VIPER: Vibrational probes for redox enzyme exploration

Redox enzymes are at the heart of a wide range of bioenergetic processes essential for all living organisms. They operate under mild conditions with high specificity, low overpotential and high turnover rates. Deciphering "how redox enzymes operate" is a prerequisite before harnessing their outstanding properties. Amongst a plethora of biophysical techniques, Surface Enhanced InfraRed Absorption (SEIRA) spectroelectrochemistry shines at probing redox enzymes in situ and in operando revealing information on secondary structure elements, orientation, catalytic mechanisms, redox chemistry and functionality.

However, despite this outstanding potential, SEIRA spectroelectrochemistry faces limitations mainly arising from the nature of the biological sample itself. In fact, it is nearly impossible to rely exclusively on intrinsic infrared markers of the protein to elucidate structural or environmental variations at a local level. Consequently, evaluating the immobilised redox enzyme orientation or observing protein dynamics is difficult as it is hampered by the global response of the protein amide bands. Similarly, monitoring catalysis at the redox active site is restricted to only a handful of enzymes.

The VIPER project aims at lifting off those limitations for: (1) improving SEIRA resolution and (2) broadening the range of investigable redox enzymes. The methodology will rely on the introduction of site-specific Vibrational Probes (VPs), into a model redox enzyme (Laccase). Vibrational probes are small molecules which vibrational mode is sensitive to its immediate environment in terms of electrostatics and H-bonding. Laccase catalytic activity couples the one-electron oxidation of a wide range of organic and inorganic substrates to the four-electron reduction of molecular oxygen into water. We will decipher the orientation - activity relationships of laccase upon immobilisation onto electrodes. We will subsequently monitor its structural dynamics at a localised level and ultimately probe the various states of its catalytic cycle. This interdisciplinary project will pave the way for the spectroelectrochemical characterisation of poorly or yet uncharacterised redox enzymes.