The amazing phycobilisome

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Review

The amazing phycobilisome☆ Noam Adir , Shira Bar-Zvi, Dvir Harris ⁎

Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa 32000, Israel

ARTICLE INFO

ABSTRACT

Keywords: Energy transfer Cyanobacteria Protein structure Tetrapyrrole Spectroscopy Evolution

Cyanobacteria and red-algae share a common light-harvesting complex which is different than all other complexes that serve as photosynthetic antennas – the Phycobilisome (PBS). The PBS is found attached to the stromal side of thylakoid membranes, filling up most of the gap between individual thylakoids. The PBS self assembles from similar homologous protein units that are soluble and contain conserved cysteine residues that covalently bind the light absorbing chromophores, linear tetra-pyrroles. Using similar construction principles, the PBS can be as large as 16.8 MDa (68×45×39nm), as small as 1.2 MDa (24 × 11.5 × 11.5 nm), and in some unique cases smaller still. The PBS can absorb light between 450 nm to 650 nm and in some cases beyond 700 nm, depending on the species, its composition and assembly. In this review, we will present new observations and structures that expand our understanding of the distinctive properties that make the PBS an amazing light harvesting system. At the end we will suggest why the PBS, for all of its excellent properties, was discarded by photosynthetic organisms that arose later in evolution such as green algae and higher plants.

1. Historical background The Phycobilisome (PBS) was first identified by Gantt and Conti in the 1960's [1,2]. Excellent reviews on the early and more recent history of the study of the PBS have been written by Tandeau de Marsac [3], Grossman [4,5], Glazer [6,7], Ikeuchi [8] and others [9,10]. Our group has also contributed reviews that deal with the structure and function of the PBS [9,11–15]. In the 1980's, the first structures of the PBPs were obtained by Huber and co-workers using X-ray crystallography [16–22]. These were the first structures of light-harvesting components to be determined to high resolution. Negatively stained (NS) PBS particles were examined by transmission electron microscopy from the 1970's by many groups [23–26], leading to the classic PBS model of core cylinders surrounded by rods radiating out in orderly fashion. Variations on the classic structure were suggested based on additional experiments (such as crystallography) or due to geometric considerations [7–9,14]. 2. The classic PBS structure In the 1970s and 80s, NS electron microscopy was the only tool that could provide structural insight into the structure of the entire PBS, due to its notably large dimensions, as tools which enable single particle averaging and three-dimensional reconstruction were not available at

that time. Thus, screening of particles was mostly a subjective operation, with particles of apparent symmetry and completeness singled out as representing the proposed true structure of the PBS. It should be noted here that isolation of the PBS from all sources requires the presence of high concentrations of phosphate (or citrate) buffer [25], proposed to mimic in some respect the molecular crowding in-between the thylakoid membranes. Even in the presence of phosphate, some PBS complexes quickly disassemble, and dilution of the sample before application to EM grids has an effect on complex stability. Thus, reported EM studies usually show some aggregates with less than perfect symmetry. In any case, what appear to be the most intact complexes were shown to have a core substructure that contains 2–5 cylinders (each cylinder made up of 4 discs) which was associated with the most redshifted PBP component, allophycocyanin (APC) [27,28]. The core has been envisioned to be positioned and tethered in some fashion above the membrane-bound Photosystem II (PSII) complexes. In a later EM study, Sui and co-workers isolated PBS attached to objects of the correct dimensions of a PSII-dimer, indicating that this interaction indeed occurs [29]. Another substructure of the PBS are the rods, which radiate out from the core. This arrangement has recently been confirmed by cryo-electron tomography of Synechocystis cells [30]. These rods were shown to be composed of higher energy absorbing PBPs: phycocyanin (PC, proximal to the core) and in some cases phycoerythrin (PE) or phycoerythrocyanin (PEC), distal to PC [7]. The rod dimensions are

This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce. Corresponding author. E-mail address: [email protected] (N. Adir).

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https://doi.org/10.1016/j.bbabio.2019.07.002 Received 20 April 2019; Received in revised form 19 June 2019; Accepted 9 July 2019 0005-2728/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Noam Adir, Shira Bar-Zvi and Dvir Harris, BBA - Bioenergetics, https://doi.org/10.1016/j.bbabio.2019.07.002

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similar to those of the core cylinders leading to the early proposal that all PBPs are assembled in a similar fashion. Each PBP assembly initiates with the formation of a stable complex between two homologous subunits termed α and β with molecular weights of 16–18 kDa. All PBP (αβ) heterodimers are referred to in the literature as monomers and are quite stable. Monomers self-assemble into (αβ)3 trimeric rings which then undergo further assembly into [(αβ)3]2 hexamers. The precise structures of many PBPs, assembled into trimers or hexamers have been obtained by X-ray crystallography at different resolutions [9,13,31]. Only recently, the structure of PE from the red-algae Phormidium sp. A09DM was resolved to 1.14 Å, the highest resolution obtained so far for a PBP [32]. 2–4 hexamers then further assemble into the rods and core cylinders. The mode of attachment and arrangement of the rods to core could not be directly identified in the low resolution NS TEM studies, however other studies attempted to identify the interaction interfaces between these sub-structures [33]. A variation of the classical PBS structures was identified in Gleobacter violaceus, where the rods are bundled together and positioned in one site above the core [34]. In the process of isolation of the PBS, additional proteins were identified within the complexes, that were proposed to stabilize the complex and were thus called linker proteins (LPs) [35]. In most cases, the LPs lack bound chromophores and were suggested to reside solely within the rods or core cylinders inner cavities. In addition to stabilization, it was proposed that the LPs serve to modify the excited state properties of nearby chromophores, leading to fine tuning of EET [36]. A single crystal structure of an APC trimer with its associated [37] LP has been determined [38], however as will be described in detail below, cryo-EM has now provided a much clearer picture of the positions of all LPs, as well as their composition. Coupled with biochemical isolation, the advent of comprehensive sequencing methods, allowed the association of different LPs into families. LR, stabilize and terminate rods, LRC connect rods to cores, LC stabilize and terminate core cylinders and LCM assist in attaching the cores to the thylakoid membrane. In many studies, researchers have used other terminologies, including gene product names (such as ApcE for LCM), with additional numbers for cases of multiple members of a family, or added the molecular weight as a superscript (such as LR9 for the rod capping linker) [35]. In order to obtain the G. violaceus PBS in its unique assembly (mentioned above), novel linkers are required [39]. The self-assembled protein complex described above is of course the scaffold that holds the phycobilin (PB) chromophores in their proper configuration, orientation and position, both absolute (within each subunit) and relative (with respect to PBs attached to adjacent subunits/monomers/hexamers, etc.). All of the PBs are linear tetrapyrroles that are obtained by modifying hemes by the activities of heme oxidase, ferredoxin-dependent bilin reductases and finally subunit/site specific lyases [8,37,40–42]. The lyases catalyze that covalent attachment of the specific PBs to conserved cysteine residues. The efficiency, rate and direction of EET between PBs is a result of the distance and relative orientations of the PB dipoles, all determined by their geometries, as directed and imparted by the protein scaffold, with the relative positions imparted by assembly. Each of the (αβ) monomers of APC, PC, PEC or PE contain 1 + 1, 1 + 2, 1 + 2, or 2 + 3 PBs (per subunit), respectively. APC and PC contain only the lowest energy absorbing phycocyanobilin (PCB) while PEC and PE contain more blue absorbing PBs: phycoerythrobilin (PEC), phycourobilin (PUC) or phycoviolobilin (PVB). The precise absorption/emission property of each PB is then further tuned by the phycobiliprotein (PBP) environment, presence of LP and level of assembly. Experiments performed in the presence of intact PBS and the orange carotenoid protein (OCP) have shown that energy efficiently flows throughout the complex to the terminal emitters within the core (see more details below). As the classic PBS have been described in many previous reviews, we will concentrate here on newer PBS types, that collectively show how the PBS can deal with different environmental challenges, in order to provide the optimal flux of energy to operate the needed photochemistry.

