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

Wu Xu,Amelie Laujac,Yingchun Wang,Eric Taylor,Parag R Chitnis,Sergei Savikhin,Liyan Gao,Paul Cobine

doi:10.1371/journal.pone.0024625

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.

Highlights

Photosystem I (PS I) is a protein-pigment complex in cyanobacteria and higher plants
When the recovered wild type (RWT) and mutant strains were cultured with glucose, all mutant strains except the one with F649C/G650I substitution could grow under normal light intensity (40 mmoles m22 s21) with only small differences in their growth rates
When the photoautotrophic growth of the strains was tested under normal and high light intensity, the mutants with H651C/L652M, F649C/G650I and F647C substitutions could not grow under photoautotrophic conditions, which is consistent with the observations of cell growth on plates

Summary

Introduction

Photosystem I (PS I) is a protein-pigment complex in cyanobacteria and higher plants. It mediates the light-driven electron transfer from plastocyanin to ferredoxin [1]. Monomers of cyanobacterial PS I complex contain twelve protein subunits, ninety six chlorophyll a molecules, twenty two bcarotenes, two phylloquinones, four lipid molecules, and three [4Fe-4S] clusters. The PsaA and PsaB proteins of PS I form the core that binds the P700, which is a dimer of chlorophyll a and a9 molecules, and the chain of electron acceptors A0 (a chlorophyll a molecule), A1 (a phylloquinone) and FX (a [4Fe-4S] cluster). Arabidopsis plants mutated in both plastocyanin-coding genes and with a functional cytochrome c6 cannot grow photoautotrophically because of a complete blockade in light-driven electron transport, demonstrating that in Arabidopsis only plastocyanin can donate electrons to photosystem I in vivo [10]

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