Photosystem II: the reaction center of oxygenic photosynthesis.

Photosystem II (PSII) uses light energy to split water into chemical products that power the planet. The stripped protons contribute to a membrane electrochemical potential before combining with the stripped electrons to make chemical bonds and releasing O2 for powering respiratory metabolisms. In this review, we provide an overview of the kinetics and thermodynamics of water oxidation that highlights the conserved performance of PSIIs across species. We discuss recent advances in our understanding of the site of water oxidation based upon the improved (1.9-Å resolution) atomic structure of the Mn4CaO5 water-oxidizing complex (WOC) within cyanobacterial PSII. We combine these insights with recent knowledge gained from studies of the biogenesis and assembly of the WOC (called photoassembly) to arrive at a proposed chemical mechanism for water oxidation.

Cyanobacterial photosystem II reaction center design in tobacco chloroplasts increases biomass in low light.

The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 µE m-2 s-1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.

Exceptional Quantum Efficiency Powers Biomass Production in Halotolerant Algae Picochlorum sp.

The green algal genus Picochlorum is of biotechnological interest because of its robust response to multiple environmental stresses. We compared the metabolic performance of P. SE3 and P. oklahomense to diverse microbial phototrophs and observed exceptional performance of photosystem II (PSII) in light energy conversion in both Picochlorum species. The quantum yield (QY) for O2 evolution is the highest of any phototroph yet observed, 32% (20%) by P. SE3 (P. okl) when normalized to total PSII subunit PsbA (D1) protein, and 80% (75%) normalized per active PSII, respectively. Three factors contribute: (1) an efficient water oxidizing complex (WOC) with the fewest photochemical misses of any organism; (2) faster reoxidation of reduced (PQH2)B in P. SE3 than in P. okl. (period-2 Fourier amplitude); and (3) rapid reoxidation of the plastoquinol pool by downstream electron carriers (Cyt b6f/PETC) that regenerates PQ faster in P. SE3. This performance gain is achieved without significant residue changes around the QB site and thus points to a pull mechanism involving faster PQH2 reoxidation by Cyt b6f/PETC that offsets charge recombination. This high flux in P. SE3 may be explained by genomically encoded plastoquinol terminal oxidases 1 and 2, whereas P. oklahomense has neither. Our results suggest two distinct types of PSII centers exist, one specializing in linear electron flow and the other in PSII-cyclic electron flow. Several amino acids within D1 differ from those in the low-light-descended D1 sequences conserved in Viridiplantae, and more closely match those in cyanobacterial high-light D1 isoforms, including changes near tyrosine Yz and a water/proton channel near the WOC. These residue changes may contribute to the exceptional performance of Picochlorum at high-light intensities by increasing the water oxidation efficiency and the electron/proton flux through the PSII acceptors (QAQB).

Cyclic electron flow around photosystem II in silico: How it works and functions in vivo.

To date, cyclic electron flow around PSI (PSI-CEF) has been considered the primary (if not the only) mechanism accepted to adjust the ratio of linear vs cyclic electron flow that is essential to adjust the ratio of ATP/NADPH production needed for CO2 carboxylation. Here we provide a kinetic model showing that cyclic electron flow within PSII (PSII-CEF) is essential to account for the accelerating rate of decay in flash-induced oscillations of O2 yield as the PQ pool progressively reduces to PQH2. Previously, PSII-CEF was modeled by backward transitions using empirical Markov models like Joliot-Kok (J-K) type. Here, we adapted an ordinary differential equation methodology denoted RODE1 to identify which microstates within PSII are responsible for branching between PSII-CEF and Linear Electron Flow (LEF). We applied it to simulate the oscillations of O2 yield from both Chlorella ohadii, an alga that shows strong PSII-CEF attributed to high backward transitions, and Synechococcus elongatus sp. 7002, a widely studied model cyanobacterium. RODE2 simulations reveal that backward transitions occur in microstates that possess a QB- semiquinone prior to the flash. Following a flash that forms microstates populating (QAQB)2-, PSII-CEF redirects these two electrons to the donor side of PSII only when in the oxidized S2 and S3 states. We show that this backward transition pathway is the origin of the observed period-2 oscillations of flash O2 yield and contributes to the accelerated decay of period-4 oscillations. This newly added pathway improved RODE1 fits for cells of both S. elongatus and C. ohadii. RODE2 simulations show that cellular adaptation to high light intensity growth is due to a decrease in QB availability (empty or blocked by Q2-B), or equivalently due to a decrease in the difference in reduction potential relative to QA/QA-. PSII-CEF provides an alternative mechanism for rebalancing the NADPH:ATP ratio that occurs rapidly by adjusting the redox level of the PQ:PQH2 pool and is a necessary process for energy metabolism in aquatic phototrophs.

