An Analysis of the Redox Properties and Stability of Chlamydomonas reinhardtii Cytochrome F, Cytochrome C6, and Mutants thereof

This body of work presents mutagenesis studies conducted on two c-type cytochromes from Chlamydomonas reinhardtii. Cytochrome f, a unique c-type cytochrome, is investigated in regards to its redox potential, the dependence of the redox potential, and the thermal stability of the protein. The mutations made were Y1F, Y9F, Y160F, Y160L, R156L, and R156K. The residues that were mutated surround the heme. It was found that, relative to the wild-type, only the Y160L and R156 mutants showed any difference in midpoint potential at pH 7. Wild-type and mutants both had a midpoint potential that was dependent upon pH indicating that none of the investigated residues are responsible for the alkaline transition seen in cytochrome f. The stability of each of the mutants also did not vary from that of the wild-type protein. The arginine mutants were unsuitable to be investigated by current methods in regards to the pH dependence of the midpoint potential and stability. Cytochrome c6, a typical class I, c-type cytochrome, and the mutants K29I and K57I were investigated in the same manner as was cytochrome f along with CD spectral studies. The K29 and K57 residues are found in the vicinity of the heme. In regards to the midpoint potential of the mutants at pH 7, they were found to be lower than that for the wild-type protein. The midpoint potential for both the wild-type and mutants was found to be independent of pH as far as pH 10. The melting temperature of the mutants, when examined, was also lower than that of the wild-type indicating a lower relative stability of the mutants when compared to the wild-type. CD spectroscopy was done to investigate if an aromatic residue in the vicinity of the heme is responsible for the negative Cotton effect seen in the oxidized spectrum. The residue in Chlamydomonas reinhardtii cytochrome c6 is a tryptophan that is not co-facially oriented with respect to the heme. The absence of the Cotton effect provides further evidence that for the presence of a negative Cotton effect there must be an aromatic residue in the vicinity and it must be co-facially oriented with respect to the heme