3. Recently reported structures and assemblies of the PBS – nonclassical PBS structures a. The PBS from the red algae Griffithsia pacifica. As described above, visualization of the entire PBS structure was obtained at low resolution by NS-TEM, while high resolution structures of more smaller components, trimers, hexamer and rods [36] were obtained by X-ray crystallography. An improvement in the clarity of the structures was a direct result of the improvements in cryo-EM techniques. Higher resolution EM structures were obtained by Sui and coworkers from the cyanobacterium Nostoc flagelliforme at 2.8 Å [43] followed by the PBS from the cyanobacterium Anabaena sp. PCC 7120 at 2.1 Å [29]. The later study showed a much more complex arrangement of the rods surrounding a tri-cylindrical core. It also provided strong evidence for the positions of LPs, which till then had only been visualized by X-ray crystallography in a single trimeric structure of APC [38], or as isolated partial protein [44]. Other fragments of the linker proteins were determined by NMR and deposited in the Protein Data Bank (https://www.rcsb.org/), however these structures have not yet been fully described in the literature. The group led by Sui and Zhang made a tremendous breakthrough in the understanding of the PBS by their successful determination of the entire complex from the red-algae G. pacifica by cryo-EM [45]. In this study, the revolution of modern cryo-EM methods [46] enabled the determination of the entire structure to 3.5 Å. The structure represents the largest PBS complex described to date, with a molecular weight of over 16.8 MDa (Fig. 1A). The structure contains 862 unique protein chains and over 2000 chromophore molecules. 72 LPs were modeled within the core cylinders and rods. The G. pacifica PBS structure (GpPBS) provides answers to many of the open questions about the assembly of the PBS. In most cases, the GpPBS structure confirms, or at least strongly corroborates structural elements that were suggested by other structural methods. However, the GpPBS is also a unique structure for a number of reasons. It is the first reported PBS structure that contains core cylinders with < 4 trimers. The two basal cylinders have only three trimers, while the single top cylinder has only two trimers. One could envision a number of scenarios that might result in such a unprecedented core: i) The GpPBS could represent a new form of core, not previously identified; ii) The core could have lost four trimers (one on each basal cylinder and two on the top cylinder) during isolation; iii) In organisms that contain fourtrimer cylinders, the tight packing of the PBS complexes along the cylinder axis [30] leads to direct interactions between the ends of the cylinders, and during separation, some complexes gain additional trimers from adjacent PBS complexes (but actually have fewer trimers, as in GpPBS). One aspect of the arrangement of the trimers in the GpPBS core is the lack of the ApcC linker protein on one side of the basal cylinders, and total lack of ApcC from the top cylinder. However, the carboxytermini of the two ApcE linker protein thread into the trimeric aperture, perhaps serving in the role of capping or terminating linkers, in a fashion similar to CpcD in rods [47]. Comparative studies with other red-algal PBS complexes will be required to identify the reasons for the unique assembly of this core. The core is surrounded by 14 rods, many more than typically associated with a tricylindrical core type PBS [29]. In fact, there has been no previous description of so many rods in any form of PBS, including pentacylindrical PBS types [26,48]. Another anomaly of the GpPBS is the presence of additional individual subunits and individual hexamers attached to the periphery of the complex. What could be the role of these components, not integrated into rods, is certainly an exciting new question. A more complete picture of the PBS packing in this organism may reveal potential inter-complex functional or structural links that may require these additional components. Cryo-tomography has recently revealed apparent links between adjacent PBS complexes [30]. 2

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Fig. 1. New PBS structures. A. The structure of entire 16.8 MDa PBS complex from G. pacifica (PDB code 5Y6P, [45]) determined by cryo-EM. Each color represents a different subunit (not all subunits are visible). The membrane bound reaction centers would be found directly below the PBS. The third dimension of the PBS (into the plane of the paper) is 45 nm. B. Cartoon representation of the structure of the ApcE (LCM) linker protein in the G. pacifica PBS core. The APC proteins (in transparent cartoon representation) form three cylinders, two on the bottom (each containing three trimers) and one on the top (with two APC trimers). The two copies of ApcE (in purple and brown) contain a ApcA-like phycobiliprotein domain (black ovals), followed by three linking domains that wind through the bottom cylinders up to the top cylinder. The panel on the right is rotated counterclockwise by 90°, showing how the ApcE juts out of the top cylinder. C. Model of aligned A. marina PBS. The AmPBS is composed of four hexamers containing PC, in a single rod (1.2 MDa), with multiple rods aligned in parallel fashion between thylakoid membranes [61]. The structure of A. marina PC was determined by X-ray crystallography (PDB code 5OOK, [64]) showing that the PC subunits in the AmPBS are from two isomers of both α and β subunits. Here we schematically show the minor α and β subunits in green and red, respectively. The major subunits are in purple. The actual positions of the minor subunits are not yet known and are shown here for illustration purposed only. Molecular graphics were created using Chimera X (http://www.rbvi.ucsf.edu/ chimerax).

The GpPBS structure also provides a roadmap for elucidating energy transfer through multitudes of pathways, leading from PE or PC to the core APC, and within the core to the terminal emitters, ApcD and ApcE. As has been suggested from crystal structures of smaller assemblies [36,49] and coupled cross-linking/mass spectrometry [33], the closest center to center distance between PB chromophores within the rod PC or cylinder APC substructures is ~20 Å (between adjacent monomers within the same trimer). The PE components are the most PB rich of all of the PBPs, with five chromophores per monomer. The α subunits of PE have an additional chromophore (PEB) at position 139 that is highly solvent exposed. PE crystal structures [32] reveal the potential to make closer inter-PB distances, predicted to be between 10.5 and 12.5 Å, if the rods bearing this chromophore were assembled in orientation bearing similarities to the crystal packing. However, until recently this possibility was only based on the crystal packing of structures obtained by crystallization of isolated trimers or hexamers. With the complete GpPBS structure, some (but not all) of these close inter-PB distances have been confirmed. At these short distances, the potential for stronger excitonic coupling (via the description given by the modified Redfield theory, [50]) increases in the most solvent-exposed, (possibly-dynamic) PEB binding regions. This faster dynamics could assist in the efficient transfer of EET from distal parts of the PBS rods to the core. The energy transfer distances between all other components (hexamer-hexamer, rod-core, core-RC) are significantly longer (32–40 Å), which one would expect result in reduced efficiency of EET. However, at normal growth illumination intensities, the quantum efficiency of EET is nearly unity. Amongst all of the important details revealed in the GpPBS structure is the almost complete description of the LPs. Within the electron density the authors were able to place all four core linkers, 16 rod-core linkers and 52 rod linkers. Analysis of the contacts between LPs shows a wealth of variation. Some LPs interact directly with adjacent LPs within the rods structures, in a fashion reminiscent of a structural backbone. Other LPs appear to branch out of the rod to make contact with other

rods, in a fashion that might stabilize the PBS. Attachment of rods to the core are facilitated by the RC type LPs, of which there are three types, one that links PC containing rods, and two others that link rods that contain only PE. The RC LPs use a similar mechanism for association to core cylinders, but at different positions, and with differences in the precise interactions. The RC LPs all have C-terminal helices that associate with the α subunits of core trimers. The structure revealed a new class of previously unknown LPs (LRC4–6) whose role is attachment of the unique subunits mentioned above to the core. These LPs have significantly different structures and they interact with the core with long extended helices and unstructured elements. The most important of all of the LPs is also the longest, referred to in the literature as either LCM (core-membrane) or ApcE. This LP has a PBP domain and is thus integrated into a core trimer, replacing an α subunit (αLCM). The bound PCB chromophore has the most red-shifted emission and is thus considered the terminal emitter that leads to EET to the RC. The total length of LCM is dependent on the number of core cylinders. In GpPBS, following the α LCM domain, three sequential regions link together the three core cylinders. Two-fold symmetry in the core leads to two such LCM stabilizing interactions on the three cylinders. The LCM/ core interactions are shown in Fig. 1b. Prediction of the actual EET pathways is typically based on recognizing each chromophore as a separate absorbing/emitting entity (as predicted by the Förster resonance energy transfer (FRET) mechanism [51,52]). This however may not be entirely correct, as there have been suggestions that excitonic coupling of some form may occur, even at 20–30 Å distances, enabling the PBS to perform EET at high rates/efficiencies [12,28,53–56]. In addition to the roles of the LPs in modifying the structure of the PBP units [38], linking the PBP units [35] and changing the absorption/emission properties of selected chromophores [36,54], an additional role may be in creating the environment necessary for stronger coupling. This hypothesis is further corroborated as recent findings have demonstrated that γ33, PE3

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associated linker protein chromophores (PUB and PEB) from another red algae, Gracilaria chilensis, do not demonstrate energy transfer between them, inferring that they might facilitate energy transfer between units of the PBP [57].

described in detail above, it is certainly possible that PC612 may be found at alternative positions surrounding the core. Additional details on the AmPBS, with respect to bathochromic shifting to match the absorbance of Chl d, will be detailed below.

b. Acaryochloris marina. Until recently, all of the cyanobacterial species studied contained only Chl a as the main chromophore of both PSII and PSI. These species also had PBS that conformed to one of the types of canonical forms: hemidiscoidal arrangement of 6–8 rods surrounding a core of 2–5 cylinders. Discovery of new cyanobacterial species, isolated from a variety of environments, have led to the discovery species that contain Chl's with absorption in the near-IR region, Chl d or Chl f, with novel PBS types. One of the first of these non-canonical PBSs was that of Acaryochloris marina. This cyanobacterium was first isolated in the 1990's [58] and was shown to grow in association with didemnid ascidians exposed to only weak white light. It was then shown that the major pigment is Chl d, which has a red-shifted absorption peak when compared to the typical Chl a pigments. Indeed, cultures grown in labs showed slow growth in red-light. The presence of PBS like antenna's in A. marina was explored, and when identified and isolated, they were shown to be composed of PC and a small amount of APC, forming single 4hexamer rods (Fig. 1C). Surprisingly, in the original measurements of isolated A. marina PBS (AmPBS), absorption and emission characteristics were quite similar to “typical” PBS. This indicated that instead of possessing the strong overlap between PBS emission and reaction center Chl absorption found in typical PBS, here the overlap was very small. Growth in low white light dramatically increases the amount of PBS, suggesting that the cyanobacteria do utilize the PBS for LH [59,60]. The AmPBS was located within the cells as patches of quasi-aligned rods, not equivalently distributed throughout the cell [61]. Higher resolution NS micrographs confirmed the packed rod structure and indicated that each rod contains four hexamers [62]. Previous studies had already suggested that the AmPBS rods contain 3 PC hexamers and a single heterohexamer consisting of a PC trimer and an APC trimer [63]. It was thus proposed that the APC trimer is closest to the PSII within the membrane.

