Strains of Synechocystis sp. PCC 6803 with Altered PsaC

Photosystem I (PS I) is a transmembraneal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A 0 ), a phylloquinone (A 1 ), and a [4Fe-4S] iron-sulfur cluster F X , all located on the core subunits PsaA and PsaB, to iron-sulfur clusters F A and F B , located on subunit PsaC. Earlier, it was attempted to determine the function of F X in the absence of F A/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging F X . We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A 1 occurred. The photoreduction of F X was largely inhibited, as seen from direct measurement of the extent of electron transfer from A 1 to F X . From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaCdeficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on F X by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of F X by serine in site-directed mutation C565S/D566E in subunit PsaB caused an ϳ10-fold slow down of electron transfer from the quinone to F X without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that F X might have a major role in controlling electron transfer through PS I.

Section: Discussionmentioning

confidence: 86%

Section: Photosystem I (Ps I)mentioning

confidence: 97%

Section: Forward Electron Transfer From a 1 To F X In Psacmentioning

confidence: 99%

See 1 more Smart Citation

Control of Electron Transport in Photosystem I by the Iron-Sulfur Cluster FX in Response to Intra- and Intersubunit Interactions

Gong

Agalarov

Brettel

et al. 2003

“…The protocols for the isolation of the cyanobacterial thylakoid membranes and the purification of the n-dodecyl ␤-D-maltoside PS I complexes are described in Ref. 16.…”

Section: Isolation Of Thylakoid Membranes and Purification Of Dm-ps Imentioning

confidence: 99%

Strains of Synechocystis sp. PCC 6803 with Altered PsaC

Jung

Vassiliev

Yu³

et al. 1997

Self Cite

A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.

“…Growth of Cells, Oxygen Evolution Rates, and Chlorophyll Content in Whole Cells-Cell growth rates, oxygen evolution rates, and chlorophyll content in the whole cells were determined as described (18). For large-scale culture, wild-type and mutant cells were grown under lightactivated heterotropic growth conditions in carboys with 15 liters of BG-11 medium supplemented with 5 mM glucose.…”

Section: Methodsmentioning

confidence: 99%

The Cysteine-proximal Aspartates in the FX-binding Niche of Photosystem I

Vassiliev

Jung

et al. 1999

Self Cite

The F X electron acceptor in Photosystem I (PS I) is a highly electronegative (E m ‫؍‬ ؊705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566 PsaB and D557 PsaB on electron transfer through F X by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566A PsaB , D557A PsaB , D566K PsaB , and D557K PsaB ) or in pairs (D557A PsaB /D566A PsaB and D557K PsaB /D566A PsaB ). All mutants except for D557K PsaB /D566A PsaB grew photoautotrophically, but the growth of D557K PsaB and D557A PsaB /D566A PsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557K PsaB and D557A PsaB / D566A PsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP ؉ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of backreaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors F A and F B in PS I complexes from all mutants. The EPR spectrum of F X was identical to that in the wild type in all but the single and double D566A PsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through F X .