Bicarbonate activation of the monomeric photosystem II-PsbS/Psb27 complex.

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.

Near ambient N2 fixation on solid electrodes versus enzymes and homogeneous catalysts.

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 revolutionize fertilizer production and even provide an energy-dense, truly carbon-free fuel. This Review provides a coherent comparison of recent progress made in dinitrogen fixation on solid electrodes, homogeneous catalysts and 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.

Impact of energy limitations on function and resilience in long-wavelength Photosystem II.

Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two types of cyanobacterial PSII can use chlorophyll d (Chl-d) and chlorophyll f (Chl-f) to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII, and Chl-a-PSII. We show that: (i) all types of PSII have a comparable efficiency in enzyme turnover; (ii) the modified energy gaps on the acceptor side of Chl-d-PSII favour recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; (iii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favouring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.

Algae, plants and cyanobacteria perform a process called photosynthesis, in which carbon dioxide and water are converted into oxygen and energy-rich carbon compounds. The first step of this process involves an enzyme called photosystem II, which uses light energy to extract electrons from water to help capture the carbon dioxide. If the photosystem absorbs too much light, compounds known as reactive oxygen species are produced in quantities that damage the photosystem and kill the cell. To ensure that the photosystem works efficiently and to protect it from damage, about half of the energy from the absorbed light is dissipated as heat, while the rest of the energy is stored in the products of photosynthesis. The standard form of photosystem II uses the energy of visible light, but some cyanobacteria contain different types of photosystem II, which do the same chemical reactions using lower energy far-red light. One type of far-red photosystem II is found in Acaryochloris marina, a cyanobacterium living in stable levels of far-red light, shaded from visible light. The other type is found in a cyanobacterium called Chroococcidiopsis thermalis, which can switch between using its far-red photosystem II when shaded from visible light and using its standard photosystem II when exposed to it. Being able to work with less energy, the two types of far-red photosystem II appear to be more efficient than the standard one, but it has been unclear if there were any downsides to this trait. Viola et al. compared the standard photosystem II with the far-red photosystem II types from C. thermalis and A. marina by measuring the efficiency of these enzymes, the quantity of reactive oxygen species produced, and the resulting light-induced damage. The experiments revealed that the far-red photosystem II of A. marina is highly efficient but produces elevated levels of reactive oxygen species if exposed to high light conditions. On the other hand, the far-red photosystem II of C. thermalis is less efficient in collecting and using far-red light, but is more robust, producing fewer reactive oxygen species. Despite these tradeoffs, engineering crop plants or algae that could use far-red photosynthesis may help boost food and biomass production. A better understanding of the trade-offs between efficiency and resilience in the two types of far-red photosystem II could determine which features would be beneficial, and under what conditions. This work also improves our knowledge of how the standard photosystem II balances light absorption and damage limitation to work efficiently in a variable environment.

Bicarbonate-controlled reduction of oxygen by the QA semiquinone in Photosystem II in membranes.

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 O2, forming superoxide, but this reaction has not yet been demonstrated experimentally. Here, using chlorophyll fluorescence in plant PSII membranes, we show that O2 does oxidize [Formula: see text] at physiological O2 concentrations with a t1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O2 to a binding site near [Formula: see text], with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, [Formula: see text] could only reduce O2 when bicarbonate was absent from its binding site on the nonheme iron (Fe2+) and the addition of bicarbonate or formate blocked the O2-dependant decay of [Formula: see text] These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from [Formula: see text] to O2 occurs when the O2 is bound to the empty bicarbonate site on Fe2+ A protective role for bicarbonate in PSII was recently reported, involving long-lived [Formula: see text] triggering bicarbonate dissociation from Fe2+ [Brinkert et al, Proc. Natl. Acad. Sci. U.S.A. 113, 12144-12149 (2016)]. The present findings extend this mechanism by showing that bicarbonate release allows O2 to bind to Fe2+ and to oxidize [Formula: see text] This could be beneficial by oxidizing [Formula: see text] and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.

Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II.

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.

Bicarbonate-Mediated CO2 Formation on Both Sides of Photosystem II.

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 QA quinone leads to the release of HCO3- from its binding site on the non-heme iron and (ii) the Em potential of the QA/QA•- couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the Em of QA decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with 13C 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.

The primary donor of far-red photosystem II: ChlD1 or 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 ±â€¯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.

Energetics of the exchangeable quinone, QB, in Photosystem II.

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH2), is released into the membrane from the QB site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the QB site. Despite their functional importance, the thermodynamic properties of the PQ in the QB site, QB, in its different redox forms have received relatively little attention. Here we report the midpoint potentials (Em ) of QB in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: Em QB/QB•- ≈ 90 mV, and Em QB•-/QBH2 ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, QB•-, is stabilized thermodynamically; 2) the resulting Em QB/QBH2 (∼65 mV) is lower than the Em PQ/PQH2 (∼117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for QBH2 release into the pool; 3) PQ is ∼50× more tightly bound than PQH2; and 4) the difference between the Em QB/QB•- measured here and the Em QA/QA•- from the literature is ∼234 meV, in principle corresponding to the driving force for electron transfer from QA•- to QB The pH dependence of the thermoluminescence associated with QB•- 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 QB in PSII are comparable to those in the homologous purple bacterial reaction center.

A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii.

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.