As the issues of global warming continue to drive the global energy transition, the demand for clean energy carriers like hydrogen continues to grow. Whilst the industrial hydrogen production routes we employ today are estimated to release 1.3 billion tonnes of carbon dioxide into the atmosphere each year, Nature has evolved highly efficient [FeFe]-hydrogenase metalloenzymes capable of producing hydrogen under mild conditions and with minimal energy input. Their remarkable behavior has intrigued scientists for decades.
In particular, their reversible, bidirectional activity and high catalytic rates make [FeFe]-hydrogenases promising candidates for integration into scalable electrocatalytic hydrogen-producing technologies. However, their structural fragility, large size, and extreme sensitivity to oxygen are currently limiting their practical application.
This thesis presents a platform for the electrochemical investigation of a model [FeFe]-hydrogenase, ``\textit{Cp}I'', from the nitrogen-fixing soil bacterium \textit{Clostridium pasteurianum}. The first part of this work describes the development of a simple procedure for fabricating a mesoporous indium tin oxide (ITO) film, within which the enzyme can be immobilized to achieve high-performance hydrogen evolution. Using a multi-analytical approach, the interaction between the enzyme and electrode is examined, and the influence of the film morphology on enzyme performance is determined.
With a view toward developing scalable hydrogenase electrodes, we used the ITO electrode as a platform to address additional challenges that may come to hinder their eventual application in future biotechnologies. In particular, we simplified the electrode functionalization procedure to circumvent the need for enzyme purification, which is a time-intensive and costly step in enzyme preparation. Additionally, we observed that the mesoporous ITO film affords the immobilized [FeFe]-hydrogenase a degree of protection against oxygen-induced damage.
We hope this thesis advances our understanding of how [FeFe]-hydrogenases function within electrocatalytic systems and that the mesoporous ITO electrode developed in this work can serve as a useful platform for investigating other redox metalloenzymes in the future.
