The multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2O to N2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c552 concentration dependence, we estimate the following kinetic parameters: Kmc552 = 50.2 ± 9.0 μM and Vmaxc552 = 1-8 ± 0.6 units/mg. The N2O concentration dependence indicates a KmN2O of 14.0 ± 2.9 μM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c552 is used as the electron donor (pKa = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by 1H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.