A new CuZ active form in the catalytic reduction of N₂O by nitrous oxide reductase from Pseudomonas nautica

The final step of bacterial denitrification, the two-electron reduction of N₂O to N₂, is catalyzed by a multicopper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting nonactive state (1Cu^II/3Cu^I) to the fully reduced state (4Cu^I), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N₂O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ°, in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 ± 0.9) × 10⁵ M^-1 S ^-1 was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ° species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/ inactivation mechanism of the enzyme are discussed.