Electron transfer complex between nitrous oxide reductase and cytochrome c₅₅₂ from Pseudomonas nautica: Kinetic, nuclear magnetic resonance, and docking studies

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: K_mc552 = 50.2 ± 9.0 μM and V_maxc552 = 1-8 ± 0.6 units/mg. The N₂O concentration dependence indicates a K_mN2O 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 (pK_a = 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.