Within the BioPac project, we aim to understand the molecular details of hydrogenase function in order to design and improve next generation biofuel cells. Hydrogenases are key enzymes for the enzymatic conversion of molecular hydrogen into protons and electrons, representing a promising replacement of chemical catalysts in fuel cell devices. To obtain a working biofuel cell, we have been studying the Aquifex Aeolicus (Aa) hydrogenase enzyme because of its resistance to oxygen and carbon monoxyde. Moreover, the presence of a putative transmembrane helix and protein-DDM interaction represent two challenging features to efficiently immobilize the protein on the electrode surface. Using classical molecular dynamics simulations in explicit solvent, we compare the properties of the enzyme from Desulfovibrio fructosovorans, a much studied bacterium, with the one from Aa. We focus mainly on surface properties with the aim to optimize immobilisation on an electrode surface. We also analyze differences among both enzymes as they might provide insight into the origin of oxygen resistance. Moreover, REFT simulations have been used to address both the DDM-protein interaction and the transmembrane helix conformational landscape. In the experimental counterpart of the project, a first working fuel cell prototype was realized yielding 300 μW cm−2 power. Insights on the interaction between hydrogenase model and electrode will drive the experimental design of an improved electrode increasing the efficiency of the association and hence of electron transfer.