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Fuel cell is a device that can directly convert chemical energy into electrical energy. For low-temperature fuel cells, catalysts are required. Fuel cells using Pt-based or other non-biological materials as catalysts are known as conventional fuel cells. Inspired from Nature, enzymes can be used as catalysts in fuel cells known as enzyme-based fuel cells. The conventional and enzymatic fuel cells share the same underlying electrochemical principles, while enzyme-based fuel cells have their intrinsic advantages and disadvantages due to enzyme properties. The objective of this thesis is to investigate the current/voltage/ power/stability capabilities of enzyme-based membrane-less H2 fuel cells in order to design the enzymatic fuel cells with improved performance. This thesis presents a facile, effective method for the construction of 3D porous carbon electrodes. The 3D porous carbon electrodes are constructed by compacting suitable carbon nanomaterials into discs. The 3D porous carbon electrodes, with large roughness, high specific surface area, and optimized pore size distribution, are able to increase the loading density of enzymes, that is, reaction sites per unit geometric electrode area. The high loading density of enzymes can result in the high current/power density of the enzyme-based membrane-less H2 fuel cells. Moreover, the large enzyme loading can bring about the improvement in fuel cell stability because current becomes limited by mass transport of dissolved gases rather than enzyme immobilization so that neither inactivation nor desorption of enzymes would influence the current output. Based on one type of 3D porous carbon electrodes, the maximum power density of enzyme-based membrane-less H2 fuel cells has increased to the mW•cm2 level by at least one order of magnitude and the half-life has also increased from several hours to one week. This thesis presents a method for the increase in power density otherwise limited by low cathodic currents due to meagre O2 in non-explosive H2-rich H2-air mixtures. The power density of enzyme-based membrane-less H2 fuel cells can be increased by re-proportioning cathode/anode geometric area ratio to balance the cathodic and anodic currents under such an unusual H2-air mixture. This thesis also demonstrates that the 3D porous carbon electrode can improve the apparent O2 tolerance of anodic catalysts – hydrogenases, which are very important for the fuel cell performance. The degrees of apparent O2 tolerance for both O2-tolerant and O2-sensitive [NiFe]-hydrogenases are greatly increased based on the 3D porous carbon electrodes, so that even an O2-sensitive [NiFe]-hydrogenase can be used as an anodic catalyst in the enzyme-based membrane-less H2 fuel cell under a non-explosive H2-rich H2-air mixture. This thesis presents a design of a test bed in which series and parallel connections of sandwich-like electrode stacks can be varied. The fuel cell test bed has demonstrated low-loss interconnects and efficient stack configuration. Operated under a non-explosive H2-air mixture containing only 4.6% O2 at 20 °C, the maximum volume power density of the fuel cell test bed exceeds 2 mW•cm3, capable of powering electronic gadgets, which is a good demonstration of electricity that originates from the buried active sites of enzymes and is transmitted by long-range electron hopping in accordance with Marcus theory.

Nitrogenase is a metalloenzyme that plays a key role in biological nitrogen fixation by catalysing the reduction of dinitrogen to ammonia. Study of nitrogenase is particularly challenging because of its unique electron transfer and catalytic components. This Thesis describes the development of a mediated electron transfer system for the MoFe protein of nitrogenase, in order to overcome the complexity of electron transfer by the native reductant Fe protein coupled to hydrolysis of ATP. A series of redox mediators was employed including Eu^III/II-polyaminocarboxylate complexes, which have reduction potentials in a very negative range. In the presence of the redox mediators, the wild type MoFe protein exhibits a catalytic current due to protein-catalysed proton reduction. With this mediated electron transfer method, the potential of proton reduction by nitrogenase was determined for the first time. The redox mediator system was also applied in an infrared (IR) spectroelectrochemical study of CO binding to the wild type and β-98^His variant MoFe protein. The first IR evidence was provided for ATP-independent CO binding to the active site of the MoFe protein, in both the wild type and the variant. The peak wavenumbers and time-dependent changes in intensity found in this study are consistent with the result of previous CO coordination with nitrogenase obtained by electron transfer from the Fe protein driven by ATP. This strongly suggests that this mediated electron transfer approach can deliver low potential electrons into the MoFe protein and reduce the active site FeMoco to the substrate binding level. Moreover, this technique allows electrocatalytic activity of the protein to be monitored and the change in redox activity can be correlated directly to the potential. With the same technique, a study of cyanide binding was performed on different variant MoFe proteins of nitrogenase. The redox properties of the isolated cofactor of Mo- and V-dependent nitrogenase were investigated in parallel to the study of the protein-bound cofactors. It was found that FeVco, the active site from V-nitrogenase, exhibited different redox properties compared to that of Mo-nitrogenase. This might account for the unexpected activity in CO reduction that was reported previously for V-nitrogenase.