Adjustment of bioenergetic enzymes to quinones – ADAPT2Q

Bioenergetic chains that fuel cellular metabolism have experienced dramatic changes since life appeared on Earth, with a diversification of environmental energy sources and the evolution of numerous dedicated enzymes. The mechanism of energy conservation, however, has remained nearly constant. It predominantly involves ATP synthases operating on a transmembrane proton motive force created mainly by the diffusion of liposoluble H+/electron carriers (quinones/quinols (Q/QH2)) in the membrane between bioenergetic enzymes carrying Q/QH2-binding sites. These Q/QH2 have diversified over the course of evolution and exhibit today a wide chemical and redox variability. The evolutionary history of this variability and the adaptations of enzymes to the different Q/QH2 are far from understood. Our hypothesis proposes that bioenergetic enzymes have adapted over time their Q/QH2-binding sites either for a specific Q/QH2 or for several Q/QH2-types, depending on the thermodynamic constraints of their redox reactions. When the thermodynamic constraint of the co-reaction with Q/QH2 and environmental substrate allows for it, the enzymes would have evolved their Q/QH2-binding site to accommodate any type of Q/QH2, whereas when the co-reaction doesn’t allow for it, enzymes would have evolved by shaping their Q/QH2-site for a specific Q/QH2. Here we aim at testing this hypothesis by working on four structurally distinct enzymes, i.e. the respiratory nitrate reductase Nar, the cytochrome bd oxidase, the Rieske/cytb complex and the alternative arsenite oxidase Arx. The two first ones are furthermore thermodynamically not constrained while the two last ones are. They embrace therefore the enzymatic diversity in bioenergetics. The choice of these four enzymes maximizes the expected information that can be obtained in the time allocated to this proposal. By combining cutting-edge bioinformatics, biochemistry, biophysics, molecular modeling, enzyme engineering and organic synthesis we will address four objectives: (1) establish the level of wild-type protein specificity towards Q/QH2-types, (2) identify amino acids which are part of wild-type Q/QH2-sites and those interacting with Q/QH2, (3) reveal the evolutionary link between Q/QH2-site structure and Q/QH2-type availability and (4) change the Q/QH2-specificity of the enzymes by protein engineering. The first output from ADAPT2Q is the synthesis of new hydrophilic and spin-labelled Q/QH2-analogs that will benefit the entire community working on Q/QH2-enzymes. The second output from ADAPT2Q is an unprecedented global view of the evolution events in the Q/QH2-biogenesis pathways across the prokaryotic world that will be useful to all researchers interested in bioenergetics. But the major benefit from ADAPT2Q will be a comprehensive molecular view of the interplay between Q and their partner enzymes, as well as the evolutionary history of this interplay over the past 3 billion years.