A theoretical study on nitric oxide reductase activity in a ba3-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba3-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a3 and is partly reduced having some nitroxyl anion character (3NO−), and it is thus activated toward the attack by the second NO. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to CuB is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the NO bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the NO bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the NO bond and form N2O at a feasible rate. The cleavage of the NO bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.