In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

Copper(II)-Assisted Degradation of Pheophytin a by Reactive Oxygen Species.

The central ion Mg2+ is responsible for the differences between chlorophyll a and its free base in their reactivity toward metal ions and thus their resistance to oxidation. We present here the results of spectroscopic (electronic absorption and emission, circular dichroism, and electron paramagnetic resonance), spectroelectrochemical, and computational (based on density functional theory) investigations into the mechanism of pheophytin, a degradation that occurs in the presence of Cu ions and O2. The processes leading to the formation of the linear form of tetrapyrrole are very complex and involve the weakening of the methine bridge due to an electron withdrawal by Cu(II) and the activation of O2, which provides protection to the free ends of the opening macrocycle. These mechanistic insights are related to the naturally occurring damage to the photosynthetic apparatus of plants growing on metal-contaminated soils.

In vitro bioaccessibility evaluation of chlorophyll pigments in single and binary carriers.

Chlorophyll was extracted and microencapsulated using different carrier agents. Subsequently, in vitro digestion was performed, and the bioaccessibility of chlorophyll in the different encapsulation systems was carried out. The zeta potential, particle size, and PDI were significantly modified after the micellarization of digested microcapsules. I-W-Chl presented with the highest total chlorophyll recovery and micellarization rate of 54% and 43%, respectively. In the aqueous micellar fraction, the different encapsulation systems had total chlorophylls, pheophytins, and pheophorbides ranging from 13 to 49%, 42 - 77%, and 3 - 22% respectively. The bioaccessibility of total chlorophyll pigment ranging from 7% to 20% is given in the following order: I-W-Chl > WPI-Chl > Z-Chl > Ca-Chl > SCChlV > SCChlC. The result established in this study shows that the carrier agent type could inhibit or mediate the bioaccessibility of chlorophyll with the potential to be an efficient delivery system for health promoting compounds.

Screening of dephytinization reaction of chlorophyll pigments with citrus acetone powder by UPLC-DAD-MS.

The preparation of dephytylated chlorophyll standards is inefficient and the process is complicated, which hinders chlorophyll determination and related bioactive property investigation. In this paper, chlorophyll derivatives from four phytylated chlorophylls (chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b) before and after the enzymatic reaction were qualitatively and quantitatively characterized by UPLC-DAD-MS. A simple index was proposed to characterize chlorophyll pigments from their oxidized counterparts by the λmax of the typical peak of chlorophyll derivatives in UV-visible spectrum and their signal intensity ratios. The optimal reaction conditions for the enzymatic reaction of four chlorophyll pigments were optimized, and kinetic models were fitted. The results showed that the optimal temperatures for the enzymatic reactions of chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b were 30, 30, 60, and 60°C, respectively, and their optimal reaction time was 2, 3, 1, and 3 h, respectively. Kinetic models were fitted under optimal reaction conditions to study the Km and Vm values of the enzymatic reactions. PRACTICAL APPLICATION: Dephytylated chlorophylls, such as chlorophyllide and pheophorbide, are frequently determined in food industry and are always required to be prepared in lab with acetone powder from plant tissue. Moreover, chlorophyll pigments are easy to undergo oxidations, which make the characterization of dephytylated chlorophyll pigments more complicated and difficult. In this paper, four types of phytylated chlorophylls were investigated respectively about the dephytinization process with the citrus acetone powder, and the reaction mixture was analyzed with UPLC-DAD-MS, which can provide an important reference for relevant chlorophyll determination studies and the development of chlorophyll identification protocols.

In vitro bioaccessibility evaluation of pheophytins in gelatin/polysaccharides carrier.