c. Organisms with Chl f. The two examples above have presented a wealth of organizational changes that can occur within the PBS that can apparently lead to optimal light harvesting for the parent organism, under the changing environmental conditions they experience. During the past decade, the limits of oxygenic photosynthesis have been exceeded with the discovery of Chl f containing organisms [68–70]: Leptolyngbya sp. JSC-1 [71] (LepJ), Synechococcus sp. PCC 7335 [72,73] (Syn 7335), Aphanocapsa sp. KC1 [74,75] and Halomicronema hongdechloris [76,77] (HalH). Exchange of Chl a with Chl f requires significant remodeling of the photosynthetic apparatus, as evidenced from changes in gene expression, which enables the replacement of protein isoforms whose sequences enable correct assembly and function under these new conditions [73]. In LepJ, transfer to far-red light growth illumination results in the replacement of the typical hemidiscoidal PBS (with a pentacylindric core) with to a PBS containing a bicylindric core containing red-shifted APC and rods with both PC and PE. A similar type of complete remodeling of the PBS also occurs in HalH upon transfer of the organism to light beyond 700 nm [76]. In this species, the PBS is reduced to a simple double trimer cylinder, containing APC whose absorption and emission maxima are significantly red-shifted, to 712 nm and 728 nm, respectively [76], without rods. In the case of Syn7335PBS, novel bicylindrical cores containing red-shifted APC isoforms with absorption and emission maxima at 711 nm and 730 nm, respectively are expressed. However, unlike both LepJ and HalH, exposure to far-red light does not completely eliminate the previously formed tricylindrical PBS that contain PE, PC and APC. The source of red-shifted emission has been associated in these unique PBS forms with the expression of special isoforms of the terminal emitter ApcE. In the case of Syn7335, the novel PBS also contains other unique APC isoforms that are not found within the typical PBS – as it was shown that these APCs do not covalently bind the phycobilin chromophore, thus extending the conjugation by having one additional double bond, red-shifting this system [78]. In addition to this major change in the conjugation, red-shifted emissions are also due to changes in the PCB environment, imparted by the residues lining and surrounding the chromophore binding pocket – and not due to changes in the PB itself, as described above for the A. marina PC.

In order to better understand the AmPBS, we aimed at obtaining high resolution crystal structures of isolated components as well as the entire tetra-hexameric PBS. In the process of complex and component isolation, we utilized mass spectrometry-based proteomics (LC MS/MS) [49,64]. Two observations were readily obtained by these measurements. The first is that the PC component of the AmPBS is heterogeneous in composition, comprised of different amounts of two isoforms of both the α and β subunits. The second observations is that the amount of APC expressed by the cells under the laboratory conditions that they were grown (low white light) was negligible to a degree that in most measurements it was absent. This observation was puzzling, as it meant that most (if not all) of the PBS was composed of the higher energy PC component, leading to further diminished overlap with the reaction centers. The crystal structure of trimeric A. marina PC was determined to 2.1 Å resolution (PDB code 5OOK), and confirmed the presence of both subunit isoforms in the trimers, superimposed within the asymmetric unit (which is a single (αβ) monomer) [64]. It also revealed that asparagine 72 of the β subunit is not methylated, a conserved post-translational modification found in practically all other PBPs. It has been suggested that the presence of γ-N-methyl-βAsn72, adjacent to the β84 PCB chromophore, is required to facilitate directed energy transfer throughout the PBS and also may have a protective role [65,66]. Only a single instance of lack of methylation has been reported previously, in a sub-fraction of PC from T. vulcanus [67]. This subfraction (termed PC612 for its blue shifted absorption), was co-isolated with the PBS core APC fraction and was suggested to serve as an intermediate fraction between the rods and core. With the recent determination of the extremely complex PBS from G. pacifica [45]

4. What makes a perfect LHC? Photosynthesis links the energy of the sun to high-energy storable chemical compounds that run the processes of life. The natural flux of sunlight changes over the course of the day, the season, the geographical location and the amount of coverage (by water or other organisms). Thus, a successful photosynthetic system will have to increase the probability of providing a steady flow of excitation energy to drive a constant flow of electrons through the reaction centers, under any condition the organism experiences. If absorption and excitation energy transfer (EET) become limiting or in large excess, back reactions or damaging chemical intermediates may harm the apparatus. It has been estimated that a flow of electrons that can drive all of the enzymatic processes of product synthesis at the observed rate of 102–104 s−1 through a single photosynthetic unit (antennae + reaction centers) is required by most photosynthetic organisms [79]. Calculation of the absorption cross section and energy transfer rates leads to an estimated maximum of about 4 photons s−1 needed to be absorbed by each antenna pigment containing several dozen pigments per photosynthetic unit [80]. Since the occurrence of massively increased light flux due to environmental changes (wind, clouds or background shading on leaves/ 4

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cells may make them experience 100-fold light fluctuation in a matter of seconds) is a real possibility [81,82]. EET may exceed the rate of chemistry, harming the integrity of the system. The problem of massively reduced light flux may be an even greater problem (due to time of day, clouds, physical coverage, depth of water column, etc.), so the active pigment bed must actually be large enough to successfully drive photosynthesis. In order to protect the system, dynamic processes must be carefully linked to these large pigment beds. What then are the parameters that must be optimized to obtain a “perfect” light harvesting complex (LHC)? One might assume that such a perfect LHC would have simultaneously optimized both the physical and the biochemical characteristics. On the physical side, the LHC should have maximal spectral coverage obtained by a sufficient number of pigments (equivalent or different) that are efficiently electronically coupled. On the biochemical side, the LHC requires a specific protein scaffold that provides the correct environment for the pigments, directs EET in the correct direction, enables the assembly and disassembly of the system, depending on the immediate needs or conditions and provides the intrinsic chemistries that will enable fast and slow dynamic responses to changes in its substrate – light. As described in this volume, photosynthetic organisms have evolved to thrive in almost every terrestrial and aquatic environment that is exposed to sunlight. The types of LHCs that drive the different types of photosynthesis can be roughly divided into four types [50,83,84]: 1) membrane bound bacteriochlorophyll (bChl) or chlorophyll (Chl)-based (eukaryotic plants and algae, some prokaryotic oxygenic species and purple bacterial LHCs); membrane external bChl-aggregates (chlorosomes in green sulfur or green filamentous bacteria); membrane external protein bound tetrapyrrole-based phycobilisomes (PBS, cyanobacteria and red-algae); and 4) minor LHC variants, such as the antennas of glaucophytes. We can assume that each LHC type represents an optimized system for the general environment that the species usually occupies. However since light intensity and quality is rarely constant (except for in the lab), no static LHC can be optimal at all times. Thus, a second layer of functionalities must exist that modifies, attenuates, enhances or quenches the light harvesting and energy transfer process. Each LHC must thus provide structural elements that enable the dynamic activation of protection mechanisms. Cyanobacteria and red-algae are the most wide-spread of all photosynthetic organisms, so we might conclude that their major LHC, the PBS is the most generally optimal LHC system. And yet, prochlorophytes, green algae and plants, species that evolved from cyanobacteria or red-algae, lost the capability to express functional phycobiliproteins (PBP). The general characteristics of the PBS that make it so successful include: i) self-assembly to very large dimensions without losing the ability to perform EET to single transfer points to the reaction centers (RCs); ii) attachment to the outside of the thylakoid membrane allowing for packing of large amounts of PBS in-between thylakoid membranes without losing high efficiency EET to RCs. Due to the potential for lateral EET, the increased antenna bed enhances the potential photon capturing ability per RC; iii) water-soluble proteins with covalently linked chromophores, imparting high stability, mobility and functional flexibility (multiple EET pathways) as it is more accessible for other water-soluble regulators [85] and can be more easily disassembled in certain states of stress [86]. In this review, we will describe the details of these characteristics especially in light of new observations and structures, illuminating the functionalities of the PBS. In the end, we will also try to suggest reasons for its evolutionary demise.

