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Title: The interaction between cytochrome f and plastocyanin from higher plants
Author: Fisher, N. E.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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The aims of this study were to investigate the nature of the interaction between cytochrome f and plastocyanin isolated from higher plants. The reaction kinetics for these redox partners isolated from two homologous systems (spinach and turnip) were investigated. Limited proteolysis of purified spinach cytochrome f with proteinase K generated a form of the protein that was essentially kinetically identical to soluble, monomeric turnip cytochrome f in the kinetics of its reaction with plastocyanin. The second order rate constant for the reaction between oligomeric, nonproteolysed spinach cytochrome f and spinach plastocyanin (7.19 x 107 M-1 s-1) was 1.9-fold less than that observed for the reaction between proteinase K-treated spinach cytochrome f and spinach plastocyanin (13.7 x 107 M-1 s-1). The reactivity of cytochrome f when present in isolated cytochrome bf complex towards plastocyanin (k2 = 7.78 x 107 M-1 s-1 for the spinach plastocyanin/spinach bf reaction and 8.68 x 107 M-1 s-1 for the turnip plastocyanin/turnip bf reaction) was very similar to that of unproteolysed spinach cytochrome f. The difference in rate between proteolysed and non-proteolysed cytochrome f in its reaction with plastocyanin is likely to be due to the occlusion of a region of the cytochrome involved in interacting with plastocyanin, caused by aggregation of the non-proteolysed form of the protein. This may also explain the decrease in rate observed for the reaction between bf complex and plastocyanin compared to the reaction with soluble cytochrome f. The reaction kinetics for homologous (turnip cytochrome f/ turnip plastocyanin) and heterologous (turnip cytochrome f/spinach plastocyanin) systems were very similar (k2 = 14.31 x 107 and 12.9x 107 M-1 s-1 respectively at pH 6.0. and 100 mM ionic strength). It is concluded that heterologous higher plant systems are valid for examining the reaction between cytochrome f and plastocyanin on the condition that the cytochrome is isolated from a cruciferous source. Mutation of Y83 (a residue suggested to be essential for the electron transfer activity of the protein) to serine, glutamate and tryptophan generated structurally unstable mutants which lost copper during the purification process. Similar instability was observed when the surface exposed copper ligand H87 was mutated to cysteine. Introduction of leucine at position 90 (adjacent to H87), replacing alanine, resulted in a stable mutant.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available