Regulation of light energy conversion between linear and cyclic electron flow within photosystem II controlled by the plastoquinone/quinol redox poise.

Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H+o/H+i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA-) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H+o/H+i) and the PQ pool redox poise via e-/H+ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q-H+)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P+QA-) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.

Evidence for a robust photosystem II in the photosynthetic amoeba Paulinella.

Paulinella represents the only known case of an independent primary plastid endosymbiosis, outside Archaeplastida, that occurred c. 120 (million years ago) Ma. These photoautotrophs grow very slowly in replete culture medium with a doubling time of 6-7 d at optimal low light, and are highly sensitive to photodamage under moderate light levels. We used genomic and biophysical methods to investigate the extreme slow growth rate and light sensitivity of Paulinella, which are key to photosymbiont integration. All photosystem II (PSII) genes except psb28-2 and all cytochrome b6 f complex genes except petM and petL are present in Paulinella micropora KR01 (hereafter, KR01). Biophysical measurements of the water oxidation complex, variable chlorophyll fluorescence, and photosynthesis-irradiance curves show no obvious evidence of PSII impairment. Analysis of photoacclimation under high-light suggests that although KR01 can perform charge separation, it lacks photoprotection mechanisms present in cyanobacteria. We hypothesize that Paulinella species are restricted to low light environments because they are deficient in mitigating the formation of reactive oxygen species formed within the photosystems under peak solar intensities. The finding that many photoprotection genes have been lost or transferred to the host-genome during endosymbiont genome reduction, and may lack light-regulation, is consistent with this hypothesis.

Dedication to Ron Pace.

Ron Pace (6 July 1946 to 4 January 2021) was a scientist of deep intellectual pursuits, an eager debater of the laws of nature, and an admired teacher, whose generous character and humorous spirit was a gift to his colleagues, collaborators, and students.

Bridging the gap between Kok-type and kinetic models of photosynthetic electron transport within Photosystem II.

Historically, two modeling approaches have been developed independently to describe photosynthetic electron transport (PET) from water to plastoquinone within Photosystem II (PSII): Markov models account for losses from finite redox transition probabilities but predict no reaction kinetics, and ordinary differential equation (ODE) models account for kinetics but not for redox inefficiencies. We have developed an ODE mathematical framework to calculate Markov inefficiencies of transition probabilities as defined in Joliot-Kok-type catalytic cycles. We adapted a previously published ODE model for PET within PSII that accounts for 238 individual steps to enable calculation of the four photochemical inefficiency parameters (miss, double hit, inactivation, backward transition) and the four redox accumulation states (S-states) that are predicted by the most advanced of the Joliot-Kok-type models (VZAD). Using only reaction kinetic parameters without other assumptions, the RODE-calculated time-averaged (e.g., equilibrium) inefficiency parameters and equilibrium S-state populations agree with those calculated by time-independent Joliot-Kok models. RODE also predicts their time-dependent values during transient photochemical steps for all 96 microstates involving PSII redox cofactors. We illustrate applications to two cyanobacteria, Arthrospira maxima and Synechococcus sp. 7002, where experimental data exists for the inefficiency parameters and the S-state populations, and historical data for plant chloroplasts as benchmarks. Significant findings: RODE predicts the microstates responsible for period-4 and period-2 oscillations of O2 and fluorescence yields and the four inefficiency parameters; the latter parameters are not constant for each S state nor in time, in contrast to predictions from Joliot-Kok models; some of the recombination pathways that contribute to the backward transition parameter are identified and found to contribute when their rates exceed the oxidation rate of the terminal acceptor pool (PQH2); prior reports based on the assumptions of Joliot-Kok parameters may require reinterpretation.