The type of carrier agent could impact pheophytin stability and bioaccessibility. Hence, it is important to have an elaborate understanding on the extent and type of pheophytin transformation during in vitro digestion of microcapsules. Four kinds of protein/polysaccharides complex were used to fabricate pheophytin microcapsules and investigated for pigments bioaccessibility. With different carriers, pheophytin pigments showed new characteristics influencing particle size and zeta potential during in vitro digestion. Pheophytin b was widely transformed to pheophorbide b, confirming pheophorbidation of the b series in proper condition. No 151-hydroxy lactone chlorophyll or pheophytin derivatives were detected, indicating some protective effect of microencapsulation. Pheophytins loaded in gelatin-pectin complex exhibited a relatively higher recovery rate, micellarization rate, and bioaccessibility index. The result presented in this study shows that the type of carrier agent could initiate the removal of phytyl groups in pheophytins and also inhibit or mediate their bioaccessibility.

Karenia brevis Extract Induces Cellular Entry through Distinct Mechanisms in Phagocytic RAW 264.7 Macrophages versus Non-Phagocytic Vero Cells.

Marine algae extracts are an important area of potential drug discovery; however, nearly all studies to date have used non-fluorescent-based methods to determine changes in target cell activity. Many of the most robust immunological and cellular analyses rely on fluorescent probes and readouts, which can be problematic when the algae extract is fluorescent itself. In this study, we identified the fluorescent spectrum of an isolated extract from the marine dinoflagellate Karenia brevis, which included two fluorescing components: chlorophyll α and pheophytin α. When excited at 405 nm and 664 nm, the extract emitted fluorescence at 676 nm and 696 nm, respectively. The extract and its fluorescing components, chlorophyll α and pheophytin α, entered phagocytic RAW 264.7 macrophages and non-phagocytic Vero kidney cells through distinct mechanisms. When incubated with the extract and its main components, both the RAW 264.7 macrophages and the Vero cells accumulated fluorescence as early as 30 min and continued through 48 h. Vero kidney cells accumulated the K. brevis fluorescent extract through a dynamin-independent and acidified endosomal-dependent mechanism. RAW 264.7 macrophages accumulated fluorescent extract through a dynamin-independent, acidified endosomal-independent mechanism, which supports accumulation through phagocytosis. Furthermore, RAW 264.7 macrophages downregulated cell-surface expression of CD206 in response to extract stimulation indicating activation of phagocytic responses and potential immunosuppression of these immune cells. This study represents the first characterization of the cellular update of K. brevis extracts in phagocytic versus non-phagocytic cells. The data suggest the importance of understanding cellular uptake of fluorescing algae extracts and their mechanism of action for future drug discovery efforts.

HPLC-MS2 Analysis of Chlorophylls in Green Teas Establishes Differences among Varieties.

Green teas are nonfermented teas, the quality of which is measured by the green color. However, this category encompasses a high number of tea varieties that differ in cultivation and processing. For example, leaf or stem/bubble tea, plants cultivated under a light or shadow regime, powdered or unpowdered tea, etc. These variables determine the different qualities among green teas (Matcha, Sencha, Gyokuro, etc.) and consequently their different values on the market. Our purpose is to determine if these variables can exert an influence on the chlorophyll profile and to establish a characteristic profile for specific green teas. With such an aim, we analyzed the chlorophyll profiles of 6 different green tea varieties via HPLC-hr ESI/APCI-MS2 and identified up to 17 different chlorophyll compounds. For the first time, 132-hydroxy-chlorophylls, 132-hydroxy-pheophytins, and 151-hydroxy-lactone-pheophytins have been identified in green teas. Shadow teas (Matcha and Sencha) and light-regimen green teas can be statistically differentiated by the total chlorophyll content and the a/b ratio. However, only Matcha tea contains a higher proportion of chlorophylls a and b among the green tea varieties analyzed, justifying the higher quality and price of this variety. Other chlorophyll metabolites (pheophytins, pyropheophytins, and oxidized chlorophylls) are indicative of the various processing and storage conditions.

Transgenerationally Transmitted DNA Demethylation of a Spontaneous Epialleles Using CRISPR/dCas9-TET1cd Targeted Epigenetic Editing in Arabidopsis.

CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.

Phytoplankton bloom stages estimated from chlorophyll pigment proportions suggest delayed summer production in low sea ice years in the northern Bering Sea.

