Objective
The general objective of the project is the detailed understanding of the structure of the cytochrome (cyt) bf complex at the molecular level and of its function in the electron transfer and energy conservation. The goals are divided up into three closely related subjects. (i) The structure on the molecular level: New subunits of the intact complex had to be identified and new large scale preparation techniques of material for crystallization had to be established. (ii) The internal function of the subunits is approached by design of mutated subunits in the alga Chlamydomonas reinhardtii with emphasis on binding sites of plastoquinone, subunit interactions and biogenesis. (iii) The interaction of the cyt bf complex within the framework of the energy converting electron transport chains is analyzed by time-resolved measurements.
Structure on the molecular level. Two-dimensional crystals and three-dimensional crystalloids were obtained from Chlamydomonas reinhardtii by participant 4. The factors preserving the dimeric state of the complex and a projection map of the negatively stained complex has been established at a resolution of 8 Å. The heme of cyt f and the FeS center of the Rieske protein can be localized. A large scale purification of the dimeric cyt bf complex from lettuce has been improved by participant 5 and resulted in crystalline material. The interaction between the five iron centers and their ligands has been resolved by Moessbauer spectroscopy (participant 1).
Characterization of subunits. Three small subunits of the Cyt bf complex PetG, PetL, (previously ycf7), and PetM have been characterized by participant 4. The complex contains one copy of each of these small and the four larger subunits and forms active dimers. The midpoint potential and the EPR characteristics of the redox centers have been determined by participant 2. A chlorophyll a molecule has been demonstrated to be an authentic component by participant 4. The composition of the heliobacterial complex shows similarities to the cyt bf complex of cyanobacteria and plants (participant 2).
Structure-function relation analyzed by site-directed mutagenesis. Molecular genetics for the cyt bf complex has been established in Chlamydomonas reinhardtii by participant 6. A minus petL mutant shows the importance of this subunit in the transmembrane transfer of electrons or protons. A mutant of cyt f without membrane anchoring is functional in linear electron transport. Mutations of the cyt b subunit show that the heme binding is essential for assembly of the complex. Mutation of a residue being the target of RNA editing in other organisms provides the clue for the understanding of this process. Biogenesis of cyt b is controlled by several nuclear genes. The biogenesis of cyt f involves two independent processes of maturation which have been analyzed in depth. Other mutations in collaboration between participant 6, 5 and 3 allowed to identify residues critical for the electron flow through the plastoquinol oxidizing site, Q0. The electron transfer site of cyt f has been probed by 15 mutants of plastocyanin by participant 1.
Interaction of the cyt bf complex in the electron transport chain. The operative midpoint potential of the Rieske FeS-protein has been analyzed in chloroplasts and in intact algae in a joint effort by participant 6, 3, and 1. The electron transport kinetics and the formation of the electric field by the cyt bf complex has been analyzed by participant 3 with emphasis on the Q-cycle hypothesis. The oxidation of the cyt bf complex is limited by the release of oxidized plastocyanin from photosystem (participant 1). The functional characteristics of the heliobacterial complex resemble those of cyt bc1 complexes (participant 2). A range of potent inhibitors of the cyt bf complex like stigmatellin was exploited by participant 5. Participant 2 found that the inhibitor DBMIB interacts with the Rieske protein. The effect of DCCD on the electrogenic electron transfer within the cyt bf complex indicates that the movement of proton precedes the electrogenic transfer associated with the oxidation of cyt b6 (participant 3).
MAJOR SCIENTIFIC BREAKTHROUGHS:
Two-dimensional crystals and small three-dimensional crystalloids, first structural information;
identification of three new small subunits of the cyt bf complex;
mutation of amino acids of several subunits functional important for electron transfer and biogenesis;
function and redox potential of the Rieske-FeS protein in situ;
separate detection of the proton and electrogenic electron transfer.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- natural sciencesbiological sciencesmolecular biologymolecular genetics
- natural sciencesbiological sciencesmicrobiologyphycology
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural sciencesbiological sciencesgeneticsmutation
- natural scienceschemical sciencesorganic chemistryamines
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Topic(s)
Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
79104 Freiburg
Germany