towards the photochemical reaction centers; and 3) control of all processes to maximize both photochemistry and system protection. 1) As already indicated above, different PBS forms have different absorption cross sections. The maximal range covered by the PBS occurs in PBS that contain PE, PC and APC, resulting in an absorption range of > 250 nm (450–700 nm) [83] that can be extended even further into the near IR spectral region, as described above. Thus the PBS has the potential to match the total width of absorption (considering the far-red APC variants) provided by Chl a + b in the combined Soret and Qy bands. The molar extinction coefficient of the bound PB chromophores also compare nicely with those of Chl's: Chl a and b have extinction coefficients of 1.1 and 1.6 × 105 in their Soret bands (respectively, in diethyl ether) [87], while the PBs have coefficients that are similar or greater (on a single chromophore basis) than those of the Chl's. For instance, an APC trimer (with six PCB chromophores) has a molar extinction coefficient of 7.3 × 105 (~1.2 × 105/PCB). As seen in the GpPBS, an intact complex can contain > 2000 PBs, all funneling absorbed solar energy to the dimeric PSII or trimeric PSI. Even more typical (and significantly smaller) PBS complexes, such as those found in Synechocystis or T. vulcanus, will contain almost 400 PBs. A typical amount of Chl molecules attached to LHCs in green algae or plant PSII is on the order of 200–250 Chl's [88,89]. A similar number of LHC Chl molecules are associated with PSI [90]. From these considerations, in the smallest PBS complexes, such as those in A. marina (1.2 MDa), or the Chl f containing organisms described above, the pigment bed will be significantly smaller. However, the packing of adjacent complexes may provide lateral energy transfer, enabling the actual number of pigments that can funnel energy to the RCs to be greatly increased [14,30]. From all of these considerations, it can be suggested that PBS containing organisms will have an excellent opportunity to harvest light throughout the visible spectrum, and will be highly successful at greatly reduced light intensities. This is further evidenced by a significant increase in the amount of PBS in organisms grown in low light [60].

5. The PBS as an excellent LHC

2) The efficiency of EET within different LHCs has been measured for different organisms, at different levels of isolation/assembly and under different conditions of illumination [83,91]. This is of course also true for the PBS [7,92,93]. The rates of EET between units are orders of magnitude faster than the relaxation times, as obtained by time-resolved fluorescence measurements. Recent high quality spectroscopic measurements have identified the existence of multiple functional domains or compartments in Synechocystis, Synechococcus WH 7803 and A. marina [94–96]. Decomposition of the entire pathways into compartments facilitates characterization of the various options that the PBS affords to EET to prevent energy loss. The existence of back transfer of energy from lower to higher energy compartments, providing additional chances for energy to reach the correct final destination. As already mentioned above, time-resolved absorption measurements have revealed sub-ps kinetics of EET between different components (compartments), indicating the existence of excitonic coupling that would facilitate the overall efficiency, outcompeting the non-desirable relaxation pathways [54,96]. At typical growth light fluencies, the overall efficiency has been estimated to be close to 100%. This is even more impressive considering the close packing of PBSs along the thylakoid membrane, which allows lateral energy migration over long distances to an open RC. [30,62].

As described above, the three major requirements of an antenna complex are: 1) an absorption cross section that enables a high rate of photon capture over a wide spectral coverage; 2) efficient EET at rates that are orders of magnitude greater than competing processes that are photochemically unproductive (heat or fluorescence) and directed

3) The third functionality that we associate with LHCs are their response to changes in the incoming light energies by wavelength (spectra) and intensity (flux). A superior LHC will provide the needed driving force for the RCs at a maximum range of the solar spectrum. As already described above, the PBS, compensates nicely 5

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for the diminished absorption by Chl's (of all types) in the range of 500–600 nm. Chromatic acclimation is a predominant mechanism present in some cyanobacterial species, enabling spectral fine-tuning of PBS to cope with changes in radiation wavelength. It was shown that rod PBP (PC and PE) expression, can be altered upon different light conditions (green/red) [97,98] This response is dependent on photoreceptors that are activated by the absorption of specific wavelengths and initiate metabolic cascades that result in the correct PBP expression [99,100]. The high efficiency of the PBS prevents loss of absorbed energy at low light intensities, allowing the species containing PBS antennas to survive at extremely low light. On the other hand, the large cross-section for absorption of the PBS and its propensity to exquisitely transfer its absorbed energy towards PSII may become a two-edged sword. As high light irradiation yields over-excitation of P680, which exposes the photosynthetic organism to the formation of reactive oxygen species (ROS), which induces cell stress that can lead to cell death [85,101]. To prevent such an adverse scenario, cyanobacteria have a number of potential pathways [14]. One important mechanism is to utilize non-photochemical quenching (NPQ), deflecting the excited electron relaxation pathway towards heat dissipation [102], which will be described in depth below.