Decreased sea ice cover in the northern Bering Sea has altered annual phytoplankton phenology owing to an expansion of open water duration and its impact on ocean stratification. Limitations of satellite remote sensing such as the inability to detect bloom activity throughout the water column, under ice, and in cloudy conditions dictate the need for shipboard based measurements to provide more information on bloom dynamics. In this study, we adapted remote sensing land cover classification techniques to provide a new means to determine bloom stage from shipboard samples. Specifically, we used multiyear satellite time series of chlorophyll a to determine whether in-situ blooms were actively growing or mature (i.e., past-peak) at the time of field sampling. Field observations of chlorophyll a and pheophytin (degraded and oxidized chlorophyll products) were used to calculate pheophytin proportions, i.e., (Pheophytin/(Chlorophyll a + Pheophytin)) and empirically determine whether the bloom was growing or mature based on remotely sensed bloom stages. Data collected at 13 north Bering Sea stations each July from 2013-2019 supported a pheophytin proportion of 28% as the best empirical threshold to distinguish a growing vs. mature bloom stage. One outcome was that low vs. high sea ice years resulted in significantly different pheophytin proportions in July; in years with low winter-to-spring ice, more blooms with growing status were observed, compared to later stage, more mature blooms following springs with abundant seasonal sea ice. The detection of growing blooms in July following low ice years suggests that changes in the timing of the spring bloom triggers cascading effects on mid-summer production.

Chlorophyll Inhibits the Digestion of Soybean Oil in Simulated Human Gastrointestinal System.

Nowadays, much available processed and highly palatable food such as cream products and fried and convenient food, which usually showed a high energy density, had caused an increase in the intake of dietary lipids, further leading to significant growth in the prevalence of obesity. Chlorophyll, widespread in fruits and vegetables, was proven to have beneficial effects on alleviating obesity. This study investigated the effects of chlorophyll on the digestive characteristics of lipids under in vitro simulated adult and infant gastrointestinal systems. Chlorophyll decreased the release rate of free fatty acid (FFA) during in vitro adult and infant intestinal digestion by 69.2% and 60.0%, respectively. Meanwhile, after gastrointestinal digestion, chlorophyll changed the FFA composition of soybean oil emulsion and increased the particle size of oil droplets. Interestingly, with the addition of chlorophyll, the activity of pancreatic lipase was inhibited during digestion, which may be related to pheophytin (a derivative of chlorophyll after gastric digestion). Therefore, the results obtained from isothermal titration calorimetry and molecular docking further elucidated that pheophytin could bind to pancreatic lipase with a strong affinity of (4.38 ± 0.76) × 107 M-1 (Ka), while the binding site was amino acid residue Trp253. The investigation not only explained why chlorophyll inhibited digestive enzyme activity to reduce lipids digestion but also provided exciting opportunities for developing novel chlorophyll-based healthy products for dietary application in preventing obesity.

Influence of food composition on chlorophyll bioaccessibility.

Chlorophylls are ingested and effectively absorbed by our organism daily, but the effect of food composition on its bioaccessibility is unknown. Therefore, the present research analyses the chlorophyll bioaccessibility of ten commercial foods (guacamole, virgin olive oil, tortellini, basil hummus, creamed spinach, vegetable pasta, green tea chocolate, avocado and kiwi juices, and pesto sauce), selected based on their different nutritional (fat, fiber, protein, and carbohydrates) and chlorophyll composition and content. The most unexpected result was to correlate chlorophyll degradation during in vitro digestion with the salt content of the digested food. Surprisingly, independently of the foods' nutritional composition or the chlorophyll content, the chlorophyll profile after in vitro digestion was formed by 90% pheophytins and 10% chlorophylls and pheophorbides. Such a pattern can only be modified when the ingested food contains a high proportion of pheophorbides (˃20%) that prevailed up to the mixed micelles.

Inactivation Mechanism of Algal Chlorophyll by Allelochemical Quercetin.

Cyanobacteria harmful algal blooms (CyanoHABs) are a global concern. Application of allelochemicals is a promising solution for cyanobacteria control, due to its high efficiency, low cost and ecological safety. Flavonoids (natural polyphenols produced by aquatic plants) are reported capable of effectively inhibiting the growth of algae; however, the molecular mechanism of algae chlorophyll inactivation is still unclear. In this study, quercetin was used as a typical flavonoid, to investigate the inactivation effect of allelochemical on Microcystis aeruginosa chlorophyll a. The absorption and fluorescence spectra showed that chlorophyll reacted with quercetin to form pheophytin, and the formation rate of pheophytin increased with increasing quercetin concentration (1 × 10-5-1 × 10-2 M). FTIR spectra and DFT calculation showed that Mg2+ complexed with the 3-OH and 4-C = O groups in the quercetin ring C so that chlorophyll was inactivated due to the loss of Mg2+ ions. Overall, this study revealed that quercetin inactivated chlorophyll a of cyanobacteria by capturing Mg2+ ions, providing insights into the molecular mechanisms of algal bloom control by allelochemicals.

Ultrafast excitation quenching by the oxidized photosystem II reaction center.

Photosystem II (PSII) is the pigment-protein complex driving the photoinduced oxidation of water and reduction of plastoquinone in all oxygenic photosynthetic organisms. Excitations in the antenna chlorophylls are photochemically trapped in the reaction center (RC) producing the chlorophyll-pheophytin radical ion pair P+ Pheo-. When electron donation from water is inhibited, the oxidized RC chlorophyll P+ acts as an excitation quencher, but knowledge on the kinetics of quenching is limited. Here, we used femtosecond transient absorption spectroscopy to compare the excitation dynamics of PSII with neutral and oxidized RC (P+). We find that equilibration in the core antenna has a major lifetime of about 300 fs, irrespective of the RC redox state. Two-dimensional electronic spectroscopy revealed additional slower energy equilibration occurring on timescales of 3-5 ps, concurrent with excitation trapping. The kinetics of PSII with open RC can be described well with previously proposed models according to which the radical pair P+ Pheo- is populated with a main lifetime of about 40 ps, which is primarily determined by energy transfer between the core antenna and the RC chlorophylls. Yet, in PSII with oxidized RC (P+), fast excitation quenching was observed with decay lifetimes as short as 3 ps and an average decay lifetime of about 90 ps, which is shorter than the excited-state lifetime of PSII with open RC. The underlying mechanism of this extremely fast quenching prompts further investigation.

Isomerization kinetics of bacteriochlorophyll b and bacteriopheophytin b under acidic conditions.

Bacteriochlorophyll (BChl) b has a unique π-conjugation system, in which the bacteriochlorin macrocycle is conjugated with the C8-ethylidene group. This π-system is converted easily to the chlorin macrocycle. However, the effects of the central magnesium in BChl b on this conversion are unclear. In this study, the isomerization kinetics of BChl b and its demetalated pigment, bacteriopheophytin (BPhe) b, was analyzed under weakly acidic conditions. BChl b exhibited faster acid-induced isomerization than BPhe b. These results were attributed to the stabilization of a cationic intermediate, whose C8-ethylidene group is protonated, during the isomerization of BChl b compared to BPhe b because of a difference in the electron densities of the π-conjugation systems between BChl b and BPhe b. High-performance liquid chromatography analyses indicated that BChl b was primarily isomerized to 3-acetyl Chl a, followed by demetalation. The reaction order was due to the slower demetalation kinetics of metallobacteriochlorins than metallochlorins. These results will be helpful for handling unstable BChl b and BPhe b. The reaction properties of BChl b and BPhe b demonstrated here will be helpful for understanding the in vivo formation of BPhe b, which acts as the primary electron acceptor in photosynthetic reaction center complexes in BChl b-containing purple photosynthetic bacteria.

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins.

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Transforming Passive into Active: Multimodal Pheophytin-Based Carbon Dots Customize Protein Corona to Target Metastatic Breast Cancer.

