Cyanobacterial Light-harvesting Complex Research Articles

AlphaFold and Structural Mass Spectrometry Enable Interrogations on the Intrinsically Disordered Regions in Cyanobacterial Light-harvesting Complex Phycobilisome

Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of proteins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for interrogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS). Here, a joint platform is built to probe such structural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor complex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.

Engineering Cells, Membranes and Light Harvesting/Photosystem II Super-Complexes in Bio-Photoelectrochemical Cells

Photosynthetic organisms, membranes and complexes are attractive starting materials for solar energy conversion (SEC). Our overall goal is to develop methods to perform SEC using these materials in simple, inexpensive and a fashion that will be non-polluting and will not compete with the growth of food materials. I will describe here how the remarkable photocatalytic activity of the photosynthetic apparatus can provide overall water splitting with oxygen and hydrogen production in Bio-Photo-Electro-Chemical (BPEC) cells via the simplest and cleanest of processes wither in the absence or presence of added electron transport molecules. With plant thylakoids, electrons are shuttled by FeCN to a transparent FTO electrode, yielding a photocurrent density of 0.5 mA·cm-2. Hydrogen evolution occurs at the cathode at a bias as low as 0.8 V. A tandem cell comprising the BPEC cell with the thylakoid membranes and a Si photovoltaic module achieves overall water splitting with solar to hydrogen conversion efficiency of 0.3% (Pinhassi et al. Nature Communications 2016). With cyanobacterial cells, following a brief treatment that does not induce loss of cell viability, electrons from both the respiratory and photosynthetic systems are transferred directly to a graphite electrode, utilizing endogenous electron carriers (Saper et al. Nature Communications 2018). The current produced can be used for hydrogen production at low additional bias for significantly longer durations than the plant thylakoids.Another route is to isolate the photosynthetic complexes and chemically connect them to the electrochemical cell. One of the issues with this strategy is the small number of light absorbing chromophores connected to the complex, lowering the photochemical efficiency. Connection of isolated light harvesting complexes can help to overcome this deficiency. Photosystem II (PSII) is the only enzyme that catalyzes light-induced water oxidation being the basis for its application as a biophotoanode in various bio-photovoltaics and photo-bioelectrochemical cells. However, the absorption spectrum of PSII limits the quantum efficiency in the range of visible light, due to a gap in the green absorption region of chlorophylls (500 - 650 nm). To overcome this limitation, we have stabilized the interaction between PSII and Phycobilisomes (PBSs) – the cyanobacterial light harvesting complex, in vitro. The PBS of different cyanobacteria were analyzed for their ability to transfer energy to Thermosynechococcus elongatus (Te) PSII by fluorescence spill-over and photocurrent action spectra. Integration of the PBS-PSII super-complexes within an Os-complex-modified hydrogel on macro-porous indium tin oxide electrodes (MP-ITO) resulted in notably improved, wavelength dependent, incident photon-to-electron conversion efficiencies (IPCE). IPCE values in the green gap were doubled from 3 % to 6 % compared to PSII electrodes without PBS and a maximum IPCE up to 10.9 % at 670 nm was achieved. Figure 1

Different roles for ApcD and ApcF in Synechococcus elongatus and Synechocystis sp. PCC 6803 phycobilisomes

The phycobilisome, the cyanobacterial light harvesting complex, is a huge phycobiliprotein containing extramembrane complex, formed by a core from which rods radiate. The phycobilisome has evolved to efficiently absorb sun energy and transfer it to the photosystems via the last energy acceptors of the phycobilisome, ApcD and ApcE. ApcF also affects energy transfer by interacting with ApcE. In this work we studied the role of ApcD and ApcF in energy transfer and state transitions in Synechococcus elongatus and Synechocystis PCC6803. Our results demonstrate that these proteins have different roles in both processes in the two strains. The lack of ApcD and ApcF inhibits state transitions in Synechocystis but not in S. elongatus. In addition, lack of ApcF decreases energy transfer to both photosystems only in Synechocystis, while the lack of ApcD alters energy transfer to photosystem I only in S. elongatus. Thus, conclusions based on results obtained in one cyanobacterial strain cannot be systematically transferred to other strains and the putative role(s) of phycobilisomes in state transitions need to be reconsidered.

Phycobilisomes Harbor FNRL in Cyanobacteria.

