett, K. Schulten, C.N. Hunter, Integration of energy and electron transfer
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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. High-resolution AFM topographs of the cytoplasmic surface of native ICM represented
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by Rba sphaeroides membrane patches (A-C) [14] and flattened thylakoid-like discs from Rps. photometricum (D-F) [15]. The Rba. sphaeroides membrane patches were prepared with 0.03%
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-dodecyl maltoside, which is at a sub-critical micelle concentration, in order to open up ICM vesicles so that the resulting ICM patches are located closer to the mica surface, thereby
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maximizing the area available to the AFM tip during tapping mode acquisition of topographs. This represents a subsolubilizing level of detergent without apparent effects on membrane
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structure [10]. ICM preparations in panels (A) and (B) are from cells grown at low light intensity (4 W/m2). (C) Cells grown at high light intensity (220 W/m2). LH2 complexes are seen as
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separate nonameric ring structures (∼6–7 nm in diameter), assuming a zigzag appearance especially in LH2-only domains (B), as a result of vertical displacement when the natively
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curved membranes adsorb onto the planar mica surface, leading to lines of raised LH2 complexes. This is also seen in panel (A) for a membrane patch also containing a row of RCLH1 dimers (∼8.5 nm RC-RC separation within dimer) with the RC-H subunit projecting from the membrane surface. Panel C shows a vast predominance of dimeric RC-LH1 complexes in patch form high light cells with only a few LH2 and monomeric RC-LH1 complexes. This predominance of RC-LH1 dimers was seen previously [16] in non-detergent treated membrane preparations representing nascent ICM growth initiation sites isolated from the upper-pigmented band. For the high- and low-light chromatophores, the LH2/LH1 molar ratios, determined as described by Sturgis et al. [17] were 0.53 and 1.23 respectively. (D-F) High-resolution AFM topographs of native Rsp. photometricum ICM preparations obtained by contact mode AFM. Here the RC-H subunit has been removed by nanodissection with the AFM tip leaving
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ACCEPTED MANUSCRIPT cytoplasmic surfaces of the L and M subunits of the RC exposed. (D) High-light-adapted (~100 W/m2) ICM [15]. M, closed monomeric RC-LH1 core complex; LH2, nonameric LH2 complex.
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The core complexes and LH2 rings are arranged randomly and are found in an ∼1:3.5 ratio. (E,F)
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High-resolution topographs of low-light-adapted (10 to 20 W/m2) ICM. In panel E, an area of
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mixed core complexes and LH2 rings is shown, with an overall core complex:LH2 ratio of ∼1:7; areas of paracrystalline hexagonal arrays of LH2 are also seen that are devoid of core complexes.
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In panel F, an area is seen with substantially more core complexes than the average distribution. In these regions, as in panel D, several core complexes are found in contact, and the overall
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∼1:3.5 core complex:LH2 ratio is retained. The arrow in panel (D) delineates a free lipid bilayer space between LH2 complexes, which is thought to be functionally important in facilitating
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membrane mobility for quinone-quinol exchange to and from the core complex [18], just as the openings in the dimeric RC-LH1 complexes at the ends of linear core chains are thought to serve
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in this function in Rba. sphaeroides [19].
Fig. 2. Proteomic analysis of chromatophore LH2 gel band derived from clear-native electrophoresis, showing the distribution of spectral counts [25]. Chromatophores were isolated after 3 and 24 h from cells undergoing acclimation from high to low light intensity. For clearnative electrophoresis, chromatophores were solubilized with n-octyl β-D-glucopyranoside (-OG) and deoxycholate (DOC), both at concentrations of 15 mM, and subjected to clear-native electrophoresis essentially as described by Wittig et al. [33]. Resolved gel bands were excised and subjected to in-gel trypsin digestion, followed by LC-MS/MS at sub-femtomol sensitivity. Tandem mass spectrometry data files were searched against the NCBI assembly of the annotated Rba. sphaeroides 2.4.1 genome [34]. Proteomic data were quantified by spectral counting, which
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ACCEPTED MANUSCRIPT measures protein abundance on the basis of the number of tandem mass spectral observation for all constituent peptides. Accordingly, spectral counts mainly reflect the ability of trypsin to act at
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potential cleavage sites and are therefore only semiquantitative in nature. Proteins that are not
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highly abundant with a large number of accessible sites can give rise to higher counts than more abundant proteins with far fewer sites; in cases where no sites are available, no counts are found,
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e. g., PufB (LH1-β polypeptide) and PucA (LH2-α), although the latter polypeptide was detected
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after chymotrypsin cleavage [25]. Since the gel lanes were loaded with equal amounts of total protein, valid comparisons can be made between the same protein components in the
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chromatophore and upper-pigmented band preparations. Since spectral count sampling statistics have high technical reproducibility, they reflect precise assessments of relative protein
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abundance for differential protein expression studies such as those reported here. The spectral count distributions shown are for clusters of orthologous groups [35], in which the usual energy
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production and conversion categories have been divided into subgroups, which account for the distinct metabolic capabilities unique to Rba. sphaeroides.
