Résumé
Deciphering which parameters control the immobilization of enzymes on solid supports is essential for the development of biotechnological devices such as biosensors, bioreactors, and enzymatic fuel cells. In this work, we used surface plasmon resonance (SPR) coupled with electrochemistry and polarization modulated infrared reflection absorption spectroscopy (PMIRRAS) to correlate the loading, the conformation, and the activity of Myrothecium verrucaria bilirubin oxidase (Mv BOD) enzymes immobilized on two oppositely charged self-assembled monolayers (SAMs) on gold electrodes. The SPR signal showed that an enzyme layer close to a monolayer was formed by spontaneous adsorption on both negatively and positively charged SAMs. A different catalytic process for O-2 reduction was obtained, however, being a direct catalysis at negative interfaces and a mediated catalysis at positive interfaces, in relation to the charge of the amino acids surrounding the surface of the Cu T1 and the dipole moment direction of Mv BOD. The stability of the enzymatic current was dependent on the SAM type. On the positively charged SAM electrode, the mediated catalytic current was stable with time. On the negatively charged SAM electrode, the direct catalytic current decreased continuously with time, leading to a decrease in the TOF (turnover frequency) from 114 to 7 s(-1), while the SPR signal remained stable, showing that the decrease in the catalytic current is not related to a desorption process. PMIRRAS studies suggested a conformational change in the tertiary structure as a result of strong electrostatic interactions between arginine residues close to the T1 Cu and the carboxylic functions on the SAM. Covalent binding, however, resulted in a great enhancement of the current stability, which can be explained by a rigidification of the enzyme layer.