Supercomplexes of IsiA and photosystem I in a mutant lacking subunit PsaL

The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI-IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI-IsiA complexes.     

Supercomplexes of IsiA and Photosystem I in a mutant lacking subunit PsaL

The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI-IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI-IsiA complexes.     

Spectroscopic properties of PSI-IsiA supercomplexes from the cyanobacterium Synechococcus PCC 7942

The cyanobacterium Synechococcus PCC 7942 grown under iron starvation assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 CP43' or IsiA light-harvesting complexes [Nature 412 (2001) 745]. Here we present a spectroscopic characterization by temperature-dependent absorption and fluorescence spectroscopy, site-selective fluorescence spectroscopy at 5 K, and circular dichroism of isolated PSI-IsiA, PSI and IsiA complexes from this cyanobacterium grown under iron starvation. The results suggest that the IsiA ring increases the absorption cross-section of PSI by about 100%. Each IsiA subunit binds about 16-17 chlorophyll a (Chl a) molecules and serves as an efficient antenna for PSI. Each of the monomers of the trimeric PSI complex contains two red chlorophylls, which presumably give rise to one exciton-coupled dimer and at 5 K absorb and fluoresce at 703 and 713 nm, respectively. The spectral properties of these C-703 chlorophylls are not affected by the presence of the IsiA antenna ring. The spectroscopic properties of the purified IsiA complexes are similar to those of the related CP43 complex from plants, except that the characteristic narrow absorption band of CP43 at 682.5 nm is missing in IsiA.     

Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.     

Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.     

THE CYANOBACTERIAL CHLOROPHYLL‐BINDING‐PROTEIN ISIA ACTS TO INCREASE THE IN VIVO EFFECTIVE ABSORPTION CROSS‐SECTION OF PSI UNDER IRON LIMITATION1

Iron availability limits primary production in >30% of the world’s oceans; hence phytoplankton have developed acclimation strategies. In particular, cyanobacteria express IsiA (iron‐stress‐induced) under iron stress, which can become the most abundant chl‐binding protein in the cell. Within iron‐limited oceanic regions with significant cyanobacterial biomass, IsiA may represent a significant fraction of the total chl. We spectroscopically measured the effective cross‐section of the photosynthetic reaction center PSI (σ_PSI) in vivo and biochemically quantified the absolute abundance of PSI, PSII, and IsiA in the model cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that accumulation of IsiA results in a ～60% increase in σ_PSI, in agreement with the theoretical increase in cross‐section based on the structure of the biochemically isolated IsiA‐PSI supercomplex from cyanobacteria. Deriving a chl budget, we suggest that IsiA plays a primary role as a light‐harvesting antenna for PSI. On progressive iron‐stress in culture, IsiA continues to accumulate without a concomitant increase in σ_PSI, suggesting that there may be a secondary role for IsiA. In natural populations, the potential physiological significance of the uncoupled pool of IsiA remains to be established. However, the functional role as a PSI antenna suggests that a large fraction of IsiA‐bound chl is directly involved in photosynthetic electron transport.     

The molecular structure of the IsiA-Photosystem I supercomplex, modelled from high-resolution, crystal structures of Photosystem I and the CP43 protein

We present the molecular structure of the IsiA-Photosystem I (PSI) supercomplex, inferred from high-resolution, crystal structures of PSI and the CP43 protein. The structure of iron-stress-induced A protein (IsiA) is similar to that of CP43, albeit with the difference that IsiA is associated with 15 chlorophylls (Chls), one more than previously assumed. The membrane-spanning helices of IsiA contain hydrophilic residues many of which bind Chl. The optimal structure of the IsiA-PSI supercomplex was inferred by systematically rearranging the IsiA monomers and PSI trimer in relation to each other. For each of the 6,969,600 structural configurations considered, we counted the number of optimal Chl-Chl connections (i.e., cases where Chl-bound Mg atoms are ≤ 25 Å apart). Fifty of these configurations were found to have optimal energy-transfer potential. The 50 configurations could be divided into three variants; one of these, comprising 36 similar configurations, was found to be superior to the other configurations in terms of its potential to transfer excitation energy to the reaction centres under low-light conditions and its potential to dissipate excess energy under high-light conditions. Compared to the assumed model [Biochemistry 42 (2003) 3180-3188], the new Chl increases by 7% the ability of IsiA to harvest sunlight while the rearrangement of the constituent components of the IsiA-PSI supercomplex increases by 228% the energy-transfer potential. In conclusion, our model allows us to explain how the IsiA-PSI supercomplex may act as an efficient light-harvesting structure under low-light conditions and as an efficient dissipater of excess energy under high-light conditions.     

Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins

In response to iron deficiency, cyanobacteria synthesize the iron stress-induced chlorophyll binding protein IsiA. This protein protects cyanobacterial cells against iron stress. It has been proposed that the protective role of IsiA is related to a blue light-induced nonphotochemical fluorescence quenching (NPQ) mechanism. In iron-replete cyanobacterial cell cultures, strong blue light is known to induce a mechanism that dissipates excess absorbed energy in the phycobilisome, the extramembranal antenna of cyanobacteria. In this photoprotective mechanism, the soluble Orange Carotenoid Protein (OCP) plays an essential role. Here, we demonstrate that in iron-starved cells, blue light is unable to quench fluorescence in the absence of the phycobilisomes or the OCP. By contrast, the absence of IsiA does not affect the induction of fluorescence quenching or its recovery. We conclude that in cyanobacteria grown under iron starvation conditions, the blue light-induced nonphotochemical quenching involves the phycobilisome OCP-related energy dissipation mechanism and not IsiA. IsiA, however, does seem to protect the cells from the stress generated by iron starvation, initially by increasing the size of the photosystem I antenna. Subsequently, the IsiA converts the excess energy absorbed by the phycobilisomes into heat through a mechanism different from the dynamic and reversible light-induced NPQ processes.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

Supramolecular organization and dual function of the IsiA chlorophyll-binding protein in cyanobacteria

A significant part of global primary productivity is provided by cyanobacteria, which are abundant in most marine and freshwater habitats. In many oceanographic regions, however, the concentration of iron can be so low that it limits growth. Cyanobacteria respond to this condition by expressing a number of iron stress inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. It was recently shown that 18 IsiA proteins encircle trimeric photosystem I (PSI) under iron-deficient growth conditions. We report here that after prolonged growth of Synechocystis PCC 6803 in an iron-deficient medium, the number of bound IsiA proteins can be much higher than previously known. The largest complexes bind 12-14 units in an inner ring and 19-21 units in an outer ring around a PSI monomer. Fluorescence excitation spectra indicate an efficient light harvesting function for all PSI-bound chlorophylls. We also find that IsiA accumulates in cyanobacteria in excess of what is needed for functional light harvesting by PSI, and that a significant part of IsiA builds supercomplexes without PSI. Because the further decline of PSI makes photosystem II (PSII) increasingly vulnerable to photooxidation, we postulate that the surplus synthesis of IsiA shields PSII from excess light. We suggest that IsiA plays a surprisingly versatile role in cyanobacteria, by significantly enhancing the light harvesting ability of PSI and providing photoprotection for PSII.     

Novel supercomplex organization of photosystem I in Anabaena and Cyanophora paradoxa

The supercomplex organization of photosystem complexes was studied in various cyanobacteria, a glaucocystophyte and a primitive rhodophyte by blue-native PAGE with a wide range of detergent concentrations. In contrast to known cyanobacteria that produced the PSI trimer, a filamentous N(2)-fixing cyanobacterium Anabaena sp. PCC 7120 and a glaucocystophyte Cyanophora paradoxa NIES 547 had a PSI tetramer and dimer but no trimer at all. This was confirmed by sucrose density gradient centrifugation. A primitive rhodophyte Cyanidioschyzon merolae had two species of PSI monomeric complex with a light-harvesting Chl complex of a different composition. These results are discussed with regard to the evolution of the PSI supercomplex.     

