A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Phylogenetic and spectroscopic insights on the evolution of core antenna proteins in cyanobacteria.

Light harvesting by antenna systems is the initial step in a series of electron-transfer reactions in all photosynthetic organisms, leading to energy trapping by reaction center proteins. Cyanobacteria are an ecologically diverse group and are the simplest organisms capable of oxygenic photosynthesis. The primary light-harvesting antenna in cyanobacteria is the large membrane extrinsic pigment-protein complex called the phycobilisome. In addition, cyanobacteria have also evolved specialized membrane-intrinsic chlorophyll-binding antenna proteins that transfer excitation energy to the reaction centers of photosystems I and II (PSI and PSII) and dissipate excess energy through nonphotochemical quenching. Primary among these are the CP43 and CP47 proteins of PSII, but in addition, some cyanobacteria also use IsiA and the prochlorophyte chlorophyll a/b binding (Pcb) family of proteins. Together, these proteins comprise the CP43 family of proteins owing to their sequence similarity with CP43. In this article, we have revisited the evolution of these chlorophyll-binding antenna proteins by examining their protein sequences in parallel with their spectral properties. Our phylogenetic and spectroscopic analyses support the idea of a common ancestor for CP43, IsiA, and Pcb proteins, and suggest that PcbC might be a distant ancestor of IsiA. The similar spectral properties of CP47 and IsiA suggest a closer evolutionary relationship between these proteins compared to CP43.

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II.

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from ß-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2-5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and ß-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a â†’ ß-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of ß-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.

Photosynthetic modulation during the diurnal cycle in a unicellular diazotrophic cyanobacterium grown under nitrogen-replete and nitrogen-fixing conditions.

Cyanobacteria are the only oxygenic photosynthetic organisms that can fix nitrogen. In diazotrophic cyanobacteria, the regulation of photosynthesis during the diurnal cycle is hypothesized to be linked with nitrogen fixation and involve the D1 protein isoform PsbA4. The amount of bioavailable nitrogen has a major impact on productivity in aqueous environments. In contrast to low- or nitrogen-fixing (-N) conditions, little data on photosynthetic regulation under nitrogen-replete (+ N) conditions are available. We compared the regulation of photosynthesis under -N and + N conditions during the diurnal cycle in wild type and a psbA4 deletion strain of the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. We observed common changes to light harvesting and photosynthetic electron transport during the dark in + N and -N conditions and found that these modifications occur in both diazotrophic and non-diazotrophic cyanobacteria. Nitrogen availability increased PSII titer when cells transitioned from dark to light and promoted growth. Under -N conditions, deletion of PsbA4 modified charge recombination in dark and regulation of PSII titer during dark to light transition. We conclude that darkness impacts the acceptor-side modifications to PSII and photosynthetic electron transport in cyanobacteria independently of the nitrogen-fixing status and the presence of PsbA4.

Introduction of cysteine-mediated quenching in the CP43 protein of photosystem II builds resilience to high-light stress in a cyanobacterium.

Photosystem (PS) II is prone to photodamage both as a direct consequence of light, and indirectly by producing reactive oxygen species. Engineering high-light tolerance in cyanobacteria with minimal impact on PSII function is desirable in synthetic biology. IsiA, a CP43 homolog found exclusively in cyanobacteria, can dissipate excess light energy. We have recently determined that the sole cysteine residue of IsiA in Synechocystis sp. PCC 6803 has a critical role in non-photochemical quenching. Similar cysteine-mediated energy quenching has also been observed in green­sulfur bacteria. Sequence analysis of IsiA and CP43 aligns cysteine 260 of IsiA with valine 277 of CP43 in Synechocystis sp. PCC 6803. In the current study, we explore the impact of replacing valine 277 of CP43 to a cysteine on growth, PSII activity and high-light tolerance. Our results imply a decline in the PSII output for the mutant (CP43V277C) presumably due to the dissipation of absorbed light energy by cysteine. Spectroscopic analysis of isolated PSII from this mutant strain also suggests a delayed transfer of excitation energy from CP43-associated chlorophyll a to PSII reaction center. The mutation makes the PSII high-light tolerant and provides a small advantage in growth under high-light conditions. This previously unexplored strategy to engineer high-light tolerance could be a step further towards developing cyanobacterial cells as biofactories.