Surface Hydrides on Fe2P Electrocatalyst Reduce CO2 at Low Overpotential: Steering Selectivity to Ethylene Glycol.

Development of efficient electrocatalysts for the CO2 reduction reaction (CO2RR) to multicarbon products has been constrained by high overpotentials and poor selectivity. Here, we introduce iron phosphide (Fe2P) as an earth-abundant catalyst for the CO2RR to mainly C2-C4 products with a total CO2RR Faradaic efficiency of 53% at 0 V vs RHE. Carbon product selectivity is tuned in favor of ethylene glycol formation with increasing negative bias at the expense of C3-C4 products. Both Grand Canonical-DFT (GC-DFT) calculations and experiments reveal that *formate, not *CO, is the initial intermediate formed from surface phosphino-hydrides and that the latter form ionic hydrides at both surface phosphorus atoms (H@Ps) and P-reconstructed Fe3 hollow sites (H@P*). Binding of these surface hydrides weakens with negative bias (reactivity increases), which accounts for both the shift to C2 products over higher C-C coupling products and the increase in the H2 evolution reaction (HER) rate. GC-DFT predicts that phosphino-hydrides convert *formate to *formaldehyde, the key intermediate for C-C coupling, whereas hydrogen atoms on Fe generate tightly bound *CO via sequential PCET reactions to H2O. GC-DFT predicts the peak in CO2RR current density near -0.1 V is due to a local maximum in the binding affinity of *formate and *formaldehyde at this bias, which together with the more labile C2 product affinity, accounts for the shift to ethylene glycol and away from C3-C4 products. Consistent with these predictions, addition of exogenous CO is shown to block all carbon product formation and lower the HER rate. These results demonstrate that the formation of ionic hydrides and their binding affinity, as modulated by the applied potential, controls the carbon product distribution. This knowledge provides new insight into the influence of hydride speciation and applied bias on the chemical reaction mechanism of CO2RR that is relevant to all transition metal phosphides.

Symbiosis extended: exchange of photosynthetic O2 and fungal-respired CO2 mutually power metabolism of lichen symbionts.

Lichens are a symbiosis between a fungus and one or more photosynthetic microorganisms that enables the symbionts to thrive in places and conditions they could not compete independently. Exchanges of water and sugars between the symbionts are the established mechanisms that support lichen symbiosis. Herein, we present a new linkage between algal photosynthesis and fungal respiration in lichen Flavoparmelia caperata that extends the physiological nature of symbiotic co-dependent metabolisms, mutually boosting energy conversion rates in both symbionts. Measurements of electron transport by oximetry show that photosynthetic O2 is consumed internally by fungal respiration. At low light intensity, very low levels of O2 are released, while photosynthetic electron transport from water oxidation is normal as shown by intrinsic chlorophyll variable fluorescence yield (period-4 oscillations in flash-induced Fv/Fm). The rate of algal O2 production increases following consecutive series of illumination periods, at low and with limited saturation at high light intensities, in contrast to light saturation in free-living algae. We attribute this effect to arise from the availability of more CO2 produced by fungal respiration of photosynthetically generated sugars. We conclude that the lichen symbionts are metabolically coupled by energy conversion through exchange of terminal electron donors and acceptors used in both photosynthesis and fungal respiration. Algal sugars and O2 are consumed by the fungal symbiont, while fungal delivered CO2 is consumed by the alga.

Water and vapor transport in algal-fungal lichen: Modeling constrained by laboratory experiments, an application for Flavoparmelia caperata.

Algal-fungal symbionts share water, nutrients, and gases via an architecture unique to lichens. Because lichen activity is controlled by moisture dynamics, understanding water transport is prerequisite to understand their fundamental biology. We propose a model of water distributions within foliose lichens governed by laws of fluid motion. Our model differentiates between water stored in symbionts, on extracellular surfaces, and in distinct morphological layers. We parameterize our model with hydraulic properties inverted from laboratory measurements of Flavoparmelia caperata and validate for wetting and drying. We ask: (1) Where is the bottleneck to water transport? (2) How do hydration and dehydration dynamics differ? and (3) What causes these differences? Resistance to vapor flow is concentrated at thallus surfaces and acts as the bottleneck for equilibrium, while internal resistances are small. The model captures hysteresis in hydration and desiccation, which are shown to be controlled by nonlinearities in hydraulic capacitance. Muting existing nonlinearities slowed drying and accelerated wetting, while exaggerating nonlinearities accelerated drying and slowed wetting. The hydraulic nonlinearity of F. caperata is considerable, which may reflect its preference for humid and stable environments. The model establishes the physical foundation for future investigations of transport of water, gas, and sugar between symbionts.

'Birth defects' of photosystem II make it highly susceptible to photodamage during chloroplast biogenesis.

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water-oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane-inlet mass spectrometry and O2 -polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these 'PSII birth defects' in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de-etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O2 -polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, QB -inhibitor binding, and thermoluminescence studies indicate that the decline of the high-light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability QA - → QB during de-etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer-range energy transfer.

Rewiring of Cyanobacterial Metabolism for Hydrogen Production: Synthetic Biology Approaches and Challenges.

With the demand for renewable energy growing, hydrogen (H2) is becoming an attractive energy carrier. Developing H2 production technologies with near-net zero carbon emissions is a major challenge for the "H2 economy." Certain cyanobacteria inherently possess enzymes, nitrogenases, and bidirectional hydrogenases that are capable of H2 evolution using sunlight, making them ideal cell factories for photocatalytic conversion of water to H2. With the advances in synthetic biology, cyanobacteria are currently being developed as a "plug and play" chassis to produce H2. This chapter describes the metabolic pathways involved and the theoretical limits to cyanobacterial H2 production and summarizes the metabolic engineering technologies pursued.

Photosystem II-cyclic electron flow powers exceptional photoprotection and record growth in the microalga Chlorella ohadii.

The desert microalga Chlorella ohadii was reported to grow at extreme light intensities with minimal photoinhibition, tolerate frequent de/re-hydrations, yet minimally employs antenna-based non-photochemical quenching for photoprotection. Here we investigate the molecular mechanisms by measuring Photosystem II charge separation yield (chlorophyll variable fluorescence, Fv/Fm) and flash-induced O2 yield to measure the contributions from both linear (PSII-LEF) and cyclic (PSII-CEF) electron flow within PSII. Cells grow increasingly faster at higher light intensities (µE/m2/s) from low (20) to high (200) to extreme (2000) by escalating photoprotection via shifting from PSII-LEF to PSII-CEF. This shifts PSII charge separation from plastoquinone reduction (PSII-LEF) to plastoquinol oxidation (PSII-CEF), here postulated to enable proton gradient and ATP generation that powers photoprotection. Low light-grown cells have unusually small antennae (332 Chl/PSII), use mainly PSII-LEF (95%) and convert 40% of PSII charge separations into O2 (a high O2 quantum yield of 0.06mol/mol PSII/flash). High light-grown cells have smaller antenna and lower PSII-LEF (63%). Extreme light-grown cells have only 42 Chl/PSII (no LHCII antenna), minimal PSII-LEF (10%), and grow faster than any known phototroph (doubling time 1.3h). Adding a synthetic quinone in excess to supplement the PQ pool fully uncouples PSII-CEF from its natural regulation and produces maximum PSII-LEF. Upon dark adaptation PSII-LEF rapidly reverts to PSII-CEF, a transient protection mechanism to conserve water and minimize the cost of antenna biosynthesis. The capacity of the electron acceptor pool (plastoquinone pool), and the characteristic times for exchange of (PQH2)B with PQpool and reoxidation of (PQH2)pool were determined.

