Quinone reduction and supercomplex formation: the two enigma of Rieske/cytb complexes – BIsP

-Development of a purification procedure for the suerpcomplex of Geobacillus (choice of detergent, chromatography)
-development of a purification procedure for the Rieske/cytb complex of heliobatceria.
- development and construction of a themostatable cuvette holder for the JTS equipped with exciting LEDs at 800nm
-Study of the heliobacteria photosynthetic reaction chain in vivo with the JTS
-test the influence of culture and light conditions on the redox state of the heliobacteria reactions chain in vivo
-enrichment and charcterization of a ferredoxin of heliobacteria
-Heterologous expression of the Rieske subunit from H. modesticaldum. Redox titration followed by EPR spectroscopy
-development of tagging enzymes from bacillus and Geobacillus
- Construction of Qi site mutants in Chlamydomonas
- development of in vivo studies of the respiratory chain of Geobacillus by flash induced de-binding of CO from the active site of the oxydase
- crystallization assays of the supercomplex from Geobacillus
- investigation of the supercomplex from Geobacillus by electron microscopy

-purification of the supercomplex from G. stearothermophilus: the obtained preparation so far allowed us to determine spectra and redox midpoint potentials of all co-factors, important parameters for the comprehension of enzyme function. In particular unusually low redox midpoint potentials were found for the b-hemes which will be an important fact to be taken into consideration in the framework of a Q-cycle.
-PFO from Heliobacteria is involved in oxidation of pyruvate but also able to produce pyruvate from acetate and CO2
-determination of the redox midpoint potential of the Rieske protein and one of the ferredoxins of H. modesticaldum. These potentials were the missing values in the redox characterization of the photosynthetic reaction chain of Heliobacteria and are valuable for the interpretation of in vivo experiments.
-crystallization of complex II from Geobacillus

-Achievement of a more homogeneous preparation of the supercomplex from Geobacillus for crystallization
-development of an activity test for the supercomplex from Geobacillus
-back to the membrane system to study heme ci in the presence of its natural quinones under different redox conditions
- deconvolution and interpretation of in vivo kinetics as measured on Heliobacteria with the redox midpoint potentials of all the individual co-factors now at hand
Characterization of heme ci from the Qi site mutants of Chlamydomonas by EPR spectroscpy

Bergdoll, L., Point, E., Bayann, F., Picot, D. congrès du GfB 2013 : ‘Study of a respiratory supercomplex from the low GC firmicutes Geobacillus sterothermophilus’

F.Baymann : EBEC 2012 ‘Evolution of Rieske/cytb complexes

Energy transduction is constitutive to all living organisms and the electrochemical proton gradient is a central intermediate in the bioenergetics of any living cell. It is sustained mainly by photosynthesis and respiration through an elaborated, fine-tuned network of electron-driven proton transfers. Classical concepts of optimised catalysis have recently been challenged by hypotheses that focus on the necessity to hinder short circuit reactions and radical formation. Since the destructive consequences of an ill-tuned bioenergetic system directly or indirectly cause pathophysiological phenomena, a deeper understanding of this long lasting controversy has far-reaching consequences.
The appearance of oxygen on Earth has increased the challenge for redox proteins, since this highly oxidising molecule may adopt several redox states, some of which are very reactive and can cause damage to proteins as well as to DNA and lipids. One source for the production of reactive oxygen species are the redox sites in proteins where electron transfer from a one-electron donor to a two-electron acceptor or the reverse takes place. Two-electron carriers are in general highly reactive in their singly reduced state and may react with oxygen. Proteins and organisms therefore need to protect themselves from these harmful side reactions.
One class of bioenergetic enzymes offers a unique system to investigate these questions: the Rieske/cytochrome b (Rieske/cytb) complexes present in respiration and photosynthesis, in aerobic and anaerobic organisms and in high potential (ubiquinone based) or low potential (menaquinone based) bioenergetic chains.
The best characterized member of this family, mitochondrial complex III has been shown to suppress ROS formation by stabilizing a semiquinone (SQ) in its quinone reducing site through modulation of the redox potentials of the Q/SQ/QH2 couples. Decades of research have shown that the plastidic and cyanobacterial counterpart of complex III, i.e. the cytochrome b6f complex does not employ this strategy and that the electrochemical equilibrium of individual redox steps differs from those of complex III. The structure of the cytochrome b6f complex from the green alga has been solved by us (Stroebel et al., Nature, 2003) and has revealed a unique quinone reducing site featuring heme with a free coordination site on the iron (heme ci). We have further investigated this site to decipher its mechanism (Alric et al. PNAS, 2005, Baymann et al., PNAS 2007) by combination of molecular studies with in vivo spectroscopy, but so far have not found a conclusive answer.
Phylogentically the b6f complex is part of a cluster of closely related enzymes present in anoxygenic phototrophs and non-phototrophs. In order to progress in our understanding of the exceptional properties of the b6f complex, this project proposes to extend our studies to non-cyanobacterial or -plastidic members of this cluster and more specifically to the anoxygenic phototrophic Heliobacteria and the aerobic O2-respiring Bacilli. All these organisms have been shown (Ducluzeau et al BBA 2008) or are likely to contain heme ci and are expected to help understanding the quinone reduction mechanism in these enzymes. Furthermore, since the Rieske/cytb complexes in these species are (Bacilli) or putatively are ((Heliobacteria) organized in supercomplexes, our project also has the potential to elucidate the make-up of a fully functional unit by crystallographic means.