Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

<p>Abstract</p> <p>Background</p> <p>The eukaryotic green alga, <it>Chlamydomonas reinhardtii</it>, produces H₂ under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium <it>Shewanella oneidensis</it> as a new and efficient system for expression and maturation of HydA1 from <it>Chlamydomonas reinhardtii</it>.</p> <p>Results</p> <p>Based on codon usage bias and hydrogenase maturation ability, the bacterium <it>S. oneidensis</it>, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for <it>C. reinhardtii </it>[Fe-Fe] hydrogenase expression. Hydrogen formation by <it>S. oneidensis </it>strain AS52 (Δ<it>hydA</it>Δ<it>hyaB</it>) transformed with a plasmid bearing <it>Cr</it>HydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of <it>S. oneidensis </it>is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.</p> <p>Conclusion</p> <p>In the present work a new efficient system for heterologous expression and maturation of <it>C. reinhardtii </it>hydrogenase has been developed. HydA1 of <it>C. reinhardtii </it>was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l^-1(OD₆₀₀= 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H₂mg^-1min^-1.</p

Electrochemical measurements of the kinetics of inhibition of two FeFe hydrogenases by O2 demonstrate that the reaction is partly reversible

International audienceThe mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al, Nat. Chem. 4 336 (2014)] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect

Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: Study of redox mutants

Cytochrome c550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c550 we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and –30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c550 was also constructed. It showed a lowered affinity for Cl– and Ca2+ as reported earlier for the cytochrome c550-less Synechocystis 6803 mutant, but it showed a shorter lived Formula state, rather than a stabilized S2 state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c55

Search for new physics in events with opposite-sign leptons, jets, and missing transverse energy in pp collisions at sqrt(s) = 7 TeV

A search is presented for physics beyond the standard model (BSM) in final states with a pair of opposite-sign isolated leptons accompanied by jets and missing transverse energy. The search uses LHC data recorded at a center-of-mass energy sqrt(s) = 7 TeV with the CMS detector, corresponding to an integrated luminosity of approximately 5 inverse femtobarns. Two complementary search strategies are employed. The first probes models with a specific dilepton production mechanism that leads to a characteristic kinematic edge in the dilepton mass distribution. The second strategy probes models of dilepton production with heavy, colored objects that decay to final states including invisible particles, leading to very large hadronic activity and missing transverse energy. No evidence for an event yield in excess of the standard model expectations is found. Upper limits on the BSM contributions to the signal regions are deduced from the results, which are used to exclude a region of the parameter space of the constrained minimal supersymmetric extension of the standard model. Additional information related to detector efficiencies and response is provided to allow testing specific models of BSM physics not considered in this paper.Comment: Replaced with published version. Added journal reference and DO

Crystallization and electron paramagnetic resonance characterization of the complex of photosystem I with its natural electron acceptor ferredoxin.

The formation of a transient complex between photosystem I and ferredoxin is involved in the process of ferredoxin photoreduction in oxygenic photosynthetic organisms. Reduced ferredoxin is an essential redox intermediate involved in many assimilatory processes and is necessary for the reduction of NADP(+) to NADPH. Single crystals from a complex of photosystem I with ferredoxin were grown using PEG 400 and CaCl(2) as precipitation agents. The crystals diffract x-rays to a resolution of 7-8 A. The space group was determined to be orthorhombic with the unit cell dimensions a = 194 A, b = 208 A, and c = 354 A. The crystals contain photosystem I and ferredoxin in a 1:1 ratio. Electron paramagnetic resonance (EPR) measurements on these crystals are reported, where EPR signals of the three [4Fe-4S] clusters F(A), F(B), F(X), and the [2Fe-2S] cluster of ferredoxin were detected. From the EPR spectra observed at three particular orientations of the crystal in the magnetic field, the full orientation pattern of the F g-tensor was simulated. This simulation is consistent with the presence of 12 magnetically inequivalent F clusters per unit cell with the C(3) axis of the PSI trimers oriented at (23 degrees, 72 degrees, 77 degrees ) to the unit cell axes

The ferredoxin docking site of photosystem I

The reaction center of photosystem I (PSI) reduces soluble ferredoxin on the stromal side of the photosynthetic membranes of cyanobacteria and chloroplasts. The X-ray structure of PSI from the cyanobacterium Synechococcus elongatus has been recently established at a 2.5 A resolution [Nature 411 (2001) 909]. The kinetics of ferredoxin photoreduction has been studied in recent years in many mutants of the stromal subunits PsaC, PsaD and PsaE of PSI. We discuss the ferredoxin docking site of PSI using the X-ray structure and the effects brought by the PSI mutations to the ferredoxin affinity

ESEEM study of the phyllosemiquinone radical A· in N- and N-labeled photosystem I

The phyllosemiquinone radical of the photosystem I reaction center has been studied by electron spin echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of ESEEM data of the semiquinone in N- and N- labeled PSI and numerical simulations demonstrate the existence of two protein nitrogen nuclei coupled to the semiquinone. One of the 14N couplings is characterized by a quadrupolar coupling constant eqQ/4h of 0.77 MHz, an asymmetry parameter η of 0.18, and a hyperfine coupling tensor with an almost pure isotropic hyperfine coupling, i.e. (A(xx), A(yy), A(zz)) = (1.3, 1.3, 1.5 MHz). The second nitrogen coupling is characterized by a quadrupolar coupling constant eqQ/4h of 0.45 MHz, an asymmetry parameter η of 0.85, and a weak hyperfine coupling tensor with a dominant anisotropic part, i.e. (A(xx), A(yy),A(zz)) = (-0.2, -0.2, 1.5 MHz). On the basis of a comparison of the N-ESEEM data with N-NQR and N-ESEEM data from the literature, the first coupled nitrogen is assigned to the indole nitrogen of a tryptophan residue. The coupling of the second nitrogen is much weaker and therefore more difficult to assign. However, the simulated spectrum best describes an amino nitrogen of a histidine, although the amide group of an asparagine or glutamine cannot be ruled out. The possible origins of the nitrogen hyperfine coupling are discussed in terms of the amino acid residues thought to be close to the semiquinone in PSI