Cyanobacterial phycobilisomes (PBSs) are photosynthetic antenna complexes that harvest light energy and supply it to two reaction centers (RCs) where photochemistry starts. PBSs can be classified into two types, depending on the presence of allophycocyanin (APC): CpcG-PBS and CpcL-PBS. Because the accurate protein composition of CpcL-PBS remains unclear, we describe here its isolation and characterization from the cyanobacterium Synechocystis sp. strain 6803. We found that ferredoxin-NADP+ oxidoreductase (or FNRL), an enzyme involved in both cyclic electron transport and the terminal step of the electron transport chain in oxygenic photosynthesis, is tightly associated with CpcL-PBS as well as with CpcG-PBS. Room temperature and low-temperature fluorescence analyses show a red-shifted emission at 669 nm in CpcL-PBS as a terminal energy emitter without APC. SDS-PAGE and quantitative mass spectrometry reveal an increased content of FNRL and CpcC2, a rod linker protein, in CpcL-PBS compared to that of CpcG-PBS rods, indicative of an elongated CpcL-PBS rod length and its potential functional differences from CpcG-PBS. Furthermore, we combined isotope-encoded cross-linking mass spectrometry with computational protein structure predictions and structural modeling to produce an FNRL-PBS binding model that is supported by two cross-links between K69 of FNRL and the N terminus of CpcB, one component in PBS, in both CpcG-PBS and CpcL-PBS (cross-link 1), and between the N termini of FNRL and CpcB (cross-link 2). Our data provide a novel functional assembly form of phycobiliproteins and a molecular-level description of the close association of FNRL with phycocyanin in both CpcG-PBS and CpcL-PBS.IMPORTANCE Cyanobacterial light-harvesting complex PBSs are essential for photochemistry in light reactions and for balancing energy flow to carbon fixation in the form of ATP and NADPH. We isolated a new type of PBS without an allophycocyanin core (i.e., CpcL-PBS). CpcL-PBS contains both a spectral red-shifted chromophore, enabling efficient energy transfer to chlorophyll molecules in the reaction centers, and an increased FNRL content with various rod lengths. Identification of a close association of FNRL with both CpcG-PBS and CpcL-PBS brings new insight to its regulatory role for fine-tuning light energy transfer and carbon fixation through both noncyclic and cyclic electron transport.

Discovery of carotenoid red-shift in endolithic cyanobacteria from the Atacama Desert

The biochemical responses of rock-inhabiting cyanobacteria towards native environmental stresses were observed in vivo in one of the Earth’s most challenging extreme climatic environments. The cryptoendolithic cyanobacterial colonization, dominated by Chroococcidiopsis sp., was studied in an ignimbrite at a high altitude volcanic area in the Atacama Desert, Chile. Change in the carotenoid composition (red-shift) within a transect through the cyanobacteria dominant microbial community (average thickness ~1 mm) was unambiguously revealed in their natural endolithic microhabitat. The amount of red shifted carotenoid, observed for the first time in a natural microbial ecosystem, is depth dependent, and increased with increasing proximity to the rock surface, as proven by resonance Raman imaging and point resonance Raman profiling. It is attributed to a light-dependent change in carotenoid conjugation, associated with the light-adaptation strategy of cyanobacteria. A hypothesis is proposed for the possible role of an orange carotenoid protein (OCP) mediated non-photochemical quenching (NPQ) mechanism that influences the observed spectral behavior. Simultaneously, information about the distribution of scytonemin and phycobiliproteins was obtained. Scytonemin was detected in the uppermost cyanobacteria aggregates. A reverse signal intensity gradient of phycobiliproteins was registered, increasing with deeper positions as a response of the cyanobacterial light harvesting complex to low-light conditions.

The proteolysis adaptor, NblA, is essential for degradation of the core pigment of the cyanobacterial light-harvesting complex.

The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided insitu evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.

Effect of partial or complete elimination of light‐harvesting complexes on the surface electric properties and the functions of cyanobacterial photosynthetic membranes

Influence of the modification of the cyanobacterial light-harvesting complex [i.e. phycobilisomes (PBS)] on the surface electric properties and the functions of photosynthetic membranes was investigated. We used four PBS mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less), CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE(-) (PSI-less with detached PBS). Modifications of the PBS content lead to changes in the cell morphology and surface electric properties of the thylakoid membranes as well as in their functions, such as photosynthetic oxygen-evolving activity, P700 kinetics and energy transfer between the pigment-protein complexes. Data reveal that the complete elimination of PBS in the PAL mutant causes a slight decrease in the electric dipole moments of the thylakoid membranes, whereas significant perturbations of the surface charges were registered in the membranes without assembled PBS-PSII macrocomplex (BE mutant) or PSI complex (PSI-less mutant). These observations correlate with the detected alterations in the membrane structural organization. Using a polarographic oxygen rate electrode, we showed that the ratio of the fast to the slow oxygen-evolving PSII centers depends on the partial or complete elimination of light-harvesting complexes, as the slow operating PSII centers dominate in the PBS-less mutant and in the mutant with detached PBS.