stabilizing hydrogen bonding between the β1-ring' carbonyl of the carotenoid and W288 and Y201 at the CTD as a result of possibly entering the S* excited state due to photo-excitation on a pico-second timescale, as was recently suggested by Bandara et al. [111]. Then, within 50 ns and while retaining a C6-C7 trans configuration, the carotenoid adopts a meta-stable state, burrowing further into the NTD. Later, within ~1 μs, the NTD undergoes slight structural change in some helical elements. This change weakens the meta-stable state, resulting in a further slide of the carotenoid into the NTD, finalized after 10 μs. It is only then, on a timescale exceeding 0.5 ms, that domain separation unfolds via destabilization of the NTE from the CTD, finally yielding the OCPR state. As for PBS-OCP interaction, recent work from the group of Moerner has demonstrated, using single molecule spectroscopy, that the PBS quenching is characterized not by a single quenching mode, but rather two quenching modes (denoted Q1 and Q2), showing that the fluorescence is initially reduced to 11% of initial brightness, then to 6% of initial brightness [112]. It was speculated, that these states are a result of two OCPs binding events and not a single OCP that shifts between two binding modes. Upon relaxation of light conditions, cyanobacteria need to restore standard energy transfer scheme from the PBS to PSII and it does so by exploiting a third component, called the fluorescence recovery protein (FRP), that facilitates detachment of OCP from the PBS and expedites the back conversion from OCPR state to OCPO state [85,113,114]. This process was recently analyzed using biochemical, structural and spectroscopic means by the groups of Sluchanko, Friedrich and Maksimov [115]. It was shown that a dimeric FRP binds two OCP molecules in consecutive binding events, yielding an OCP-FRP-FRP-OCP meta-stable complex, which quickly results in two OCP-FRP sub-fractions. Structurally, this process is generated by first compacting the OCP molecules to mimic the OCPO state. The second compacting event (OCP-FRP-FRP binds the second OCP) induces steric clashes between the NTDs of the two OCP molecules, forcing the meta-stable complex to collapse into two FRP-OCP moieties. It is only then the carotenoid slides back into the CTD, forming hydrogen bonds with W288 and Y201, resulting in the OCPO state. Very recent natural in-situ architecture of the canonical Synechocystis PBSs was determined using cryo electron tomography. This has revealed that the PBSs are aligned in a linear array, even forming contacts (as indicated by the presence of electron density) with one another at the core-core and at the rod-rod levels [30]. Putting together this array of PBSs, the two OCPs per one PBS and the OCP-FRPFRP-OCP complex, we hypothesize that an essentially polymeric complex may enable pseudo-concerted photo-recovery of at least two PBSs, possibly more (Fig. 3).With the suggestion that there is significant lateral energy transfer between neighboring PBSs, this multi photo-recovery concept further strengthens the idea that addressing the PBS as a single complex might not accurately describe the nature of cyanobacterial antenna system. We further strengthening the claim that a higher assembly level of PBSs should be considered, generating the “real”, in-vivo, multi-PBSs complex. Lastly, the modularity of OCP' two main domains, the CTD and the NTD, has been recently probed via their newly-found homologs, which

6. Non photochemical quenching of the PBS The orange carotenoid protein (OCP) is the key player enabling NPQ in cyanobacteria. The OCP is a water-soluble, 35 kDa protein that encapsulate a carotenoid pigment. OCP can be roughly divided into five main domains (from N-terminus to C-terminus): i. N-terminal extension (NTE); ii. N-terminal domain (NTD); iii. Flexible loop; iv. C-terminal domain (CTD) and v. C-terminal tail (CTT) [85]. The OCP has two states – the inactive OCPO and the photoactivated OCPR. In-vitro, it was shown that both strong white or blue light can trigger the conversion from the inactive to the active state. Only upon adopting the OCPR state, can the OCP interact with the PBS core domain to induce a highly effective (~90%) quenching efficiency [103–107]. On the protein dynamics level, it has been shown that upon photoactivation, the NTE (which flanks and interacts with the CTD in the inactive form) detaches from the CTD, enabling the CTD and NTD to separate completely, while remaining connected via the flexible loop. While the three-dimensional structure of the OCPO was solved using X-ray crystallography (Fig. 2A) [104,108], the full OCPR remains elusive to date. However, the X-ray structure of the carotenoid-binding NTD segment was solved and termed RCP (Red carotenoid protein; Fig. 2B). The RCP structure demonstrated that when the CTD is absent, the carotenoid translocates by 12 Å, deep into a designated cavity in the NTD (as opposed to its original position, being equally shared by the NTD and CTD in the OCPO state). The carotenoid is thereby completely surrounded by the RCP amino-acid scaffold [109]. Recent work from the groups of Kirilovsky and Kennis has provided significant insight into the photoactivation dynamic sequence on a wide time-scale (from picoseconds to milliseconds) [110]. It was found that the OCPO-to-OCPR transition is initiated via the breakage of the

Fig. 2. X-ray structures of OCP states. (A) OCPO structure (PDB code 3MG1) – NTE in blue, NTD in red, flexible loop in BLACK, CTD in orange, CTT in wheat, carotenoid in cyan. (B) RCP structure (PDB code 4XB4) – NTD in red, carotenoid in cyan. Molecular graphics in Figs. 2 and 3 created by Pymol (the Pymol Molecular Graphics System, version 2.0, Schrödinger, LLC).

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into ~5 Å, possibly forming a J-type aggregate that has new spectroscopic features (resulting in the 680 nm shoulder). CTDH was shown to have a key role in carotenoid uptake (from membranes) and delivery (to HCPs) [118,122]. The three-dimensional structure of Anabaena apoCTDH (without bound carotenoid) revealed that there are two types of dimeric interactions for CTDH – back-to-back (as seen in the asymmetric unit of PDB code 6FEJ) and head-to-head (as seen between unit cells of PDB code 6FEJ). While the latter was expected, as it shares the same preface as in OCP, the former was somewhat surprising and emphasized, together with complementary techniques, that the equilibrium between the states is crucial for the capability of carotenoid mobility. Moreover, when comparing the apoCTDH structure to the only other CTD(/H) structure, the CTD in the OCPO state (with bound carotenoid), a major structural change was observed in the CTT (Fig. 4C), which was later confirmed to be a facilitator of carotenoid uptake and delivery through mutagenesis. The cardinal debate around the quenching mechanism of the PBS, remains. Various explanations for the apparent quenching were suggested, all agree that the quenching site is at the PBS core [103,114,123,124]. Some have argued that the bulk APC (APC660) is the site of quenching [124,125], while others have claimed that it is the terminal emitters (APC680) which act as the last stop of energy transfer, prior to dissipation [126,127]. In addition to this disagreement, the actual photo-physical relaxation pathway is also unclear, as it was proposed that it could be either due to charge transfer, energy transfer or even excitonic interactions between the affected PB and the nearby carotenoid (shelled by the NTD scaffold) [124,128,129]. Alternatively, the possibility of OCP intrinsically affecting the nearby PB and thus alter its relaxation pathways, driving it towards a dark, quenched mode was also raised [106,112]. This thought could be perhaps further corroborated by the apparent dark-mode (coined “blinking”) the PBS demonstrates as a function of light intensity [130,131]. We speculate that a combination of all of these mechanisms might yield the highly efficient quenching the OCP provides.