Fig. 3. Proteomic analysis of upper-pigmented (A) and chromatophore fractions (B) in the clear native electrophoresis RC-LH1 enriched gel band. Membrane fractions were isolated from cells after a 24-h adaptation to a light intensity of 100 W/m2 [32]; the respective membrane fractions were solubilized with digitonin and
, and the resolved gel bands were subjected to in-
gel trypsin digestion and LC-MS/MS. The distributions shown are for clusters of orthologous groups [35], modified as described in the legend of Fig. 2.
Fig. 4. Effects of CCCP on the LH, RC and related energy transduction complexes of the ICM as
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ACCEPTED MANUSCRIPT revealed by proteomic analysis of clear native electrophoresis gel bands resolved from the isolated chromatophore fraction. Chromatophores were solubilized with -OG/DOC and isolated
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gel bands were subjected to in-gel trypsin digestion and LC-MS/MS [32].
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Fig. 5. Proteomic analysis of upper-pigmented fraction isolated from CCCP-treated cell suspensions undergoing ICM induction showing effect of CCCP on general membrane assembly
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factor levels (A,B). Following removal from sucrose gradients, the upper-pigmented band was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and after entering, gel
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bands were excised for in-gel trypsin digestion followed by LC-MS/MS in which a 135-min LC
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gradient was used for optimal peptide resolution.
Fig. 6. (A, B) LH1-only membrane patches prepared using sub-critical micelle concentrations of
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-dodecylmaltoglucoside, showing aberrant complexes as depicted in 3-D [53]. (A) LH-1 only membrane patch showing an incomplete LH1 ring (blue arrow), LH1 arcs (cyan arrows) and captive proteins (red arrows)(see also E, F). (B) LH-1 only membrane showing aberrant complexes consisting of a spiral structure and an open ring enclosed within the white and blue boxes, respectively. The noise levels were reduced in these topographs by using a low-pass filter. (C) Membrane patch from a LH1-limited mutant [53], showing a 3-D representation of an intact RC-LH1 complex (magenta arrow), an isolated RC (white arrow), an RC-LH1 complex without the H-subunit (blue arrow), and an empty LH1 ring (red arrow). (D-F) AFM topographs of LH1 complexes reconstituted in 2D LH1 crystals adsorbed onto mica, prepared from an LH1-only Rba. sphaeroides strain (Topographs kindly provided by Prof. C.N. Hunter). While the majority of complexes consisted of empty ()16 LH1 rings of ~115 Å diameter (D), the complexes
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ACCEPTED MANUSCRIPT shown in E. and F. were among several that resemble putative assembly intermediates with a membrane protein entrapped in their interior, tentatively identified as LhaA, an LH1 assembly
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factor, which like the Lac permease belongs to the major facilitator superfamily, forming part of
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its putative ―Chl delivery branch‖ 52]. The space filling image (G) of the Lac permease [81], closely resembles the large transmembrane protein enclosed within the LH1 annulus (E, F),
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apparently representing the assembly factor still in association with its LH1 substrate. It is
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possible that entrapment of LhaA within the LH1 ring arose from the lack of RCs in this strain, which may normally displace this complex specific assembly factor during the core assembly
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process in which the RC is believed to act as a template [53].
