A dinuclear manganese complex {[(Mn2L)-L-II,IIII(mu-OAc)(2)]-ClO4} has been synthesised, where L is the dianion of 2-{[bis-(pyrid-2-ylmethyl)amino]methyl}-6-{[(3,5-di-tert-butyl-2- hydroxybenzyl)(pyrid-2-ylmethyl)amino]methyl)-4-methylphenol, an unsymmetric binucleating ligand with two coordinating phenol groups. The two manganese ions, with a Mn-Mn distance of 3.498 angstrom, are bridged by the two bidentate acetate ligands and the 4-methylphenolate group of the ligand, resulting in a N3O3 and N2O4 donor set of Mn-II and Mn-II, respectively. Electrochemically [Mn2(II,III)L(mu-OAc)(2)](+) is reduced to [(Mn2L)-L-II,II(mu-OAc)(2)] at E-1/2(1)=-0.53 V versus Fc(+/0) and oxidised to [(Mn2L)-L-III,III(mu-OAC)(2)](2+) at E-1/2(2)=0.38 V versus Fc(+/0). All three redox states have been characterised by EPR, IR and UV/Vis spectroscopy. Subsequent oxidation of [(Mn2L)-L-II,III(mu-OAc)(2)](2+) [E-1/2(3)=0.75 V vs. Fc(+/0)] in dry acetonitrile results in an unstable primary product with a lifetime of about 100 ins. At high scan rates quasireversible voltammetric behaviour is found for all three electrode processes, with particularly slow electron transfer for the II,III/II,II [k(o)(1) = 0.002 cms(-1) and III,III/II,III [k(o)(2) = 0.005 cms(-1)] couples, which can be rationalised in terms of major distortions of the Mn-II centres. In aqueous media the bridging acetates are replaced by water-derived ligands. Deprotonation of these stabilises higher valence states, and photo-induced oxidation of the manganese complex results in a (Mn2L)-L-IlI,IV complex with oxo or hydroxo bridging ligands, which is further oxidised to an EPR-silent product. These results demonstrate that a larger number of metal-centred oxidations can be compressed in a narrow potential range if build up of charge is avoided by charge-compensating reactions.
Nostoc punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc. A protocol for isolating thylakoid membranes was developed to examine the biochem. and biophys. aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N. punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analyzed by measuring oxygen evolution activity, fluorescence spectroscopy and ESR spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centers were active in water oxidn. in the final membrane prepn. Anal. of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b6/f could be obsd. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.
Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes.We could identify asignificant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled PhotosystemII complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.
Cyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N. punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708 nm) or phycobilisomes (560. nm) specifically. In heterocysts that were pre-illuminated at 708. nm, fluorescence from the phycobilisome terminal emitter was observed in the 77 K emission spectrum. However, illumination with 560. nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750 nm, indicating that the 560 nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560 nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708 nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity.
To model the structural and functional parts of the water oxidizing complex in Photosystem 11, a dimeric manganese(II,11) complex (1) was linked to a ruthenium(II)tris-bipyridine (Ru-II(bpy)3) complex via a substituted L-tyrosine, to form the trinuclear complex 2 [J. Inorg. Biochem. 78 (2000) 15]. Flash photolysis of 1 and Ru-II(bpy), in aqueous solution, in the presence of an electron acceptor, resulted in the stepwise extraction of three electrons by Ru-III(bpy), from the Mn-2(II,II) dimer, which then attained the Mn-2(III,IV) oxidation state. In a similar experiment with compound 2, the dinuclear Mn complex reduced the photo-oxidized Ru moiety via intramolecular electron transfer on each photochemical event. From EPR it was seen that 2 also reached the Mn-2(III,IV) state. Our data indicate that oxidation from the Mn-2(II,II) state proceeds stepwise via intermediate formation of Mn-2(II,III) and Mn-2(III,III). In the presence of water, cyclic voltammetry showed an additional anodic peak beyond Mn-2(II,III/III,III) oxidation which was significantly lower than in neat acetonitrile. Assuming that this peak is due to oxidation to Mn-2(III,IV), this suggests that water is essential for the formation of the Mn-2(III,IV) oxidation state. Compound 2 is a structural mimic of the water oxidizing complex, in that it links a Mn complex via a tyrosine to a highly oxidizing photosensitizer. Complex 2 also mimics mechanistic aspects of Photosystem 11, in that the electron transfer to the photosensitizer is fast and results in several electron extractions from the Mn moiety. (C) 2002 Elsevier Science Inc. All rights reserved.
