Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

Abstract

We have utilized the unicellular cyanobacterium Synechocystis sp. PCC 6803 to incorporate site-directed amino acid substitutions into the photosystem I (PSI) reactioncenter protein PsaB. A cysteine residue (position 565 of PsaB) proposed to serve as a ligand to the [4Fe-4S] center Fx was changed to serine, histidine, and aspartate. These three mutants--C565S, C565H, and C565D--all exhibited greatly reduced accumulation of PSI reaction-center proteins and failed to grow autotrophically, indicating that this cysteine most likely does coordinate Fx, which is crucial for PSI biogenesis. Interestingly, the strain C565S accumulated significantly more PSI than the other two cysteine mutants and displayed photoreduction of the [4Fe-4S] terminal electron acceptors FA and FB. Mutations were also introduced into a leucine zipper motif of PsaB, proposed to participate in reaction-center dimerization. The mutants L522V, L536M, and L522V/L536M all exhibited wild-type characteristics and grew autotrophically, whereas the L522P mutation prevented PSI accumulation. These data do not provide support for a major structural role of the leucine zipper in reaction-center dimerization or in assembly of Fx. However, the amino acid substitutions incorporated were conservative and might not have perturbed the leucine zipper.

Radicals and Radical Pairs in Photosynthesis

“…While the internal structure is still not as well resolved as that of the purple photosynthetic bacterial RC (26), i t already gives a picture of the redox chain that is responsible for the primary charge separation process, as well as the Chl pigments that form the inner antenna of the complex (38, 41, 151). The recently developed possibility to generate site-directed mutants of the PS I proteins of cyanobacteria (152,153) and Chlamyclornonas reinhardti; (154j made it possible to test specific conserved amino acid sites for their function in pigment coordination and overall structure.…”

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

“…PCC 6803. This change did not prevent the assembly of PS I or the low temperature photoreduction of F A or F B (11). The mixed-ligand [4Fe-4S] clusters in the F X site of C565S PsaB and C556S PsaB (12) were found to be relatively inefficient in transferring electrons from A 1 to F A /F B (13).…”

“…Site-directed mutations were made with an Amersham Pharmacia Biotech in vitro mutagenesis system. Incorporation of mutations was verified by restriction mapping and by DNA sequencing, and the mutated variations of pLS3531 (11) were then used to transform strain ⌬B-RCPT. Spectinomycin-resistant colonies were selected under light-activated heterotropic growth conditions as described (17).…”

“…For oxygen evolution measurements, the light from a slide projector was passed through a copper sulfate solution and filtered with a red cutoff filter ( Ͼ 640 nm), providing saturating photon flux density of 480 mol m Ϫ2 s Ϫ1 . PS I-mediated oxygen uptake rates were determined using the isolated thylakoid membranes as described (11).…”

Topography of the Photosystem I Core Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

“…The H loop is on the p side of the thylakoid membrane in the model. It is only one transmembrane helix from the I loop which contains the F X -binding motif and interacts with PsaC on the n side (41,42). From the arrangement of helices in the 4-Å model of PS I, both I and H loops should be located in the center of the PS I core.…”