Flux balance analysis of photoautotrophic metabolism: Uncovering new biological details of subsystems involved in cyanobacterial photosynthesis.

We have constructed and experimentally tested a comprehensive genome-scale model of photoautotrophic growth, denoted iSyp821, for the cyanobacterium Synechococcus sp. PCC 7002. iSyp821 incorporates a variable biomass objective function (vBOF), in which stoichiometries of the major biomass components vary according to light intensity. The vBOF was constrained to fit the measured cellular carbohydrate/protein content under different light intensities. iSyp821 provides rigorous agreement with experimentally measured cell growth rates and inorganic carbon uptake rates as a function of light intensity. iSyp821 predicts two observed metabolic transitions that occur as light intensity increases: 1) from PSI-cyclic to linear electron flow (greater redox energy), and 2) from carbon allocation as proteins (growth) to carbohydrates (energy storage) mode. iSyp821 predicts photoautotrophic carbon flux into 1) a hybrid gluconeogenesis-pentose phosphate (PP) pathway that produces glycogen by an alternative pathway than conventional gluconeogenesis, and 2) the photorespiration pathway to synthesize the essential amino acid, glycine. Quantitative fluxes through both pathways were verified experimentally by following the kinetics of formation of 13C metabolites from 13CO2 fixation. iSyp821 was modified to include changes in gene products (enzymes) from experimentally measured transcriptomic data and applied to estimate changes in concentrations of metabolites arising from nutrient stress. Using this strategy, we found that iSyp821 correctly predicts the observed redistribution pattern of carbon products under nitrogen depletion, including decreased rates of CO2 uptake, amino acid synthesis, and increased rates of glycogen and lipid synthesis.

The strontium inorganic mutant of the water oxidizing center (CaMn4O5) of PSII improves WOC efficiency but slows electron flux through the terminal acceptors.

Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5-8× slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45°C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S=5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S=1/2 form.

The Oxygen quantum yield in diverse algae and cyanobacteria is controlled by partitioning of flux between linear and cyclic electron flow within photosystem II.

We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB(-) and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB(-) acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.

Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.

Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.

To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations--both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))--were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.

Water Oxidation by the [Co4O4(OAc)4(py)4](+) Cubium is Initiated by OH(-) Addition.

The cobalt cubium Co4O4(OAc)4(py)4(ClO4) (1A(+)) containing the mixed valence [Co4O4](5+) core is shown by multiple spectroscopic methods to react with hydroxide (OH(-)) but not with water molecules to produce O2. The yield of reaction products is stoichiometric (>99.5%): 41A(+) + 4OH(-) → O2 + 2H2O + 41A. By contrast, the structurally homologous cubium Co4O4(trans-OAc)2(bpy)4(ClO4)3, 1B(ClO4)3, produces no O2. EPR/NMR spectroscopies show clean conversion to cubane 1A during O2 evolution with no Co(2+) or Co3O4 side products. Mass spectrometry of the reaction between isotopically labeled µ-(16)O(bridging-oxo) 1A(+) and (18)O-bicarbonate/water shows (1) no exchange of (18)O into the bridging oxos of 1A(+), and (2) (36)O2 is the major product, thus requiring two OH(-) in the reactive intermediate. DFT calculations of solvated intermediates suggest that addition of two OH(-) to 1A(+) via OH(-) insertion into Co-OAc bonds is energetically favored, followed by outer-sphere oxidation to intermediate [1A(OH)2](0). The absence of O2 production by cubium 1B(3+) indicates the reactive intermediate derived from 1A(+) requires gem-1,1-dihydoxo stereochemistry to perform O-O bond formation. Outer-sphere oxidation of this intermediate by 2 equiv of 1A(+) accounts for the final stoichiometry. Collectively, these results and recent literature (Faraday Discuss., doi:10.1039/C5FD00076A and J. Am. Chem. Soc. 2015, 137, 12865-12872) validate the [Co4O4](4+/5+) cubane core as an intrinsic catalyst for oxidation of hydroxide by an inner-sphere mechanism.