The preparation of donor (D)-photosensitizer (S) arrays, consisting of a manganese complex as D and a ruthenium tris(bipyridyl) complex as S has been pursued. Two new ruthenium complexes containing coordinating sites for one (2a) and two manganese ions (3a) were prepared in order to provide models for the donor side of photosystem II in green plants. The manganese coordinating site consists of bridging and terminal phenolate as well as terminal pyridyl ligands. The corresponding ruthenium-manganese complexes, a manganese monomer 2b and dimer 3b, were obtained. For the dimer 3b, our data suggest that intramolecular electron transfer from manganese to photogenerated ruthenium(III) is fast, k(ET) > 5 x 10(7) s(-1).
The filamentous cyanobacterium Nostoc punctiforme has several oxidative stress-managing systems, including Dps proteins. Dps proteins belong to the ferritin superfamily and are involved in abiotic stress management in prokaryotes. Previously, we found that one of the five Dps proteins in N. punctiforme, NpDps2, was critical for H2O2 tolerance. Stress induced by high light intensities is aggravated in N. punctiforme strains deficient of either NpDps2, or the bacterioferritin-like NpDps5. Here, we have investigated the capacity of NpDps2 and NpDps5 to enhance stress tolerance by homologous overexpression of these two proteins in N. punctiforme. Both overexpression strains were found to tolerate twice as high concentrations of added H2O2 as the control strain, indicating that overexpression of either NpDps2 or NpDps5 will enhance the capacity for H2O2 tolerance. Under high light intensities, the overexpression of the two NpDps did not enhance the tolerance against general light-induced stress. However, overexpression of the heterocyst-specific NpDps5 in all cells of the filament led to a higher amount of chlorophyll-binding proteins per cell during diazotrophic growth. The OENpDps5 strain also showed an increased tolerance to ammonium-induced oxidative stress. Our results provide information of how Dps proteins may be utilised for engineering of cyanobacteria with enhanced stress tolerance.
Heterocysts are specialized cells that differentiate in the filaments of heterocystous cyanobacteria. Their role is to maintain a microoxic environment for the nitrogenase enzyme during diazotrophic growth. The lack of photosynthetic water oxidation in the heterocyst puts special constraints on the energetics for nitrogen fixation, and the electron transport pathways of heterocyst thylakoids are slightly different from those in vegetative cells. During recent years, there has been a growing interest in utilizing heterocysts as cell factories for the production of fuels and other chemical commodities. Optimization of these production systems requires some consideration of the bioenergetics behind nitrogen fixation. In this overview, we emphasize the role of photosynthetic electron transport in providing ATP and reductants to the nitrogenase enzyme, and provide some examples where heterocysts have been used as production facilities.
Photosynthesis is performed by a multitude of organisms, but in P nearly all cases, it is variations on a common theme: absorption of light followed by energy transfer to a reaction center where charge separation takes place, This initial form of chemical energy is stabilized by the biosynthesis of carbohydrates. To produce these energy-rich products, a substrate is needed that feeds in reductive equivalents, When photosynthetic microorganisms learned to use water as a substrate some 2 billion years ago, a fundamental barrier against unlimited use of solar energy was overcome. The possibility of solar energy use has inspired researchers to construct artificial photosynthetic systems that show analogy to parts of the intricate molecular machinery of photosynthesis. Recent years have seen a reorientation of efforts toward creating integrated light-to-fuel systems that can use solar energy for direct synthesis of energy-rich compounds, so-called solar fuels. Sustainable production of solar fuels is a long awaited development that promises extensive solar energy use combined with long-term storage. The stoichiometry of water splitting into molecular oxygen, protons, and electrons is deceptively simple; achieving it by chemical catalysis has proven remarkably difficult. The reaction center Photosystem II couples light-induced charge separation to an efficient molecular water-splitting catalyst, a Mn4Ca complex, and is thus an important template for biomimetic chemistry. In our aims to design biomimetic manganese complexes for light-driven water oxidation, we link photosensitizers and charge-separation motifs to potential catalysts in supramolecular assemblies. In photosynthesis, production of carbohydrates demands the delivery of multiple reducing equivalents to CO2. In contrast, the two-electron reduction of protons to molecular hydrogen is much less demanding. Virtually all microorganisms have enzymes called hydrogenases that convert protons to hydrogen, many of them with good catalytic efficiency. The catalytic sites of hydrogenases are now the center of attention of biomimetic efforts, providing prospects for catalytic hydrogen production with inexpensive metals. Thus, we might complete the water-to-fuel conversion: light + 2H(2)O -> 2H(2) + O-2 This reaction formula is to some extent already elegantly fulfilled by cyanobacteria and green algae, water-splitting photosynthetic microorganisms that under certain conditions also can produce hydrogen. An alternative route to hydrogen from solar energy is therefore to engineer these organisms to produce hydrogen more efficiently. This Account describes our original approach to combine research in these two fields: mimicking structural and functional principles of both Photosystem II and hydrogenases by synthetic chemistry and engineering cyanobacteria to become better hydrogen producers and ultimately developing new routes toward synthetic biology.