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Fig. 7. Fluorescence kinetic transient elicited upon 450-nm excitation of Rba. sphaeroides cells undergoing induction of ICM development at low aeration [24] measured in a Satlantic
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Fluorescence Induction/Relaxation system (Satlantic Inc., Halifax, Nova Scotia). Measurements were made on whole cells diluted typically to ~ 30 nM BChl in growth medium to produce transients in the linear range. A blue LED (450 nm, 30-nm bandwidth) served as the excitation source. Fluorescence emission is passed through an 880-nm interference filter (50-nm bandwidth) and detected by a sensitive avalanche photodiode module. The digitized fluorescence kinetic transients obtained at 880 nm are processed by computer-assisted analysis, which translates measured signals to programmed physiological parameters, following a charge separation elicited between the RC-BChl special pair and QA. Four phases can be distinguished in the plotted transients: phase I, induction phase (strong pulse of 144 μs duration), eliciting a single-turnover flash (STF), cumulatively saturating the photosystem to permit measurement of fluorescence induction from F0 to FM; phase II, relaxation phase in which indirect modulated
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ACCEPTED MANUSCRIPT light is applied to assess the relaxation kinetics of the fluorescence yield on a 500 ms time scale, reflecting reopening of the RC; phase III, in which a 20 ms flash induces multiple turnovers
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saturating the photosystem and UQ pool (MTF, multiple turnover flash); and phase IV, applying
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indirect modulated light that records the kinetics of UQ pool reoxidation on a 1 s time scale. The transients represent signal-averaged sets of 20 traces per sample, which minimized noise levels
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of individual traces. Note the difference in the rate of fluorescence induction between the 0 and
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48 h samples, reflecting marked differences in the functional absorption cross-section. The registered variable fluorescence signal reflects the RC redox state in which a low fluorescence
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yield indicates an open RC (no charge, RC capable of performing photochemistry), while a high fluorescence yield indicates a closed RC (positively charged RC, transiently non-functional
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photochemistry). Parameters derived from the induction kinetics include: the quantum yield of the primary charge separation estimated from FV/FM where FV, the variable fluorescence, is the
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difference between the minimal fluorescence (F0) and the maximal fluorescence (FM); the functional absorption cross section (), calculated from the slope of the single turnover saturation curve, related to the rate of increase in fluorescence yield. Since the extent to which the relaxation kinetics reflected RC-BChl special pair re-reduction and/or QA re-oxidation was previously unknown, the multiphasic relaxation profile is expressed as the RC electron transfer turnover rate, designated as ET. Analysis of the induction phase (I) gave functional absorption cross-section (450 nm) values of 40.6, 71.5 and 101.5 Å2 in cells sampled at 0, 3 and 48 h, respectively, while the respective values for the RC electron transfer turnover rate (ET) of 3.1, 1.7 and 6.0 ms were obtained from the relaxation phase (II). ET represents the average of the first and second phases of the three phases derived from the exponential decay kinetics.
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ACCEPTED MANUSCRIPT Fig. 8. EM and AFM analyses of chromatophore membrane showing location of cytochrome bc1 complex labeled with gold NTA-Nanogold® [111]. (A) A negatively stained patch consisting of
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a single membrane bilayer with the gold-labeled cytochrome bc1 complexes mainly clustering
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near one edge. (B) The negatively stained features in A. were identified as the LH2 complex (green), RC–LH1 complex (blue/red), and cytochrome bc1 dimers (purple), identified from gold
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labeling. (C) Upper panel: close-up of false color 3-D depiction of AFM topograph obtained
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under liquid, showing intact, uncollapsed chromatophores labeled with nanogold. Image has been filtered with a low pass filter to reduce noise. A cluster of four putative nanogold beads
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indicating possible positions of dimeric bc1 complexes are contained within the red outline. The recessed area extending from the cluster shows a protein-free region. The pairs of colored
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asterisks denote the RC-H subunits of RC–LH1 core complexes; the 9-nm separation determined for the peaks indicated by the paired blue, red or magenta asterisks is consistent with the peak-to-
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peak separation of H-subunits in a RC–LH1 dimer complex. Bottom panel: interpretation of AFM data in which putative nanogold beads are indicated by the orange circles denoting the possible positions of the cytochrome bc1 complexes contained within the red outline in the upper panel. The dimeric RC-LH1 complexes are also shown (blue/red). The edge-to-edge separation of the pairs of gold beads was found to be 2.4 ± 0.5 nm; compatible with the structure of the dimeric Rba. sphaeroides cytochrome bc1 complex [112].
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Figure 2
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Figure 8
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Robert A. Niederman, Development and Dynamics of the Photosynthetic Apparatus in Purple Phototrophic Bacteria
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The author has no conflicts of interest associated with this study.
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