Heme-copper oxidases (HCuOs) are best known as terminal oxidases in the aerobic respiratory chain, in which they catalyze the reduction of oxygen to water. By receiving protons and electrons from opposite sides of the membrane as well as pumping protons, HCuOs contribute to the electrochemical proton gradient over the membrane that can be used for ATP synthesis. Divergent members of the HCuO superfamily are nitric oxide reductases (NORs) that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N₂O) as part of the denitrification process, an alternative respiratory pathway.

The first part of the thesis focuses on electron and proton transfer reactions that are associated with the reductive conversion of NO to N₂O and O₂ to H₂O by the NOR from Paracoccus denitrificans. Our data show that proton uptake in NOR is not electrogenic (protons and electrons are taken up from the same side of the membrane) and that no protons are pumped. Also, structural variants have been investigated and the results suggest a role for these residues in proton transfer. Further, we show that lowering the pH leads to a higher NO reduction rate, while this effect is partially counteracted by a larger degree of substrate inhibition at low pH.

The second part deals with proton transfer and electrical potential generation in the reaction between the cbb₃ oxidase from Rhodobacter sphaeroides and O₂ or NO. Our data show that NO reduction by cbb₃ oxidase is not coupled to proton translocation and that the direction of proton uptake is dependent on substrate. Our findings suggest that the proton pumping mechanism in HCuOs is incompatible with NO reduction intermediates.

Finally, experiments on structural variants of the ba₃ oxidase from Thermus thermophilus indicate a functional role for the inspected residues in proton transfer and support the suggestion that a single proton-transfer pathway is used in the ba₃ oxidase.