Novel methods for infrared spectroelectrochemical characterization of hydrogenase films

Abstract

Novel methods and materials including nanoporous gold leaf films, attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy with optically coupled wafers and biocompatible gold coated doped silicon electrodes were developed for the spectroelectrochemical study of anaerobic enzymes capable of catalyzing the reversible oxidation and reduction of hydrogen known as hydrogenase. The study of hydrogenases themselves and their interaction with inorganic substrates is essential for the future development of biohybrid, biomimetic or bio-inspired devices capable of the conversion of solar energy to hydrogen fuel. In this work, [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is studied to understand the mechanism by which enzymes activate hydrogen using base metals as opposed to noble metals. Due to the limitation of the quantity of available hydrogenase and the nature of spectroelectrochemistry, novel methods presented in this work focus on optimizing the efficiency with which proteins are used in addition to maximizing IR absorbance and electrochemical stability. Confinement of proteins using a nanoporous gold leaf film was shown to maximize the concentration of proteins near the reflection plane of an attenuated total reflectance (ATR) prism while still allowing proteins to interact with a bulk electrolyte. Optically coupling wafers to an ATR prism allowed for the rapid testing of organic functionalizations or for other treatments deemed too extreme for immediate testing with a delicate and expensive ATR crystal. Biocompatible gold coated doped silicon electrodes were designed as a method of efficiently transferring charge to an adsorbed protein while remaining transparent in the infrared. The drying of CaI films for the purpose of concentrating as much as possible on an ATR prism surface revealed IR spectra as of yet to be seen in the literature. Novel techniques were found to be useful for general spectroelectrochemistry and study of other proteins as was demonstrated with various redox couples and the common protein, myoglobin.