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In thylakoid membranes, photosystem II (PSII) monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while PSII monomers (PSIIm) from granal regions lack these subunits. Here, we have isolated and characterized these 2 types of PSII complexes in tobacco (Nicotiana tabacum). PSIIm-S/27 showed enhanced fluorescence, the near absence of oxygen evolution, and limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•− couple, which controls the charge recombination route, and this limits chlorophyll triplet-mediated 1O2 formation. These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit using a bicarbonate-mediated switch and protective mechanism.

The Mo/Fe nitrogenase enzyme is unique in its ability to efficiently reduce dinitrogen to ammonia at atmospheric pressures and room temperature. Should an artificial electrolytic device achieve the same feat, it would revolutionise fertilizers and even provide an energy dense, truly carbon-free fuel. This Review provides a coherent comparison of recent progress made in dinitrogen fixation on (i) solid electrodes, (ii) homogeneous catalysts and (iii) nitrogenases. Specific emphasis is placed on systems for which there is unequivocal evidence that dinitrogen reduction has taken place. By establishing the cross-cutting themes and synergies between these systems, we identify viable avenues for future research.

In thylakoid membranes, Photosystem II monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while Photosystem II monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes. The PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to the PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for the bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•− couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation (Brinkert K et al. (2016) Proc Natl Acad Sci U S A. 113(43):12144-12149). These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit, by using a bicarbonate-mediated switch and protective mechanism.One sentence summaryA photosystem II monomer with PsbS and Psb27 as additional subunits, is inactive as isolated but activated by bicarbonate, and is attributed to be a late-stage intermediate in photoassembly.

Significance In Photosystem II (PSII), O 2 reduction by Q A • − is often discussed but has not been demonstrated. Here, we show in PSII membranes that Q A • − can reduce O 2 to superoxide, but only when bicarbonate is absent from its binding site on the nonheme Fe 2+ . Bicarbonate’s role in PSII was recently shown to involve a regulatory/protective redox-tuning mechanism linking PSII function to CO 2 concentration. A key aspect is the presence of stable Q A • − causing release of bicarbonate from its site on Fe 2+ . Here, we show that under these conditions, O 2 binds to the empty site on the Fe 2+ and is reduced by Q A • − . This unexpected reaction may be a further indication of cross-talk between the regulation of PSII and CO 2 fixation.

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. [Formula: see text], the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O

Significance The E m values of the 2 Q B redox couples, E m (Q B /Q B •− ) and E m (Q B •− /Q B H 2 ), in Photosystem II are fundamental for understanding the enzyme function, but they remain vague. Using EPR signals arising from the semiquinone, Q B •− , we have now measured both redox couples. The semiquinone is thermodynamically stable and has a relatively high potential. This minimizes back-reactions and electrons leaking onto O 2 , explaining the remarkable stability of Q B •− . The release of Q B H 2 is thermodynamically favorable, and the binding of the substrate PQ is greatly favored over the product PQH 2 : This optimizes PSII function in the presence of a largely reduced plastoquinone pool. This work corrects and explains a recent report of anomalous values for the Q B redox couples.

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a. We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d. The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210–1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm−1 electrochromic shift due to formation of the semiquinone anion, QA•-. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: PD1, PD2 ChlD1, ChlD2, PheoD1 and PheoD2. Two of these chlorins, ChlD1 and PD2, are located at a distance and orientation relative to QA•- so as to account for the observed electrochromic shift. Previously, ChlD1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that PD2 is at least as likely as ChlD1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors PD2 as the primary donor. The pros and cons of PD2 vs ChlD1 as the location of the FRL-primary donor are discussed.TOC GraphicHighlightsPrimary Donor confirmed at 726 nmDetermination of far-red chl pigment Qy excitation positions, widths, CD and MCD amplitudesQuantification of electrochromic shifts and QA•- photoconversion yieldElectrochromic shift consistent with primary donor at either ChlD1 or PD2The CD amplitude favors the primary donor at PD2

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH(2)), is released into the membrane from the Q(B) site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the Q(B) site. Despite their functional importance, the thermodynamic properties of the PQ in the Q(B) site, Q(B), in its different redox forms have received relatively little attention. Here we report the midpoint potentials (E(m)) of Q(B) in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: E(m) Q(B)/Q(B)(•−) ≈ 90 mV, and E(m) Q(B)(•−)/Q(B)H(2) ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, Q(B)(•−), is stabilized thermodynamically; 2) the resulting E(m) Q(B)/Q(B)H(2) (∼65 mV) is lower than the E(m) PQ/PQH(2) (∼117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for Q(B)H(2) release into the pool; 3) PQ is ∼50× more tightly bound than PQH(2); and 4) the difference between the E(m) Q(B)/Q(B)(•−) measured here and the E(m) Q(A)/Q(A)(•−) from the literature is ∼234 meV, in principle corresponding to the driving force for electron transfer from Q(A)(•−) to Q(B). The pH dependence of the thermoluminescence associated with Q(B)(•−) provided a functional estimate for this energy gap and gave a similar value (≥180 meV). These estimates are larger than the generally accepted value (∼70 meV), and this is discussed. The energetics of Q(B) in PSII are comparable to those in the homologous purple bacterial reaction center.

