Site-directed mutagenesis of the subunit PsaC establishes a surface-exposed domain interacting with the photosystem I core binding site.
We have postulated that the orientation of PsaC on the photosystem I core involves electrostatic interactions between charged residues on the core binding site and the subunit [Rodday, S. M., Jun, S.-S., & Biggins, J. (1993) Photosynth. Res. 36, 1-9]. We, therefore, changed eight acidic residues on PsaC to arginine and examined the efficiency of the mutant subunits in the reconstitution of P700-Fx cores in vitro. Reconstitution of the cores by the mutant subunits was determined by analysis of the kinetics of recombination reactions between P700+ and reduced acceptors as measured optically. Restoration of complete forward electron transfer, indicative of efficient subunit binding, was estimated from the ca. 30 ms decay component in the flash transients. Slightly reduced levels of reconstitution were observed for the mutants D24R, E46R/D47R. D61R, and E72R. In contrast, mutants D9R, E27R, and D32R showed significantly lower efficiencies. The presence of the iron-sulfur centers, FA and FB, in these three mutant subunits was confirmed by low-temperature EPR spectroscopy indicating that the polypeptides had folded correctly. We conclude that the introduction of positively charged side chains at positions 9, 27, and 32 seriously disrupts PsaC binding. However, when the wild-type acidic residues in these positions were changed to alanine, only mutant D9A showed a reduced level of reconstitution, suggesting that this aspartate is the most important residue participating in the electrostatic interaction with the core. The results are discussed in relation to the photosystem I crystal structure and support an orientation of PsaC on the core such that center FB is proximal to Fx.
Rodday
S M
SM
Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Do
L T
LT
Chynwat
V
V
Frank
H A
HA
Biggins
J
J
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D000458:Cyanobacteria / Q000737:chemistry; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D045331:Photosystem I Protein Complex; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018724:Spinacia oleracea / Q000737:chemistry
D017931:DNA Primers; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011506:Proteins; C089909:photosystem I, psaB subunit; D001120:Arginine; D000409:Alanine