In the natural photosynthetic reaction center photosystem II, absorption of a photon leads to photooxidationof the primary electron donor P680, which subsequently retrieves electrons from a tyrosyl residue, functioning as aninterface to the oxygen-evolving manganese complex. In a first step toward mimicking these reactions, we havemade a Ru(II)-polypyridine complex with an attached tyrosyl moiety. The photoexcited ruthenium complex playedthe role of P680and was first oxidized by external acceptors. Combined transient absorbance and EPR studies provided evidence that the Ru(III) formed was reduced by intramolecular electron transfer from the attached tyrosine, with a rate constant of 5104s-1. Thus we show that a tyrosine radical could be formed by light-induced electrontransfer reactions, and we indicate future directions for developing a closer analogy with the photosystem II reactions.
Multicellular cyanobacteria form different cell types in response to environmental stimuli. Under nitrogen limiting conditions a fraction of the vegetative cells in the filament differentiate into heterocysts. Heterocysts are specialized in atmospheric nitrogen fixation and differentiation involves drastic morphological changes on the cellular level, such as reorganization of the thylakoid membranes and differential expression of thylakoid membrane proteins. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase by developing an extra polysaccharide layer that limits air diffusion into the heterocyst and by upregulating heterocyst-specific respiratory enzymes. In this review article, we summarize what is known about the thylakoid membrane in heterocysts and compare its function with that of the vegetative cells. We emphasize the role of photosynthetic electron transport in providing the required amounts of ATP and reductants to the nitrogenase enzyme. In the light of recent high-throughput proteomic and transcriptomic data, as well as recently discovered electron transfer pathways in cyanobacteria, our aim is to broaden current views of the bioenergetics of heterocysts. This article is part of a Special Issue entitled: Bioenergetic systems in bacteria.
In plants, solar energy is used to extract electrons from water, producing atmospheric oxygen. This is conducted by Photosystem II, where a redox ”triad” consisting of chlorophyll, a tyrosine, and a manganese cluster, governs an essential part of the process. Photooxidation of the chlorophylls produces electron transfer from the tyrosine, which forms a radical. The radical and the manganese cluster together extract electrons from water, providing the biosphere with an unlimited electron source. As a partial model for this system we constructed a ruthenium(II) complex with a covalently attached tyrosine, where the photooxidized ruthenium was rereduced by the tyrosine. In this study we show that the tyrosyl radical, which gives a transient EPR signal under illumination, can oxidize a manganese complex. The dinuclear manganese complex, which initially is in the Mn(III)/(III) state, is oxidized by the photogenerated tyrosyl radical to the Mn(III)/(IV) state. The redox potentials in our system are comparable to those in Photosystem II. Thus, our synthetic redox “triad” mimics important elements in the electron donor ”triad” in Photosystem II, significantly advancing the development of systems for artificial photosynthesis based on ruthenium−manganese complexes.
Five nitrogen fixing cyanobacterial strains have been found to contain PsaB2, an additional and divergent gene copy for the Photosystem I reaction center protein PsaB. In all five species the divergent gene, psaB2, is located separately from the normal psaAB operon in the genome. The protein, PsaB2, was recently identified in heterocysts of Nostoc punctiforme sp. strain PCC 73102. 12 conserved amino acid replacements and one insertion, were identified by a multiple sequence alignment of several PsaB2 and PsaB1 sequences. Several, including an inserted glutamine, are located close to the iron-sulfur cluster F(x) in the electron transfer chain. By homology modeling, using the Photosystem I crystal structure as template, we have found that the amino acid composition in PsaB2 will introduce changes in critical parts of the Photosystem I protein structure. The changes are close to F(x) and the phylloquinone (PhQ) in the B-branch, indicating that the electron transfer properties most likely will be affected. We suggest that the divergent PsaB2 protein produces an alternative Photosystem I reaction center with different structural and electron transfer properties. Some interesting physiologcial consequences that this can have for the function of Photosystem I in heterocysts, are discussed.