Formation of protein corona on nanomaterials surface in vivo is usually considered as an unpredictable event for a predefined targeted delivery system for malignant cancers. In most situations, these protein coronas substantially change targeting efficiency or even cause adverse reactions which both hinder the clinical translation of the cargo-delivery systems. Active customization of protein corona onto nanomaterials surfaces can benefit their biomedical performances and open up new opportunities in construction of targeted delivery systems. Herein, lipid-PEG/pheophytin carbon dots (LPCDs) are prepared from natural chlorophyll and integrate seamlessly with positron emission tomography imaging, near-infrared fluorescence imaging, and photodynamic therapy capacity. In vitro measurements demonstrate that the LPCDs can actively absorb apolipoproteins into the protein corona to enhance their uptakes in breast cancer cells. In vivo studies confirm that LPCDs can give accurate delineation of metastatic breast cancer foci from surrounding normal tissues with multimodal biomedical functions. The feasibility of using LPCDs as a multimodal imaging and cancer-targeting nanoplatform may provide impetus for developing precise yet facile protein corona-targeted delivery systems for future clinical practice.

Chlorophyll and Pheophytin Dephytylating Enzymes Required for Efficient Repair of PSII in Synechococcus elongatus PCC 7942.

The Chlorophyll Dephytylase1 (CLD1) and pheophytinase (PPH) proteins of Arabidopsis thaliana are homologous proteins characterized respectively as a dephytylase for chlorophylls (Chls) and pheophytin a (Phein a) and a Phein a-specific dephytylase. Three genes encoding CLD1/PPH homologs (dphA1, dphA2 and dphA3) were found in the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and shown to be conserved in most cyanobacteria. His6-tagged DphA1, DphA2 and DphA3 proteins were expressed in Escherichia coli, purified to near homogeneity, and shown to exhibit significant levels of dephytylase activity for Chl a and Phein a. Each DphA protein showed similar dephytylase activities for Chl a and Phein a, but the three proteins were distinct in their kinetic properties, with DphA3 showing the highest and lowest Vmax and Km values, respectively, among the three. Transcription of dphA1 and dphA3 was enhanced under high-light conditions, whereas that of dphA2 was not affected by the light conditions. None of the dphA single mutants of S. elongatus showed profound growth defects under low (50 µmol photons m-2 s-1) or high (400 µmol photons m-2 s-1) light conditions. The triple dphA mutant did not show obvious growth defects under these conditions, either, but under illumination of 1,000 µmol photons m-2 s-1, the mutant showed more profound growth retardation compared with wild type (WT). The repair of photodamaged photosystem II (PSII) was much slower in the triple mutant than in WT. These results revealed that dephytylation of Chl a or Phein a or of both is required for efficient repair of photodamaged PSII.

Electrostatic profiling of photosynthetic pigments: implications for directed spectral tuning.

Photosynthetic pigment-protein complexes harvest solar energy with a high quantum efficiency. Protein scaffolds are known to tune the spectral properties of embedded pigments principally through structured electrostatic environments. Although the physical nature of electrostatic tuning is straightforward, the precise spatial principles of electrostatic preorganization remain poorly explored for different protein matrices and incompletely characterized with respect to the intrinsic properties of different photosynthetic pigments. In this work, we study the electronic structure features associated with the lowest excited state of a series of eight naturally occurring (bacterio)chlorophylls and pheophytins to describe the precise topological differences in electrostatic potentials and hence determine intrinsic differences in the expected mode and impact of electrostatic tuning. The difference electrostatic potentials between the ground and first excited states are used as fingerprints. Both the spatial profile and the propensity for spectral tuning are found to be unique for each pigment, indicating spatially and directionally distinct modes of electrostatic tuning. The results define a specific partitioning of the protein matrix around each pigment as an aid to identify regions with a maximal impact on spectral tuning and have direct implications for dimensionality reduction in protein design and engineering. Thus, a quantum mechanical basis is provided for understanding, predicting, and ultimately designing sequence-modified or pigment-exchanged biological systems, as suggested for selected examples of pigment-reconstituted proteins.

Pigment-modified reaction centers of Chloroflexus aurantiacus: chemical exchange of bacteriopheophytins with plant-type pheophytins.

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.