Formation of a cytochrome c–nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus

Abstract

Nitrous oxide reductase (N₂OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N₂O) to dinitrogen (N₂). N₂OR contains two metal centers; a binuclear copper center, Cu_A, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, Cu_Z, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (k_obs = 150 s⁻¹) between reduced horse heart (HH) cytochrome c and the Cu_A center in fully oxidized N₂OR. When fully reduced N₂OR was mixed with oxidized cytochrome c, a similar rate of electron transfer was recorded for the reverse reaction, followed by a much slower internal electron transfer from Cu_Z to Cu_A (k_obs = 0.1–0.4 s⁻¹). The internal electron transfer process is likely to represent the rate-determining step in the catalytic cycle. Remarkably, in the absence of cytochrome c, fully reduced N₂OR is inert towards its substrate, even though sufficient electrons are stored to initiate a single turnover. However, in the presence of reduced cytochrome c and N₂O, a single turnover occurs after a lag-phase. We propose that a conformational change in N₂OR is induced by its specific interaction with cytochrome c that in turn either permits electron transfer between Cu_A and Cu_Z or controls the rate of N₂O decomposition at the active site.