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 F(A) and F(B) iron- sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14D(PsaC), C51D(PsaC), C14D/C51D(PsaC)), serine (C14S(PsaC), C51S(PsaC)), and alanine (C14A(PsaC), C51A(PsaC)), and the properties of F(A) and F(B) were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14D(PsaC)-PS I and C14S(PsaC)-PS I complexes showed high levels of photoreduction of F(A) with g values of 2.045, 1.944, and 1.852 after illumination at 15 K, but there was no evidence of reduced F(B) in the g = 2 region. The C51D(PsaC)-PSI and C51S(PSaC)-PS I complexes showed low levels of photoreduction of F(B) with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced F(A) in the g = 2 region. The presence of F(B) was inferred in C14D(PsaC)-PS I and CI4S(PsaC)- PS I, and the presence of F(A) was inferred in C51D(PsaC)-PS I and C51S(PsaC)-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 F(X) 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-PSI (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 C14A(PsaC)-PS I, C51A(PsaC)-PS I and C14D/C51D(PsaC)-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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology