Patel, Vaishali; (1997) Analysis of photosystem I mutants in Chlamydomonas reinhardtii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

FX is a [4Fe-4S] iron-sulphur centre that is believed to be bound by both PsaA and PsaB. This is a rather unusual arrangement, as [4Fe-4S] iron-sulphur centres are usually bound by one protein, rather than two. This may impose constraints on the interactions of the other subunits of PSI. The region believed to be involved in binding FX is highly conserved, and a possible role has not yet been identified for most of the amino acids within this region. In this study, the function of several amino acids of this region has been examined, by creating amino acid substitutions, and then examining the effect on PSI assembly, and electron transport. Mutant D576L is a nonphotosynthetic mutant of C. reinhardtii. D576 is an amino acid present within the FX binding region, and is believed to be important in the coordination of PsaC, which in turn coordinates the terminal electron acceptors of PSI. The role of this conserved amino acid residue was examined by substituting it with a leucine residue. This substitution caused the PSI complex to be disrupted, and also altered electron transport to FA/FB. Second site suppressor mutants of the D576L mutation were also isolated. These suppressor mutants were effected in the same way as the original D576L mutants, in terms of electron transport to FA/FB, but were able to grow photoautotrophically. This unknown spontaneous mutation is identified as a nuclear mutation. The results show that D576 is important for the binding of PsaC, and that in the suppressor mutants electron transfer rates from A1 to FX are altered, but restored to wild type values in the suppressor mutants. A library of mutations was created by randomly mutating three amino acids, Arg572, Phe573 and Pro574, of the FX binding motif in PsaA. Three mutants of C. reinhardtii were produced. These mutants were selected for their ability to grow photoautotrophically, as the aim was to create minor disruptions in the photosystem, which would still allow PSI to assemble and function. It was hoped that this would provide information on the possible functions of these amino acids. However, all three mutants appear to assemble as much PSI as wild type C. reinhardtii, and the electron transport properties were also similar. Rates of electron transport to FA/FB were not altered, compared to wild type C. reinhardtii. The role of a small chloroplast encoded subunit, PsaJ, of PSI was also examined. The function of PsaJ, which is a membrane protein, is unknown, although it has been suggested that it may coordinate PsaF. The amino acid structure of PsaJ was examined, and revealed regions of hydrophobicity, which are consistent with a membrane protein. However, unlike most membrane proteins which are composed of α-helices, the secondary structure of PsaJ is predicted to be composed of β-sheets. This may prove to have some bearing on its function. The psaJ gene was deleted, and aadA was inserted in its place. This construct was used to transform C. reinhardtii, but transformants failed to emerge.

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