title: Site-directed mutagenesis of Thermosynechococcus elongatus photosystem II: the O2-evolving enzyme lacking the redox-active tyrosine D.
abstract: Site-directed mutagenesis in the photosystem II (PSII) oxygen-evolving enzyme was achieved in the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII from this species is the focus of attention because its robustness makes it suitable for enzymological and biophysical studies. PSII, which lacks the redox-active tyrosine Tyr(D), was engineered by substituting a phenylalanine for tyrosine 160 of the D2 protein. An aim of this work was to engineer a mutant for spectroscopy, in particular, for EPR, on the active enzyme. The Tyr(D)(*) EPR signal was monitored in whole cells (i) to control the expression level of the two genes (psbD(1) and psbD(2)) encoding D2 and (ii) to assess the success of the mutagenesis. Both psbD(1) and psbD(2) could be expressed, and recombination occurred between them. The D2-Y160F mutation was introduced into psbD(1) after psbD(2) was deleted and a His-tag was attached to the CP43 protein. The effects of the Y160F mutation were characterized in cells, thylakoids, and isolated PSII. The efficiency of enzyme function under the conditions tested was unaffected. The distribution and lifetime of the redox states (S(n)() states) of the enzyme cycle were modified, with more S(0) in the dark and no rapid decay phase of S(3). Although not previously reported, these effects were expected because Tyr(D)(*) is able to oxidize S(0) and Tyr(D) is able to reduce S(2) and S(3). Slight changes in the difference spectra in the visible and infrared recorded upon the formation and reduction of the chlorophyll cation P(680)(+) and kinetic measurements of P(680)(+) reduction indicated minor structural perturbations, perhaps in the hydrogen-bonding network linking Tyr(D) and P(680), rather than electrostatic changes associated with the loss of a charge from Tyr(D)(*)(H(+)). We show here that this fully active preparation can provide spectra from the Mn(4)CaO(4) complex and associated radical species uncontaminated by Tyr(D)(*).
authors.affiliation: Service de Bioénergétique, DBJC, URA CNRS 2096, CEA Saclay, 91191 Gif-sur-Yvette, France. miwa@biochem.osakafu-u.ac.jp
authors.forename: Miwa
authors.initials: M
authors.lastname: Sugiura
authors.forename: Fabrice
authors.initials: F
authors.lastname: Rappaport
authors.forename: Klaus
authors.initials: K
authors.lastname: Brettel
authors.forename: Takumi
authors.initials: T
authors.lastname: Noguchi
authors.forename: A William
authors.initials: AW
authors.lastname: Rutherford
authors.forename: Alain
authors.initials: A
authors.lastname: Boussac
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002734:Chlorophyll / Q000378:metabolism; D000458:Cyanobacteria / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D010782:Photolysis; D045332:Photosystem II Protein Complex / Q000096:biosynthesis / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000172:deficiency / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D045332:Photosystem II Protein Complex; C422369:photosystem II, psbA subunit; C085993:psbDI protein, cyanobacteria; C066447:tyrosyl radical Y(D); D002734:Chlorophyll; D014443:Tyrosine; D010649:Phenylalanine; C025167:P-680; D010100:Oxygen