Ali, K.; (2004) Photosystem I in Chlamydomonas reinhardtii. Doctoral thesis , University of London.

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Abstract

Photosystem I (PSI) catalyses the light driven electron transfer from plastocyanin/ cytochrome c6 on the luminal side of the thylakoid membrane to ferredoxin/ flavodoxin at the stromal side via a chain of electron carriers. PSI is a multi-protein membrane complex composed of a large number of polypeptide subunits, designated PsaA to PsaO. There are key differences in subunit composition between prokaryotic and eukaryotic PSI complexes. For example, PsaG, PsaH, PsaN and PsaO are all absent from cyanobacterial PSI. In eukaryotes the genes for the PSI subunits are distributed between the nuclear and chloroplast genomes. This thesis describes a series of molecular-genetic studies using the model photosynthetic eukaryote Chlamydomonas reinhardtii, aimed at understanding various aspects of the eukaryotic PSI. The nuclear gene encoding the PsaN subunit from C. reinhardtii was cloned and characterised. The psaN gene was shown to be present as a single copy in the genome and northern analysis indicated that the expression of this gene is light-induced. Antibodies were raised to the mature PsaN protein and attempts were made to down- regulate psaN gene expression using an RNA antisense approach. Several PSI mutants were investigated using western analysis. A mutant lacking PsaJ showed significantly reduced levels of PsaN protein accumulation, while a mutant lacking PsaF showed no detectable levels of PsaN, indicating that these two subunits may interact with PsaN on the lumenal side of the PSI complex. The role of the 22 ?-carotene molecules associated with the PSI complex was investigated. Molecular and biophysical analysis of a carotenoid deficient mutant established that the PSI complex is assembled and functional despite the loss of carotenoids and the PsaN protein from the complex. The crystal structure of cyanobacterial PSI reveals two possible electron transfer branches bound to the PsaA and PsaB subunits, which display a remarkable symmetry. However, a key difference is a tryptophan residue located between the PsaB-bound phylloquinone and the iron-sulphur centre Fx, which is not conserved in PsaA. The tryptophan residue was substituted with a glycine using site-directed mutagenesis. The mutant was unable to grow photoautotrophically and biophysical analysis revealed that electron transfer on the PsaB branch was partially blocked.

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