Psb27, a photosystem II assembly protein, enables quenching of excess light energy during its participation in the PSII lifecycle.

Photosystem II (PSII), the enzyme responsible for oxidizing water into molecular oxygen, undergoes a complex lifecycle during which multiple assembly proteins transiently bind to and depart from PSII assembly intermediate complexes. Psb27 is one such protein. It associates with the CP43 chlorophyll-binding subunit of PSII to form a Psb27-PSII sub-complex that constitutes 7-10% of the total PSII pool. Psb27 remains bound to PSII assembly intermediates and dissociates prior to the formation of fully functional PSII. In this study, we compared a series of Psb27 mutant strains in the cyanobacterium Synechocystis sp. PCC 6803 with varied expression levels of Psb27: wild type (WT); psb27 genetic deletion (Del27), genetically complemented psb27 (Com27); and over-expressed Psb27 (OE27). The Del27 strain demonstrated decreased non-photochemical fluorescence quenching, while the OE27 strain showed increased non-photochemical quenching and tolerance to fluctuating light conditions. Multiple flashes and fluorescence decay analysis indicated that OE27 has the least affected maximum PSII quantum yield of the mutants. OE27 also displayed a minimal impact on the half-life of the fast component of QA- reoxidation over multiple flashes, indicating robust PSII function. We propose that the close association between Psb27 and CP43, and the absence of a fully functional manganese cluster in the Psb27-PSII complex create a PSII sub-population that dissipates excitation energy prior to its recruitment into the functional PSII pool. Efficient energy dissipation prevents damage to this pre-PSII pool and allows for efficient PSII repair and maturation. Participation of Psb27 in the PSII life cycle ensures high-quality PSII assembly.

PsbX maintains efficient electron transport in Photosystem II and reduces susceptibility to high light in Synechocystis sp. PCC 6803.

PsbX is a 4.1 kDa intrinsic Photosystem II (PS II) protein, found together with the low-molecular-weight proteins, PsbY and PsbJ, in proximity to cytochrome b559. The function of PsbX is not yet fully characterized but PsbX may play a role in the exchange of the secondary plastoquinone electron acceptor QB with the quinone pool in the thylakoid membrane. To study the role of PsbX, we have constructed a PsbX-lacking strain of Synechocystis sp. PCC 6803. Our studies indicate that the absence of PsbX causes sensitivity to high light and impairs electron transport within PS II. In addition to a change in the QB-binding pocket, PsbX-lacking cells exhibited sensitivity to sodium formate, suggesting altered binding of the bicarbonate ligand to the non-heme iron between the sequential plastoquinone electron acceptors QA and QB. Experiments using 35S-methionine revealed high-light-treated PsbX-lacking cells restore PS II activity during recovery under low light by an increase in the turnover of PS II-associated core proteins. These labeling experiments indicate the recovery after exposure to high light requires both selective removal and replacement of the D1 protein and de novo PS II assembly.

A Novel Mode of Photoprotection Mediated by a Cysteine Residue in the Chlorophyll Protein IsiA.

Oxygenic photosynthetic organisms have evolved a multitude of mechanisms for protection against high-light stress. IsiA, a chlorophyll a-binding cyanobacterial protein, serves as an accessory antenna complex for photosystem I. Intriguingly, IsiA can also function as an independent pigment protein complex in the thylakoid membrane and facilitate the dissipation of excess energy, providing photoprotection. The molecular basis of the IsiA-mediated excitation quenching mechanism remains poorly understood. In this study, we demonstrate that IsiA uses a novel cysteine-mediated process to quench excitation energy. The single cysteine in IsiA in the cyanobacterium Synechocystis sp. strain PCC 6803 was converted to a valine. Ultrafast fluorescence spectroscopic analysis showed that this single change abolishes the excitation energy quenching ability of IsiA, thus providing direct evidence of the crucial role of this cysteine residue in energy dissipation from excited chlorophylls. Under stress conditions, the mutant cells exhibited enhanced light sensitivity, indicating that the cysteine-mediated quenching process is critically important for photoprotection.IMPORTANCE Cyanobacteria, oxygenic photosynthetic microbes, constantly experience varying light regimes. Light intensities higher than those that saturate the photosynthetic capacity of the organism often lead to redox damage to the photosynthetic apparatus and often cell death. To meet this challenge, cyanobacteria have developed a number of strategies to modulate light absorption and dissipation to ensure maximal photosynthetic productivity and minimal photodamage to cells under extreme light conditions. In this communication, we have determined the critical role of a novel cysteine-mediated mechanism for light energy dissipation in the chlorophyll protein IsiA.

Stabilization of Photosystem II by the PsbT protein impacts photodamage, repair and biogenesis.

Photosystem II (PS II) catalyzes the light-driven process of water splitting in oxygenic photosynthesis. Four core membrane-spanning proteins, including D1 that binds the majority of the redox-active co-factors, are surrounded by 13 low-molecular-weight (LMW) proteins. We previously observed that deletion of the LMW PsbT protein in the cyanobacterium Synechocystis sp. PCC 6803 slowed electron transfer between the primary and secondary plastoquinone electron acceptors QA and QB and increased the susceptibility of PS II to photodamage. Here we show that photodamaged ∆PsbT cells exhibit unimpaired rates of oxygen evolution if electron transport is supported by HCO3- even though the cells exhibit negligible variable fluorescence. We find that the protein environment in the vicinity of QA and QB is altered upon removal of PsbT resulting in inhibition of QA- oxidation in the presence of 2,5-dimethyl-1,4-benzoquinone, an artificial PS II-specific electron acceptor. Thermoluminescence measurements revealed an increase in charge recombination between the S2 oxidation state of the water-oxidizing complex and QA- by the indirect radiative pathway in ∆PsbT cells and this is accompanied by increased 1O2 production. At the protein level, both D1 removal and replacement, as well as PS II biogenesis, were accelerated in the ∆PsbT strain. Our results demonstrate that PsbT plays a key role in optimizing the electron acceptor complex of the acceptor side of PS II and support the view that repair and biogenesis of PS II share an assembly pathway that incorporates both de novo synthesis and recycling of the assembly modules associated with the core membrane-spanning proteins.

Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973.

Cyanobacteria are photoautotrophic prokaryotes that serve as key model organisms to study basic photosynthetic processes and are potential carbon-negative production chassis for commodity and high-value chemicals. The development of new synthetic biology tools and improvement of current ones is a requisite for furthering these organisms as models and production vehicles. CRISPR interference (CRISPRi) allows for targeted gene repression using a DNase-dead Cas nuclease ("dCas"). Here, we describe a titratable dCas12a (dCpf1) CRISPRi system and apply it to repress key photosynthetic processes in the fast-growing cyanobacterium Synechococcus sp. UTEX 2973 (S2973). The system relies on a lac repressor system that retains tight regulation in the absence of inducer (0-10% repression) while maintaining the capability for >90% repression of high-abundance gene targets. We determined that dCas12a is less toxic than dCas9. We tested the efficacy of the system toward eYFP and three native targets in S2973: the phycobilisome antenna, glycogen synthesis, and photosystem I (PSI), an essential part of the photosynthetic electron transport chain in oxygenic photoautotrophs. PSI was knocked down indirectly by repressing the protein factor BtpA involved in stabilizing core PSI proteins. We could reduce cellular PSI titer by 87% under photoautotrophic conditions, and we characterized these cells to gain insights into the response of the strain to the low PSI content. The ability to tightly regulate and time the (de)repression of essential genes in trans will allow for the study of photosynthetic processes that are not accessible using knockout mutants.