Mutational analysis of photosystem I of Synechocystis sp. PCC 6803: the role of four conserved aromatic residues in the j-helix of PsaB

Abstract

Photosystem I is the light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic electron transfer of cyanobacteria and plants. Two histidyl residues in the symmetric transmembrane helices A-j and B-j provide ligands for the P700 chlorophyll molecules of the reaction center of photosystem I. To determine the role of conserved aromatic residues adjacent to the histidyl molecule in the helix of B-j, we generated six site-directed mutants of the psaB gene in Synechocystis sp. PCC 6803. Three mutant strains with W645C, W643C/A644I and S641C/V642I substitutions could grow photoautotrophically and showed no obvious reduction in the photosystem I activity. Kinetics of P700 re-reduction by plastocyanin remained unaltered in these mutants. In contrast, the strains with H651C/L652M, F649C/G650I and F647C substitutions could not grow under photoautotrophic conditions because those mutants had low photosystem I activity, possibly due to low levels of proteins. A procedure to select spontaneous revertants from the mutants that are incapable to photoautotrophic growth resulted in three revertants that were used in this study. The molecular analysis of the spontaneous revertants suggested that an aromatic residue at F647 and a small residue at G650 may be necessary for maintaining the structural integrity of photosystem I. The (P700⁺-P700) steady-state absorption difference spectrum of the revertant F647Y has a ∼5 nm narrower peak than the recovered wild-type, suggesting that additional hydroxyl group of this revertant may participate in the interaction with the special pair while the photosystem I complexes of the F649C/G650T and H651Q mutants closely resemble the wild-type spectrum. The results presented here demonstrate that the highly conserved residues W645, W643 and F649 are not critical for maintaining the integrity and in mediating electron transport from plastocyanin to photosystem I. Our data suggest that an aromatic residue is required at position of 647 for structural integrity and/or function of photosystem I.

Figure 1. Amino acid sequence alignments of the A-j and B-j helices of the PS I core.

The deduced protein sequences of the j-helices from the PsaA and PsaB proteins of maize (Zea mays), rice (Oryza sativa), spinach (Spinacia oleracea), Arabidopsis thaliana, Chlamydomonas reinhardtii, Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002 and Synechococcus elongatus PCC 7942 were aligned using Invitrogen software (Carlsbad, CA). P700 ligands are indicated (P700).

Figure 2. Western blotting analysis of the membranes and PS I complexes from the mutant strains.

The proteins were resolved by Tricine/urea/SDS-PAGE. The antibodies against Synechocystis sp. PCC 6803 PsaA, PsaB, PsaC, PsaF, PsaL, PsaK and PsaI were used and the immunodetection was visualized by enhanced chemiluminescence. Membranes were isolated from the mutant strains and samples containing 5 µg chlorophyll were loaded in each lane.

Figure 3. Steady-state (P700⁺ - P700) absorption difference spectra for the three revertants and the mutant of H651C.

Solid and dashed curves show mutant and wild- type spectra, respectively. The y-axis shows ΔA roughly in %-level in respect to absorption in the maximum of the 680 nm band. The optimized parameters from Gaussian fit to these and other difference spectra are given in Table S2.

Figure 4. 2.5 Å structure of the mutated residues in j helix of PsaB.

The coordinates were downloaded from protein data bank (PDB ID: 1JB0) and XtalView software was used to generate the structural diagram. The nomenclature for the corresponding amino acids was changed to show the corresponding residues in Synechocystis sp. PCC 6803.