Microscopie électrochimique à Nanocavité pour l’imagerie fonctionnelle de cascades enzymatiques. – CASCADE

This proposal describes an experimental nanoscale platform to address modern aspects of enzymatic biocatalysis. The aim of this project is to develop new experimental tools to both assemble artificial enzymatic complexes, in a way spatially controlled at the nanoscale, and to probe their functional behavior, at the scale of a few, or even individual, enzyme molecules. To this aim a new local probe electrochemical technique will be designed allowing the functional probing of enzyme clusters at the nanoscale. Addressing single, or a small number of a selected arrangement of biomolecules, is the unique way to understand the correlation between the enzyme activity and fluctuation of its behavior. In the present case, using a local probe electrochemical technique will specifically allow the functional behavior of enzyme clusters to be probed as a function of their spatial position in the enzyme assembly, i.e. as a function of their degree of diffusional coupling with neighboring enzyme populations . In living systems, the cellular organization of cooperating enzymes into supramolecular complexes is a metabolism key feature. A major advantage of such organization is the transfer of biosynthetic intermediates between catalytic sites without diffusion into the bulk phase of the cell. This so-called 'metabolic channelling' is supposed to combine an enhancement of the reaction rates with a fine tuning of metabolic pathways. Our first aim will be to experimentally challenge this model through the design of a spatially controlled assembly of artificial enzymatic complexes. Using electronic nanolithography with the help of the RTB at LAAS, we will design a nanoplateform with precise control of the positioning of two enzymes performing a redox cascade reaction. In the first combination, diffusion controlled reactions will be simulated by positioning single enzymes clusters several hundred nanometers one from each other. In the second set-up, the two enzymes will be clustered at the 10-50 nm scale in order to mimick channeling. Virus capsids will be used as templates for controlling these enzymes distributions. Since the final enzyme of the biosynthetic assembly will be a redox enzyme, i.e. glucose oxidase, which accepts a large range of soluble cofactors, electrochemistry is the technique of choice to study the kinetic behavior of the reconstructed enzymatic pathway. In order to reproduce the confined working environment of the natural enzymatic supramolecular complexes, which minimizes diffusional dispersion of the reactants, we propose to develop and to use for this study an innovative nanoelectrochemical technique which will allow a small patch of the enzyme bearing surface to be confined under a 'hollow' microlectrode. This 'nanocavity ' microlectrode will be fabricated at the apex of an AFM probe. The resulting combined nanocavity AFM-SECM probe will allow the enzyme clusters to be located on the surface and the electrochemical currents associated to the enzymatic redox catalysis to be measured. Using nanocavity microelectrodes ~ 100-500 nm in size it will be possible to 'cap' a selected population of enzymes and to probe their collective functional behavior. The local probe configuration will allow the enzyme populations to be selected based on their spatial position i.e based on their degree of diffusive coupling with neighboring enzyme clusters. Using even smaller nanocavity microelectrodes, ~ a few 10 nm in size, will make the functional behavior of individual enzyme molecules experimentally accessible. Addressing the catalytic behavior of single enzyme molecules is, in itself , a biologically relevant question, since in classical enzymology, insight into the dynamic behavior of enzymatic processes is typically derived from ensemble measurements. The kinetic behavior observed is the mean of the contribution of individual enzymes fluctuating between more or less active conformations. A way for living systems to finely tune metabolic networks is to modulate enzymes conformations (ie through phosphorylation or protein regulator interactions). Direct experimental information about the dynamics at the single-molecular level, however is sparse and has until recently been primarily deduced from molecular-dynamics simulations. Exploring the individual, or a few, nano-scale behavior of molecules in complex local environments is of great need to better understand the chemical reaction efficiency and functionality of enzyme at work. Observing a single enzyme removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamic changes, without synchronization. Such an approach should reveal itself extremely valuable for an in depth understanding of in vivo complex enzymatic mechanisms.