Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions

Abstract

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 microM DCMU. This mutant carries an R7L mutation on the alpha-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 A PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lalpha cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lalpha cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lalpha cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the alpha-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

“…Our results showed that H22K␣ mutant PSII core complexes gave rise to EPR signals (g z ϭ 6.11; g y ϭ 5.90), which are characteristic for the high spin form of cyt b 559 . These EPR signals were also observed in R7L␣ mutant PSII core complexes (36) and in PSII membranes treated with 2,3-dichloro-5,6-dicyano-p-benzoquinone (characterized with the g ϭ 6.19 and g ϭ 5.95 split signal) (37). It is generally believed that the high spin state (S ϭ 5/2) corresponds to a five-coordinate complex of Fe III PorL that has a square pyramidal structure where the Fe(III) ion is above the plane of the porphyrin (Ref.…”

“…Defects in the binding of extrinsic subunits in mutant PSII core complexes have been shown to influence the redox property of cyt b 559 (5). However, our results in Table 3 showed that H22K␣ and Y18S␣ mutant PSII core complexes contained much more LP form of cyt b 559 (ϳ79 and ϳ86%, respectively) than oxygen-evolving or Triswashed wild-type PSII core complexes (ϳ14 and ϳ30%, respectively) (36). Tris-washed wild-type PSII core complexes were depleted of all extrinsic polypeptides.…”

“…Conceivably, the alteration in the cyt b 559 local structure caused by the mutations results in the activation of the state II configuration or that mutations impede the traffic of plastoquinol/plastoquinone exchange under high turnover conditions in a manner that activates state II configuration. Alternatively, the high F 0 value and the increase of the F M value during actinic light illumination could be attributed to the increased fluorescence emission from uncoupled phycoblisomes in Y18S␣ and H22K␣ cyt b 559 mutant cells (36). Further work examining these possibilities needs to be performed.…”

“…Wild-type cyt b 559 mutant PSII core complexes gave rise to EPR signals of the low spin heme of cytochromes (g z ϭ 2.92; g y ϭ 2.26), which contained contributions from both cyt b 559 and cyt c 550 . In contrast, H22K␣ cyt b 559 mutant PSII core complexes gave rise to EPR signals that are characteristic for both the low spin heme of cyt c 550 (g z ϭ 2.91; g y ϭ 2.26) and the high spin heme of cyt b 559 (g z ϭ 6.11; g y ϭ 5.91) (36,37). In addition, EPR signals (g ϭ 3.57 and 3.30) of well characterized low spin Fe(III) hemes with His/ Lys axial ligation are not detectable in spectra of H22K␣ cyt b 559 mutant PSII core complexes (Fig.…”

Az Acaryochloris marina cianobaktérium kettes fotokémiai rendszerének D1, D2 és citokróm b559 alegységeit, valamint a Hox hidrogenázt kódoló gének kifejeződésének vizsgálata

“…Lehetséges, hogy ezek az aminosav mutációk megváltoztatják a PBP kötődési affinitását, tehát azok a PSII komplexek, amelyek a D2' izoformát tartalmazzák más, valószínűleg gyengébb PBP horgonyzási képességgel bírnak. Kiemelnénk továbbá a Gln97Arg mutációt, ami a D2 fehérjének a cit b559 alegységével érintkező felületén található aminosav cseréje, melyet a PBP kapcsolódással szintén kapcsolatba hoztak (60,143). …”