NblC, a novel component required for pigment degradation during starvation in Synechococcus PCC 7942

Adjustment of photosynthetic light harvesting to ambient conditions is essential to allow efficient energy capturing and to prevent surplus excitation and the cellular damage resulting from it. Degradation of the cyanobacterial light harvesting complex, the phycobilisome, is a general acclimation response occurring under various stress conditions. This study identifies a novel component, NblC, which mediates phycobilisome degradation under nitrogen, sulphur and phosphorus starvation. Our study indicates the requirement of NblC for efficient expression of nblA, an essential component of the degradation pathway; accumulation of nblA transcripts upon nutrient starvation was impaired in the NblC-mutant. Furthermore, expression of NblC under the control of a foreign promoter resulted in accumulation of nblA transcripts and degradation of the light harvesting complex. Transcription of nblC is induced upon nutrient starvation, suggesting the requirement of elevated levels of NblC under these conditions. Importantly, NblC could not exert its positive effect on nblA expression in the absence of the response regulator NblR. Sequence alignment suggests kinase motifs as well as homology of NblC to anti-sigma factors. Accordingly, we suggest a mode of action for this newly identified modulator, which provides new insights into regulation of gene expression in response to environmental stimuli.

Structure and light-regulated expression of phycoerythrin genes in wild-type and phycobilisome assembly mutants of Synechocystis sp. strain PCC 6701

Phycoerythrin is a major pigmented component of the phycobilisome, a cyanobacterial light-harvesting complex. It contains bilin-type chromophores that absorb and transfer light energy to chlorophyll protein complexes of the photosynthetic membranes. In many cyanobacteria, phycoerythrin expression is regulated by light wavelength in a response known as chromatic adaptation. Green light-grown cells contain higher levels of this biliprotein than do cells grown in red light. The phycoerythrin gene set from the unicellular cyanobacterium Synechocystis sp. strain PCC 6701 was cloned and sequenced, and the 5' end of the phycoerythrin mRNA was localized. The amino acid sequences of the phycoerythrin subunits from Synechocystis strain 6701 and Fremyella diplosiphon were 90% identical. As observed in F. diplosiphon, the Synechocystis strain 6701 phycoerythrin transcript accumulated to high levels in green light-grown cells and low levels in red light-grown cells. Similar nucleotide sequences, which might control gene expression, occurred upstream of the transcription initiation sites of the phycoerythrin genes in both organisms. While the phycoerythrin structure and light-regulated transcript accumulation were similar in Synechocystis strain 6701 and F. diplosiphon, the steady-state levels of phycoerythrin subunits during growth in red light were quite different for the two organisms. This observation suggests that control of phycoerythrin levels in Synechocystis strain 6701 is complex and may involve posttranscriptional processes. We also characterized the phycoerythrin genes and mRNA levels in two phycobilisome assembly mutants, UV16-40 and UV16.

Cyanobacterial light-harvesting complex subunits encoded in two red light-induced transcripts.

The major light-harvesting complex in cyanobacteria and red algae, the phycobilisome, is composed of chromophoric and nonchromophoric polypeptides. Two linked genes encoding major chromophoric components, the polypeptide subunits of phycocyanin, were isolated from the cyanobacterium Fremyella diplosiphon. Transcripts from this phycocyanin subunit gene cluster were present as major species in the cyanobacterium grown in red light, but not in cultures maintained in green light. The genes for the subunits of the red light-induced phycocyanin were transcribed together (beta-phycocyanin followed by alpha-phycocyanin) on two messenger RNA species; one contained 1600 bases while the other had 3800 bases. The latter, which encompassed the smaller transcript, contained additional sequences extending from the 3' end of the coding region of the alpha-phycocyanin gene. It may encode other light-induced components of the phycobilisome. Since phycocyanin, which effectively absorbs red light, becomes a dominant constituent of the phycobilisome in red light, these different levels may reflect an important adaptive mechanism of these organisms to their environment.