Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

• NOR steady-state kinetic experiments were conducted under NO or O2 turnover.
• Higher affinity of the enzyme for its natural substrate is demonstrated.
• New insights on the substrate inhibition profile, under NO turnover, are presented.
• NOR catalytic mechanism is revised here predicting relevant catalytic intermediates.

Nitric oxide reductase (NOR) from denitrifying bacteria is an integral membrane protein that catalyses the two electron reduction of NO to N2O, as part of the denitrification process, being responsible for an exclusive reaction, the NN bond formation, the key step of this metabolic pathway. Additionally, this class of enzymes also presents residual oxidoreductase activity, reducing O2 to H2O in a four electron/proton reaction. In this work we report, for the first time, steady-state kinetics with the Pseudomonas nautica NOR, either in the presence of its physiological electron donor (cyt. c552) or immobilised on a graphite electrode surface, in the presence of its known substrates, namely NO or O2. The obtained results show that the enzyme has high affinity for its natural substrate, NO, and different kinetic profiles according to the electron donor used. The kinetic data, as shown by the pH dependence, is modelled by ionisable amino acid residues nearby the di-nuclear catalytic site. The catalytic mechanism is revised and a mononitrosyl-non-heme Fe complex (FeBII-NO) species is favoured as the first catalytic intermediate involved on the NO reduction.

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