Reductively Activated Nitrous Oxide Reductase Reacts Directly with Substrate

Abstract

In the terminal step of bacterial denitrification, N₂O is converted to N₂ at the μ₄-sulfide bridged tetranuclear Cu_Z center of nitrous oxide reductase. The enzyme can be activated by reduced methyl viologen, with up to a 15-fold increase in specific activity. The reductively activated nitrous oxide reductase from Achromobacter cycloclastes was isolated and characterized by visible absorption and EPR spectroscopy, and both methods showed that the Cu_Z center can attain a [4Cu(I)] oxidation state. When N₂O was added to the activated, reductant-free enzyme, distinct spectral changes were observed, indicating that this state of the enzyme interacts with substrate. This was further supported by the detection of ¹⁵N-labeled product in the absence of steady-state turnover conditions. A new absorption band around 970 nm appeared following reaction of activated nitrous oxide reductase with N₂O, which may represent a catalytic intermediate state of the enzyme.