Molecular hydrogen, H-2, is an energy carrier that is increasing in popularity as an alternative fuel. Presently, the major part of commercially available H-2 is produced from fossil resources. To make H-2 a true contender to fossil fuels, a sustainable production via water splitting by renewable electricity in combination with earth-abundant catalysts or by photosynthetic microorganisms is required.
The world needs new, environmentally friendly, and renewable fuels to exchange for fossil fuels. The fuel must be made from cheap, abundant, and renewable resources. The research area of solar fuels aims to meet this demand. This paper discusses why we need a solar fuel, and proposes solar energy as the major renewable energy source to feed from. The scientific field concerning artificial photosynthesis is expanding rapidly and most of the different scientific visions for solar fuels are briefly reviewed. Research strategies for the development of artificial photosynthesis to produce solar fuels are overviewed, with some critical concepts discussed in closer detail.
Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiologica characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance,H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.
[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H(2)gas production. H(2)formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase inin vitroassays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly,i.e.in binding and delivering the precatalyst.
[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt =NH(CH2S)(2)).[FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe](adt)). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe](adt). The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred'-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster. [GRAPHICS] .
The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.
In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands similar to 340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum ofthe oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = 1/2) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = 1/2) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties ofthe iron-sulfur clusters of HupS.
The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis.
The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 produces hydrogen via nitrogenase in heterocysts upon onset of nitrogen-fixing conditions. N. punctiforme expresses concomitantly the uptake hydrogenase HupSL, which oxidizes hydrogen in an effort to recover some of the reducing power used up by nitrogenase. Eliminating uptake activity has been employed as a strategy for net hydrogen production in N. punctiforme (Lindberg et al., Int. J. Hydrogen Energy, 2002, 27, 1291-1296). However, nitrogenase activity wanes within a few days. In the present work, we modify the proximal iron-sulfur cluster in the hydrogenase small subunit HupS by introducing the designed mutation C12P in the fusion protein f-HupS for expression in E. coli (Raleiras et al., J. Biol. Chem., 2013, 288, 18345-18352), and in the full HupSL enzyme for expression in N. punctiforme. C12P f-HupS was investigated by EPR spectroscopy and found to form a new paramagnetic species at the proximal cluster site consistent with a [4Fe-4S] to [3Fe-4S] cluster conversion. The new cluster has the features of an unprecedented mixed-coordination [3Fe-4S] metal center. The mutation was found to produce stable protein in vitro, in silico and in vivo. When C12P HupSL was expressed in N. punctiforme, the strain had a consistently higher hydrogen production than the background [capital Delta]hupSL mutant. We conclude that the increase in hydrogen production is due to the modification of the proximal iron-sulfur cluster in HupS, leading to a turn of the electron flow in the enzyme.
Photoactivation of photosystem II has been studied in the FUD 39 mutant of Chlamydomonas reinhardtii that lacks the 23 kDa extrinsic subunit of photosystem II. We have taken advantage of the slow photoactivation rate of FUD 39, earlier demonstrated in Rova, E. M., et al. [(1996) J. Biol. Chem. 271, 28918-28924], to study events in photosystem II during intermediate stages of the process. By measuring the EPR multiline signal, the decay of the variable fluorescence after single flashes, and electron transfer from water to the Q(B) site, we found a good correlation between the building of a tetrameric Mn cluster, longer recombination times between Q(A)(-) and the donor side of photosystem II, and the achievement of water splitting ability. An increased rate of electron transfer from Q(A)(-) to the Q(B) Site on the acceptor side of photosystem II, mainly due to enhanced efficiency of binding of Q(B) to its Site, was found to precede the building of the Mn cluster. We also showed that Tyro was oxidized simultaneously with this increase in electron-transfer rate. Thus, it appears that photoactivation is sequential, with an increased rate of electron transfer on the acceptor side occurring together with the oxidation of Tyro in the first step, followed by the assembly of the Mn cluster. We suggest that a conformational change of photosystem II is induced early in the photoactivation process facilitating electron transfer from the primary donor to the acceptor side. As a consequence, Tyr(D), an auxiliary electron donor to P-680(+)/Tyr(z)(.), is oxidized. That this occurs before the Mn cluster is fully functional serves to protect photosystem II against donor side induced photodamage.
Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O 2 ·− ). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.
The synthesis and characterization of the title trisphenolate ligand are described. From its reaction with manganese(iii) three complexes were isolated. The crystal structures revealed one pentacoordinate monomer and two similar dimers with different solvents of crystallization. In the dimers the metal ions are hexacoordinate and connected through bridging of two phenolates. A combination of electrochemistry and EPR spectroscopy showed that, in acetonitrile, the isolated batches were all identical and mainly monomeric, indicating that the mononuclear complex is in equilibrium with the dimer and perhaps also with complexes of higher nuclearity, as suggested by the detection of both the trimer and the tetramer by electrospray ionization mass spectrometry (ESI-MS). The successful use of the monomer batch as an epoxidation catalyst indicated that a high-valent manganese-oxo species can be formed, although it is probably short-lived. This is also suggested by EPR studies of the species formed by electrochemical oxidation of the complex. Upon one-electron oxidation, a manganese(iv) species was formed, which was at least partly converted to another species containing a phenoxy radical.
As a model for redox components on the donor side of photosystem II (PS II) in green plants, a supramolecular complex 4 has been prepared. In this, a ruthenium(II) tris-bipyridyl complex which mimics the function of P680 in PS II, has been covalently linked to a tyrosine unit which bears two hydrogen-bonding substituents, dipicolylamine (dpa) ligands. Our aim is to mimic the interaction between tyrosineZ and a basic histidine residue, namely His190 in PSII, and also to use the dpa ligands for coordination of manganese. Two different routes for the synthesis of the compound 4 are presented. Its structure was fully characterized by 1H NMR, COSY, NOESY, 13C NMR, IR, and mass spectrometry. 1H NMR and NOESY gave evidence for the existence of intramolecular hydrogen bonding in 4. The interaction between the ruthenium and the substituted tyrosine unit was probed by steady-state and time-resolved emission measurements as well as by chemical oxidation. Flash photolysis and EPR measurements on 4 in the presence of an electron acceptor (methylviologen, MV2+, or cobalt pentaminechloride, Co3+) showed that an intermolecular electron transfer from the excited state of Ru(II) in 4 to the electron acceptor took place, forming Ru(III) and the methylviologen radical MV+• or Co2+. This was followed by intramolecular electron transfer from the substituted tyrosine moiety to the photogenerated Ru(III), regenerating Ru(II) and forming a tyrosyl radical. In water, the radical has a g value of 2.0044, indicative of a deprotonated tyrosyl radical. In acetonitrile, a radical with a g value of 2.0029 was formed, which can be assigned to the tyrosine radical cation. In both solvents the electron transfer is intramolecular with a rate constant kET > 1 × 107 s-1. This is 2 orders of magnitude greater than the one for a similar compound 3, in which no dpa arm is attached to the tyrosine unit. Therefore the hydrogen bonding between the substituted tyrosine and the dpa arms in 4 is proposed to be responsible for the fast electron transfer. This interaction mimics the proposed His190 and tyrosineZ interaction in the donor side of PS II.
In order to model the individual electron transfer steps from the manganese cluster to the photooxidized sensitizer P680+ in Photosystem II (PS II) in green plants, the supramolecular complex 4 has been synthesized. In this complex, a ruthenium(II) tris-bipyridine type photosensitizer has been linked to a manganese(II) dimer via a substituted L-tyrosine, which bridges the manganese ions. The trinuclear complex 4 was characterized by electron paramagnetic resonance (EPR) and electrospray ionization mass spectrometry (ESI-MS). The excited state lifetime of the ruthenium tris-bipyridine moiety in 4 was found to be about 110 ns in acetonitrile. Using flash photolysis in the presence of an electron acceptor (methylviologen), it was demonstrated that in the supramolecular complex 4 an electron was transferred from the excited state of the ruthenium tris-bipyridine moiety to methylviologen, forming a methylviologen radical and a ruthenium(III) tris-bipyridine moiety. Next, the Ru(III) species retrieved the electron from the manganese(II/II) dimer in an intramolecular electron transfer reaction with a rate constant kET > 1.0 x 10(7) s(-1), generating a manganese(II/III) oxidation state and regenerating the ruthenium(II) photosensitizer. This is the first example of intramolecular electron transfer in a supramolecular complex, in which a manganese dimer is covalently linked to a photosensitizer via a tyrosine unit, in a process which mimics the electron transfer on the donor side of PS II.
