Photocatalysis on titanium dioxide is dependent on the presence of suitable electron donors and acceptors in the system. In this paper, we tested Pyrococcus furiosus, Acetomicrobium flavidum and Clostridium pasteurianum hydrogenase as electron acceptors from irradiated titanium dioxide, with tris(hydroxymethyl)aminomethane as electron donor. The adsorption and correct orientation of the hydrogenases on the titanium dioxide surface, as well as their structural features, seems important for determining an efficient electron transfer from the irradiated powder to the enzyme. In order to obtain a comparison between oxygen and hydrogenase as electron acceptors, the hydrogen photoproduction in the presence of P. furiosus hydrogenase was compared with the oxygen reduction in the same conditions. The oxygen reduction followed a Langmuir–Hinshelwood kinetics. Hydrogenase turned out significantly faster than oxygen, with a turnover number of about 1300min−1.