Involvement of phycobilisome diffusion in energy quenching in cyanobacteria

Nonphotochemical quenching (NPQ) of excitation energy is a well-established phenomenon in green plants, where it serves to protect the photosynthetic apparatus from photodamage under excess illumination. The induction of NPQ involves a change in the function of the light-harvesting apparatus, with the formation of quenching centers that convert excitation energy into heat. Recently, a comparable phenomenon was demonstrated in cyanobacteria grown under iron-starvation. Under these conditions, an additional integral membrane chlorophyll-protein, IsiA, is synthesized, and it is therefore likely that IsiA is required for NPQ in cyanobacteria. We have previously used fluorescence recovery after photobleaching to show that phycobilisomes diffuse rapidly on the membrane surface, but are immobilized when cells are immersed in high-osmotic strength buffers, apparently because the interaction between phycobilisomes and reaction centers is stabilized. Here, we show that when cells of the cyanobacterium Synechocystis sp. PCC 6803 subjected to prolonged iron-deprivation are immersed in 1 m phosphate buffer, NPQ can still be induced as normal by high light. However, the formation of the quenched state is irreversible under these conditions, suggesting that it involves the coupling of free phycobilisomes to an integral-membrane complex, an interaction that is stabilized by 1 m phosphate. Fluorescence spectra are consistent with this idea. Fluorescence recovery after photobleaching measurements confirm that the induction of NPQ in the presence of 1 m phosphate is accompanied by immobilization of the phycobilisomes. We propose as a working hypothesis that a major component of the fluorescence quenching observed in iron-starved cyanobacteria arises from the coupling of free phycobilisomes to IsiA.     

Structural analysis of the photosystem I supercomplex of cyanobacteria induced by iron deficiency

Here we describe the three-dimensional structure of the newly discovered CP43'-photosystem I (PSI) supercomplex of cyanobacteria calculated by single-particle analysis of images obtained by electron cryomicroscopy (cryo-EM). This large membrane protein complex has a molecular mass of approximately 2 MDa and is found in cyanobacteria when grown in iron deficient media. It is composed of a reaction center trimer surrounded by 18 subunits of the chlorophyll a binding CP43'protein, encoded by the isiA gene, which increases the light harvesting capacity of PSI by approximately 70%. By modeling higher-resolution structural data obtained from X-ray crystallography into the three-dimensional (3D) cryo-EM map, we have been able to gain a better understanding of the structure and functional properties of this supermolecular complex. We have identified three separate clusters of chlorophyll molecules at the periphery of the PSI core which may aid energy transfer from the CP43' antenna ring to the reaction center. Moreover, it is shown that despite the replacement of ferredoxin with flavodoxin as an electron acceptor under iron stress conditions, the 3D map has density to accommodate the extrinsic proteins, PsaC, PsaD, and PsaE. The presence of these three proteins was also confirmed by immunoblotting.     

Function,regulation and distribution of IsiA,a membrane-bound chlorophyll a-antenna protein in cyanobacteria

Photosynthetica - IsiA is a membrane-bound Chl a-antenna protein synthesized in cyanobacteria under iron deficiency. Since iron deficiency is a common nutrient stress in significant fractions of...     

Refinement of the structural model for the Photosystem II supercomplex of higher plants

Recent X-ray structures determined for the Photosystem II (PSII) core complex isolated from cyanobacteria have provided important information for understanding the functionality of this photosynthetic enzyme including its water splitting activity. As yet, no high-resolution structure is available for PSII of plants or eukaryotes in general. However, crystal structures have been determined for some components of plant PSII which together with the cyanobacterial structure can be used to interpret lower resolution structures of plant PSII derived from electron cryomicroscopy (cryo-EM). Here, we utilise the published X-ray structures of a cyanobacterial PSII core, Light Harvesting Complex II (LHCII), PsbP and PsbQ proteins to construct a model of the plant LHCII-PSII supercomplex using a 17 A resolution 3D electron density map of the spinach supercomplex determined by cryo-EM and single particle analysis. In so doing, we tentatively identify the relative positioning of the chlorophylls within the supercomplex and consider energy transfer pathways between the different subunits. The modelling has also allowed density to be assigned to the three extrinsic proteins of plant PSII, PsbO, PsbP and PsbQ associated with the water splitting centre and concluded that although the position of PsbO is the same as in cyanobacteria, PsbP and PsbQ are located in different positions to the cyanobacterial extrinsic PsbU and PsbV proteins.     

Three-dimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.     

Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans

Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.