On the path to an energy transition away from fossil fuels to sustainable sources, the European Union is for the moment keeping pace with the objectives of the Strategic Energy Technology-Plan. For this trend to continue after 2020, scientific breakthroughs must be achieved. One main objective is to produce solar fuels from solar energy and water in direct processes to accomplish the efficient storage of solar energy in a chemical form. This is a grand scientific challenge. One important approach to achieve this goal is Artificial Photosynthesis. The European Energy Research Alliance has launched the Joint Programme “Advanced Materials & Processes for Energy Applications” (AMPEA) to foster the role of basic science in Future Emerging Technologies. European researchers in artificial photosynthesis recently met at an AMPEA organized workshop to define common research strategies and milestones for the future. Through this work artificial photosynthesis became the first energy research sub-field to be organised into what is designated “an Application” within AMPEA. The ambition is to drive and accelerate solar fuels research into a powerful European field – in a shorter time and with a broader scope than possible for individual or national initiatives. Within AMPEA the Application Artificial Photosynthesis is inclusive and intended to bring together all European scientists in relevant fields. The goal is to set up a thorough and systematic programme of directed research, which by 2020 will have advanced to a point where commercially viable artificial photosynthetic devices will be under development in partnership with industry.
Magnaporthe oryzae causes rice blast disease and has become a model organism of fungal infections. M. oryzae can oxygenate fatty acids by 7,8-linoleate diol synthase, 10R-dioxygenase-epoxy alcohol synthase, and by a putative manganese lipoxygenase (Mo-MnLOX). The latter two are transcribed during infection. The open reading frame of Mo-MnLOX was deduced from genome and cDNA analysis. Recombinant Mo-MnLOX was expressed in Pichia pastoris and purified to homogeneity. The enzyme contained protein-bound Mn and oxidized 18:2n-6 and 18:3n-3 to 9S-, 11-, and 13R-hydroperoxy metabolites by suprafacial hydrogen abstraction and oxygenation. The 11-hydroperoxides were subject to β-fragmentation with formation of 9S- and 13R-hydroperoxy fatty acids. Oxygen consumption indicated apparent kcat values of 2.8 s(-1) (18:2n-6) and 3.9 s(-1) (18:3n-3), and UV analysis yielded apparent Km values of 8 and 12 μM, respectively, for biosynthesis of cis-trans conjugated metabolites. 9S-Hydroperoxy-10E,12Z,15Z-octadecatrienoic acid was rapidly further oxidized to a triene, 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid. In conclusion, we have expressed, purified and characterized a new MnLOX from M. oryzae. The pathogen likely secretes Mo-MnLOX and phospholipases to generate oxylipins and to oxidize lipid membranes of rice cells and the cuticle.
The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (Fox-LOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11 R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid beta-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-H-2] 18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the H-2-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero3- phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18: 2n-6 to the same major products as Cg-MnLOX.Jlr Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the pro R rather than the proShydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.
The structural rearrangements triggered by oxidation of the dinuclear Mn complex [Mn2(bpmp)(u-OAc)2]+ (bpmp = 2,6-bis[bis(2-pyridylmethyl)amino]methyl-4-methylphenol anion) in the presence of water have been studied by combinations of electrochemistry with IR spectroscopy and with electrospray ionization mass spectrometry (ESI-MS). The exchange of acetate bridges for water (D2O) derived ligands in different oxidation states could be monitored by mid-IR spectroscopy in CD3CN-D2O mixtures following the Vas(C-O) bands of bound acetate at 1594.4 cm-1 (II,II), 1592.0 cm-1 (II,III) and 1586.5 cm-1 (III,III). Substantial loss of bound acetate occurs at much lower water content (<0.5% v/v) in the III,III state than in the II,II and II,III states (>10%). The ligand-exchange reactions do not initially reduce the overall charge of the complex but facilitate further oxidation by proton-coupled electron transfer as the water-derived ligands are increasingly deprotonated in higher oxidation states. In the IR spectra deprotonation could be followed by the formation of acetic acid (DOAc, ~1725 cm-1, V(C-O)) from the released acetate (1573.6 cm-1, Vas(C-O)). By the on-line combination of an electrochemical flow cell with ESI-MS several product complexes could be identified. A di-u-oxo bridged III,IV dimer [Mn2(bpmp)(u-O)2]2+ (m/z 335.8) can be generated at potentials below the III,III/II,III couple of the di-u-acetato complex (0.61 V Vs. ferrocene). The ligand-exchange reactions allow for three metal-centered oxidation steps to occur from II,II to III,IV in a potential range of only 0.5 V, explaining the formation of a spin-coupled III,IV dimer by photo-oxidation with [Ru(bpy)3]3+ in previous EPR studies.