This proposal is an investigation of the function of the cytochrome b6f-complex from Chlamydomonas reinhardbi in living cells and in isolated monomers of wild type and mutant complexes during my intended postdoc time. We expect answers to severall important questions concerning the molecular basis of the function of the complex:
i) What is the pathway of electrons and protons in the complex? ii) Which amino acids determine the midpoint potentials of the cofactors? iii) Do protonable amino acid side chains participate in the reactions of the complex?
We will adress these questions using the following methods. We will: i) characterize the mutants of Chlamydomonas reinhardtii in living cells by kinetic spectroscopy to see the effect of the mutation on the function of the complex ii) titrate the isolated protein electrochemically to determine the midpoint potentials of the cofactors and to elucidate, whether a change in the kinetic behaviour of the mutant complex is related to a change in the midopoint potentials of the cofactors. iii) combine electrochemistry and photochemistry to study the reactions in membrane sheets or vesicles containing the photosystems and the cytochrome b6f-complex.