Fig. 3. Near crystalline packing of PBSs on thylakoid membrane allows simultaneous, multi-PBS photo-recovery. PBSs (blue-to-pale-green) are stacked together on the cytosolic side of the thylakoid membrane (transparent dark green background). As one PBS possibly bind two OCP molecules via their NTDs (red, see right-most, transparent PBS) at two basal core cylinders (pale green) in a symmetric fashion, one FRP dimer can bind two closely-situated CTDs (orange) of two OCPs of bound to neighboring PBSs (two right-most PBSs). The PBS complexes have been separated for clarity but actually in close packed contact as based on cryo-tomography [30].

are also exist in the cyanobacterial genome, encoding to the individual domains. NTD homologs were denoted as HCP (helical carotenoid protein, as the NTD only contains α-helices) and CTD homologs were simply denoted as CTDH [116,117]. As it was revealed that there are at least nine different HCP clades (HCP1-9) and two CTDH clades (CTDH clade 1/2). Amongst all cyanobacterial organisms, major focus was located around the specific homologs found in Anabaena PCC7120 (Nostoc PCC7120, hereafter called Anabaena). Anabaena has four HCP homologs (HCP1-4) and only one CTDH clade (type 2, which differ from type 1 with the existence of a cysteine residue forming a disulfide bond between two CTDH monomers) [118,119]. HCP4 was found to be the most conserved homolog, in terms of primary-structure, to NTD of OCP and the only homolog (in Anabaena) capable of inducing PBS quenching. However, the HCP4 lacks the CTD regulatory domain of OCP that enables the reactivation of PBS energy transfer via OCP detachment upon relaxation of high light intensities. Once bound to the PBS, HCP4 cannot be detached, creating a terminally quenched PBS. This observation, and the fact that HCP4 is found in the same reading frame as CTDH, led to the hypothesis that a fusion event between HCP4 (NTD-like) and CTDH (CTD-like) resulted in the formation of the adaptive OCP [117,119,120]. HCP2 and HCP3 were found to be singlet oxygen quenchers (similarly to the NTD of OCP), while HCP1 was found to be a very capable carotenoid carrier, but without any apparent quenching function. Recent independent results have shed light on the three-dimensional structures of HCP1 [117] (from Anabaena) and HCP2 [121] (from Tolypothrix sp. PCC 7601) proteins (by Kerfeld and coworkers, Fig. 4A) and CTDH clade 2 protein (by our group, Fig. 4B) [122]. Structural comparison between HCP1 and HCP2 (Fig. 3A) revealed that although being highly similar (Cα RMSD = 0.88 Å), they still differ in both their dimeric interface (as in both cases, the asymmetric unit contains an HCP dimer) and in their different rotation of the terminal (β1) ring in s-trans configuration, that might contribute to their slight difference in absorption spectrum in solution. Moreover, it was shown that in the case of HCP2, the major absorption peak of the protein crystal is strongly red-shifted compared to solution (Δλ = 18 nm) as well as the appearance of a new 680 nm absorption shoulder. These phenomena were attributed to the unique crystal packing of the HCP2 dimer that both impose constraints on the terminal rings (causing the Δλ = 18 nm) and bringing the two canthaxanthin (CAN) carotenoids

7. Mechanism of EET in the PBS: does the PBS exhibit coherent effects? The arrangement of the PBs in the intact PBS (of any of the forms described above), would strongly support EET that can be described by the Förster resonance energy transfer (FRET) [51,52,92]. This is as opposed to the situation of other LHCs that have more tightly packed chromophores, as described in other chapters in this edition. Tight packing (> 10 Å) leads to strong excited state mixing [132] which is better described by Redfield (or modified Redfield) theory [132,133]. In the FRET mechanism, chromophores are weakly coupled by Coulombic interactions. A more detailed description of the EET mechanisms is beyond the scope of this review, however excellent reviews on the subject have been recently published [50,132,134]. One expects EET to proceed from higher to lower energies at rates that are inversely proportional (to 106) to the inter-chromophore distance. The situation in an intact PBS, would be however far more complex, as the number of energetically equivalent chromophores is very large. The asymmetric presence of the LPs could certainly assist in funneling the energy from rods to the core, however inter-rod EET is also highly likely, as indicated from the structures of the GpPBS and AmPBS. However, there have been experimental measurements that would appear to indicate that it is difficult to explain EET within the PBS solely by FRET. This includes strong bathochromic absorptions shifts that have been suggested to be the result of strong coupling. One example of such coupling is found in the standard (αβ)3 trimeric assembly of APC. While containing the same PCB chromophores at cysteine 84 on both subunits as found in the PC component, the absorption maximum is shifted from 620 nm (PC) to 652 nm (APC). In both PC and APC, trimerization brings the α84 from one monomer and the β84 of the adjacent monomer to within 2 nm. With multiple high-resolution crystal structures of both PC 7

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Fig. 4. X-ray structures of OCP homologs. (A) HCP1 (pink, PDB code 5FCX) and HCP2 (yellow, PDB code 6MCJ) superimposed. Cartoon is transparent to emphasize carotenoids depicted as sticks. (B) ApoCTDH structure. Back-to-back (asymmetric unit) dimer is enclosed in the black circle, head-tohead (in different asymmetric units) dimer is enclosed in the black rectangle (PDB code 6FEJ). (C) CTD of OCPO (orange, PDB code 5UI2) and ApoCTDH (PDB code 6FEJ) superimposed. CTTs are depicted as tubes to emphasize changed position as a function of carotenoid presence.

and bulk APC660 (APC trimers containing only ApcA and ApcB subunits), one can safely say that within experimental error, the configurations of the PCBs are essentially the same. The major difference between these PCBs is the identity of the amino acid residues in the first and second shells surrounding the binding pocket. Analysis of these residues showed that the APC pocket holds the two PCBs in a more constricted and hydrophobic environment, with a strongly polar (potentially charged) second shell, with in PC, both shells are polar and the pocket void contains more solvent. It was thus suggested that the bathochromic shift in APC might be a result of inducing stronger coupling between the two PCBs [53]. The mechanism by which the environment could modify the degree of coupling might be via vibronic enhancement between the protein and chromophores [135]. This bathochromic shift can be enhanced further by the hydrophobic pocket forcing the PCB to be more planar, as revealed in the structure of one of the terminal emitters ApcD (APCB) [136]. Another source of enhanced excitonic coupling was recently revealed by analysis of the AmPBS. As already detailed above, the AmPBS exhibits strongly red-shifted emission in the absence of APC [49,64,96]. Mass spectrometric and crystallographic analysis showed that the complexes were heterogeneous, containing multiple isoforms of the both subunits, with apparent changes to EET pathways induced by the intensity of light used during growth. In addition, limiting the flexibility of the complex, by temperature, crystallization or desiccation leads to a significant red-shift in emission. It was suggested that these experimental observations may mimic the quasi-crystalline form of the AmPBS in vivo [62]. Taken with the observations detailed here, we can suggest that different environmental factors can lead to stronger coupling between PBS chromophores, even at large distances. In highly assembled PBS, this enhanced coupling can lead to ultrafast kinetics, beyond what would be typically expected for inter-chromophore distances in the PBS. Such enhanced kinetics have been observed in intact, isolated PBS from T. vulcanus [54], and suggest that EET can overcome the large distances in the PBS without loss of efficiency. This includes overcoming the need to “jump” between sub-complexes: between hexamers, from rods to core and from core to the RCs at psec timescales. Disruption of proper order in the PBS, may weaken coupling, leading to hypsochromic shifts, that prevent further EET towards the core component [137,138]. The experimental evidence for vibronically enhanced electronic

coupling leads to the question whether there are also coherent (quantum) effects that take place within the PBS [134,139]. One systems that has been extensively studied and exhibits apparent quantum effects is the PC645 antenna proteins from crytophytes [140]. While one of the two subunits of this complex is a PBP (homologous to β subunits), the other subunit is of different lineage. 2D ultrafast spectroscopic methods have revealed details that can be related to quantum coherent effects [141], while more recent experiments suggest that incoherent effects are more relevant for the description of EET in this protein [142]. Whether or not such coherent effects have a role in control over EET directionality and efficiency in the PBS is still unclear. Indeed, the PBS, along with other LHCs may use EET mechanisms that are on the border between classical and quantum mechanics [139]. The robust nature of the PBS allows its use under conditions that would damage most other LHC. In recent experiments, isolated PC trimers were allowed to slowly dry on solid state surfaces [55]. The trimers self-assembled into long extended wires with dimensions of 11.5 nm width and many microns in length, forming strong packing along the axis of the tubes and between adjacent wires. Double nearfield scanning optical microscopy, with resolutions of 150 nm (excitation) and 250 nm (emission) followed EET in these wires. It could be shown that emission could be measured as far as 2 μm from the site of excitation, indicating energy transfer over ~600 individual trimers. This is quite remarkable, considering that when the wire forms, each individual trimer has a width of 3 nm but the distances between chromophores are all 25–40 Å. As the sample became more dehydrated, the lifetime for emission was shortened and red-shifted, both factors indicating stronger coupling as described above for the PBS in solution. 8. Why did the PBS fail? As described in the previous sections, the PBS has many unique aspects that differentiate between it and all of the LHCs. These unique aspects allow cyanobacteria and red algae to survive, and indeed thrive, in almost every environmental niche that is exposed to visible (and in some cases to near IR) light. The size of the complex can be easily increased or decreased according to the needs of the organism, and can transfer energy laterally (between adjacent complexes) essentially allowing energy to be transferred long distances, at rapid rates to which 8

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ever RC is available. This close packed arrangement has recently been visualized by cryo-electron tomography [30]. Experiments have shown the versatility in all aspects required of light harvesting, including exquisite control over EET to the RCs and multiple mechanisms to avoid over excitation. Taking all of these parameters into account, one might assume that some form of the PBS would be found in the eukaryotic organisms that evolved from the cyanobacteria and/or red algae: the green algae and plants. These last organisms disposed of the PBS completely. An additional group of prokaryotic photosynthetic organisms are the prochlorophytes, cyanobacterial-like bacteria that do not contain PBS, but do have some remaining genes encoding for PBPs in their genome [143]. What were the possible reasons for the demise of the PBS? One major difference between the photosynthetic organisms lacking PBS versus those with PBS is in their membrane ultrastructure. Layers of PBS attached to the cytoplasmic surfaces of the thylakoids determine the spacing between membranes. For cells of limited size, the total number of thylakoid membranes will be limited. In green algal and plant chloroplasts, the thylakoid membranes are highly heterogeneous, with appressed membranes (grana lamella) that contain most of the active PSII and unappressed membranes (stroma lamella) that contain PSI, ATP synthase and most of the cytochrome b6/f complexes. This membrane arrangement could potentially increase the amount of PSII per cell as greater than the amount of PSI, as has been recently measured for the green algae C. reinhardtii [144], while the ratio of PSI/PSII in cyanobacteria has been estimated to be between 6 and 9 [145–147]. The evolutionary process, which led to the shift from single thylakoids in cyanobacteria to the appearance of PSII rich grana lamella in the chloroplast, must have required significant selective forces. Selection for increased PSII levels may have increased the potential for linear electron flow, as opposed to cyclic electron flow, which results in the production of ATP without the production of NADPH. We still do not have a clear idea as to the benefit for separation between most of the active PSII (in the grana) from the active PSI, as this separation makes the recovery from light-stress (photoinhibition) more complicated [148]. One additional characteristic of the grana lamella is that its formation is dependent on the presence of LHCII. An increase in the amount of this transmembrane antenna also permits control over the ratio of PSII to PSI activity via LHCII-phosphorylation controlled state transitions [149]. All of these changes, that apparently provided greater fitness to the first organisms that experienced terrestrial exposure to light, may have also required the exclusion of the PBS. The suggestion that formation of the grana/stroma lamella type thylakoid in chloroplasts might improve the amount of PSII activity (either by total increase in amount, or change in ratio to PSI) might also be connected with metabolic bookkeeping. Once cyanobacteria evolved enough oxygen into the atmosphere, the formation of the ozone layer removed enough damaging UV light to permit terrestrial occupation. Now photosynthetic organisms were exposed to the full intensity of sunlight, without the dispersion by water. A certain proportion of metabolic energy would now have to be used to provide new physiologies that were not required in the sea. If the normal day to day light fluencies were strong enough to drive photosynthesis to its maximal extent, maintaining all of the biochemistry required to make such a large antenna as the PBS might have been no longer energetically beneficial. Future research into this question, including the search for new exotic eukaryotes that might contain remnants of the PBS, may help understand a very basic question in biology – can “too much of a good thing” exist in biological processes?

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