The effect of bicarbonate (HCO3-) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO3- enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO2 and H2O. The other is for the electron-acceptor side, in which (i) reduction of the Q(A) quinone leads to the release of HCO(3)(- )from its binding site on the non-heme iron and (ii) the E-m potential of the Q(A)/Q(.-) couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the E-m of Q(A) decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with C-13 isotope labeling of HCO3- allows donor- and acceptor-side formation of CO2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor Q(A) to a more positive potential and lowers the yield of singlet oxygen ((1)O(2)) production. The presence of Q(A)(−) results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against (1)O(2) in vivo under photorespiratory conditions. Here, we compared the properties of Q(A) in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between Q(A) and Q(B) in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of Q(A) to a more positive value. The yield of (1)O(2) production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of Q(A)/Q(A)(−). In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO(2) conditions.

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least-studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of −420 ± 10 and −330 ± 10 mV for the two FAD potentials and −340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

Protein film photoelectrochemistry has previously been used to monitor the activity of photosystem II, the water-plastoquinone photooxidoreductase, but the mechanistic information attainable from a three-electrode setup has remained limited. Here we introduce the four-electrode rotating ring disk electrode technique for quantifying light-driven reaction kinetics and mechanistic pathways in real time at the enzyme–electrode interface. This setup allows us to study photochemical H2O oxidation in photosystem II and to gain an in-depth understanding of pathways that generate reactive oxygen species. The results show that photosystem II reacts with O2 through two main pathways that both involve a superoxide intermediate to produce H2O2. The first pathway involves the established chlorophyll triplet-mediated formation of singlet oxygen, which is followed by its reduction to superoxide at the electrode surface. The second pathway is specific for the enzyme/electrode interface: an exposed antenna chlorophyll is sufficiently close to the electrode for rapid injection of an electron to form a highly reducing chlorophyll anion, which reacts with O2 in solution to produce O2•–. Incomplete H2O oxidation does not significantly contribute to reactive oxygen formation in our conditions. The rotating ring disk electrode technique allows the chemical reactivity of photosystem II to be studied electrochemically and opens several avenues for future investigation.

Factors governing the photoelectrochemical output of photosynthetic microorganisms are poorly understood, and energy loss may occur due to inefficient electron transfer (ET) processes. Here, we systematically compare the photoelectrochemistry of photosystem II (PSII) protein-films to cyanobacteria biofilms to derive: (i) the losses in light-to-charge conversion efficiencies, (ii) gains in photocatalytic longevity, and (iii) insights into the ET mechanism at the biofilm interface. This study was enabled by the use of hierarchically structured electrodes, which could be tailored for high/stable loadings of PSII core complexes and Synechocystis sp. PCC 6803 cells. The mediated photocurrent densities generated by the biofilm were 2 orders of magnitude lower than those of the protein-film. This was partly attributed to a lower photocatalyst loading as the rate of mediated electron extraction from PSII in vitro is only double that of PSII in vivo. On the other hand, the biofilm exhibited much greater longevity (>5 days) than the protein-film (

Significance The E m of Q A / Q A − • is the reference pegging the thermodynamics of Photosystem II (PSII) to an absolute value. This work resolves a long-standing discrepancy in the literature, removing this ambiguity at the heart of PSII bioenergetics. The discrepancy in the literature reflects the loss of bicarbonate in some titrations. This surprising finding shows that bicarbonate binding controls the E m of Q A / Q A − • , and thus the redox state of Q A influences the binding of bicarbonate. This process constitutes a previously unrecognized regulation of PSII: Q A − • triggers bicarbonate release, slowing electron transfer and tuning the potential of Q A to protect PSII against photodamage. This work provides an explanation for the existence of bicarbonate in PSII, a mystery for more than half a century.

THE WORLD ENERGY PROBLEM AND THE BIOFUEL ENERGY PROBLEM Oxygenic photosynthesis has been promoted as a system for fuel production on a global scale to replace fossil fuels. The fundamental requirement for this to be viable is that the energy output of the system must be greater than the energy input from fossil fuels. For biofuel production, this criterion is not always met. This issue is often dodged because life-cycle analyses are complex (and thus disputed) and future technological innovations can always be invoked. The second requirement is a sufficiently high rate of solar energy conversion to make the process feasible in terms of the time and space needed to produce fuel on a relevant scale. Both requirements are closely linked to the photosynthetic efficiency; i.e., the conversion efficiency of solar energy to organic material (sugar, biomass, hydrocarbons, etc.). Here, we discuss limitations on photosynthetic efficiency and approaches suggested to overcome them. We focus on biofuels produced by photosynthetic microbes as they are often considered the fuels of the future for their year-round cultivation, non-competition with food crops, higher reported photosynthetic yields, and the potential for genetic engineering to produce fuels directly (Brennan and Owende, 2010). The energy investment required for biomass production (e.g., water, nutrients, fertilizers, stirring, bubbling, containment, harvesting, processing) cancels out some or all of the energy gained from sunlight (Slade and Bauen, 2013). This is described by the energy returned as a proportion of energy invested (EROI), and this factor is the key measure of energy sustainability in life-cycle analysis (Murphy and Hall, 2010). If the EROI is >1, the system produces a fuel with net solar energy content; if 1 and a high rate of solar energy conversion) on a pilot scale seems advisable before scaling-up is considered. The energetic prerequisites rely fundamentally on the efficiency of photosynthesis. Calculations for theoretical photosynthetic efficiency agree on a maximum value for solar energy to carbon–carbon bonds in glucose of around 13%, falling to around 5% of solar energy to biomass for C3 plants, considering photorespiratory and respiratory losses (Zhu et al., 2010). The highest efficiency reported for photosynthetic microbes under controlled lab conditions is 3% for light-to-biomass [Melis, 2009; also Cuaresma et al. (2009)]. Under growth conditions more relevant to industrial settings, the efficiency is stated to be significantly lower than this (Melis, 2009). Efforts are thus being made to find ways of improving photosynthesis itself.