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.

Gram-negative bacteria represent a major public health concern due to their high resistance to antibiotics resulting in millions of human deaths world-wide each year. Their multilayered envelope contains an outer membrane (OM) that forms an effective permeability barrier shielding against noxious molecules, including several antibiotics. Being exposed to the cell surface, the OM represents a promising target for the development of new antimicrobials that can act from the exterior of the cell. The design of new antimicrobial stategies urges a better understanding of the molecular pathways of OM biogenesis. Integral OM proteins are crucial for envelope homeostasis. The beta-barrel assembly machinery (BAM) plays an essential role in OM protein assembly. The activity of BAM is regulated in space and time ensuring the constant supply of protein components to active sites of OM biogenesis. Many questions remain unresolved concerning the protein folding reaction mediated by BAM and the regulation of its activity throughout the OM. Motivated by the need to better understand the biogenesis of the bacterial OM, we have discovered that in the enterobacterium Escherichia coli, a member of gamma-proteobacteria, the lipoprotein DolP associates with the BAM complex and plays a critical role in OM homeostasis and integrity. DolP is widely conserved in gamma-, beta- and some alpha-proteobacteria contributing to the virulence of several pathogens, as well as to their ability to survive in the presence of some antibiotics. Our preliminary data reveal that DolP directly interacts with BamA, the catalytic subunit of BAM, promoting BamA folding and function. Inactivation of DolP phenocopies BamA depletion and makes cells sensitive to antibiotics that are normally excluded by Gram-negative bacteria. DolP localizes at active sites of OM biogenesis, ideally positioned to support BAM activity. The molecular mechanisms by which DolP contributes to OM assembly by the BAM complex and ensures OM integrity remain to be established. Our project uses an interdisciplinary approach to determine how DolP interacts with the BAM complex, influences its organization with partner complexes and regulatory factors, and supports its OM protein assembly activity. By employing a multiscale experimental strategy, we are investigating the molecular processes mediated by DolP i) at the cell envelope-wide scale, ii) in a chemically defined in vitro system, and iii) at the structural level. We will conduct these studies in the enterobacterial model organism E. coli and test our results in other pathogens of the gamma- and beta-proteobacterial classes. Our results will be important for the research of new antibacterial compounds that can interfere with OM integrity in Gram-negative bacterial pathogens.

We recently discovered a protein from the model marine diatom, Thalassiosira pseudonana, that belongs to a new subclass of carbonic anhydrase, iota-CA. We showed that this protein is involved in CO2-concentrating mechanisms and uses Mn2+ as a co-factor instead of Zn2+. Since we also showed that iota-CA is present in other marine diatoms and is widespread in the ocean, it could have a significant role in global biogeochemical cycling. The use of Mn2+ instead of Zn2+ may reflect its availability or affect the enzyme properties. The overarching scientific objective of this project is to elucidate the role of iota-CA in photosynthesis of marine diatoms, extending the analysis to a contrasting diatom, Phaeodactylum tricornutum, where the use of genetic tools are better established. This objective will be achieved within 3 workpackages combining a range of in vitro and in vivo methods. The aim of workpackage 1 is to unravel the detailed molecular mechanisms of the function, and architecture of iota-CA from the information yielded by its protein structure using a combination of structural, biophysical and biochemical methods. The aim of workpackage 2 is to elucidate the catalytic mechanisms of iota-CA, and the role of Mn2+ instead of Zn2+. The aim of the third workpackage is to determine the effect of iota-CA inactivation on diatoms growth and physiology using genetic engineering tools and proteomic analyses, to determine the contribution of iota-CA to the metabolism of the diatoms.

Hydrogenases are the enzymes that oxidize or produce H2 at a NiFe or FeFe active site. The principles of their active site mechanisms have been understood by combining a number of techniques over decades of investigations by the international scientific community, including the partners of this project. This knowledge is now crucial to help chemists produce the solar-fuel catalysts that we need to mitigate global warming and to reduce our dependence on fossil fuels. However, hydrogenases are very diverse in terms of structures and catalytic properties, and to become able to make more useful catalysts, one needs to understand what gives certain hydrogenases the properties that are particularly sought after: high turnover frequencies in either direction of the reaction in response to even a small departure from thermodynamic equilibrium (i.e., at small overpotential), and preference for the reaction with H2 over O2 when the catalyst must operate in air. This goal is now within reach, because the partners of the project have set the methods that can be used to produce and compare original enzymes, study them using advanced kinetic techniques, and re-engineer them with the goal of transferring certain catalytic properties between homologous enzymes. This project will combine the expertise of the three partners in biophysics (notably electrochemical kinetics), protein engineering and structural biology to produce and study very original hydrogenases. Identifying the structural features that confer useful natural catalytic properties to enzymes and understanding the molecular mechanisms by which this occurs will give invaluable knowledge about how ideal catalysts work, and how to improve synthetic catalysts.

Prokaryotes have evolved sophisticated strategies to regulate intra-cellular copper concentration since it is essential for cell survival to import copper and incorporate it at the active site of key enzymes, but a higher intra-cellular concentration is toxic. These copper-concentration regulation mechanisms can be insufficient in environments with high amount of copper, leading to cell death. This bactericidal property of copper has been exploited since the Middle Age and regains interest nowadays to substitute antibiotics. Besides, copper is also used by the immune system to eliminate pathogenic organisms. Nevertheless, how copper or copper complexes induces cell death is not understood. The ChapCop project aims at a better understanding at both the cellular and the molecular levels of the mechanisms responsible for copper-induced toxicity and the resistance strategies implemented by bacteria to resist to copper stress. This project is based on our recent preliminary results showing (i) that a high concentration of copper induces the denaturation of the stable structures of many proteins leading to the formation of protein aggregates and (ii) that some molecular chaperones are able to decrease copper-induced protein aggregation. Our working hypotheses is that molecular chaperones with structural zinc sites may play a major role in bacterial survival. Copper is likely to damage these zinc sites, leading to a structural transition enabling chaperone activation against copper-induced aggregation. Using Escherichia coli as a model, two main objectives will be pursued. 1- The relative impact of free copper or of various copper-complexes known for their anti-bacterial activities on protein aggregation will be determined. Copper-sensitive proteins that tend to aggregate under a variety of conditions will be identified as well as copper complexes with the most potent bactericidal effect. 2- The role of holdase chaperones in condition of copper stress will be assessed in vivo and in vitro on model proteins as well as on physiological clients, with a special focus on the Zn-containing holdases chaperones Hsp33 and DnaJ. The ChapCop project gathers five partners with complementary expertise in copper handling, (bio)chemical reactivity, proteostasis, and molecular chaperones. Our interdisciplinary approach relying on bioinorganic chemistry, microbiology, biochemistry and structural biology will provide major breakthroughs on the understanding of strategies dedicated to maintaining bacterial proteome integrity during toxic copper exposure and per se, will define the bacterial strategies to survive to copper-stress.