Copper-dependent nitrous oxide reductase (N2OR) is the only known metabolic enzyme to reduce the inert greenhouse gas N2O to N2 in the final step of microbial denitrification [1, 2]. It utilizes the dinuclear CuA site for electron transfer to the unique [4Cu:2S] cluster CuZ [3], where substrate reduction takes place . Structural and spectroscopic studies have revealed the architecture and flexibility of both metal centers and can be reconciled with a current mechanistic proposal [4-6]. Yet, to reduce linear N2O to a bent anion radical in a first step, an activation energy barrier of 250 kJ·mol–1 must be overcome and much remains to be understood about how this is achieved [7].
N2OR exists in two forms, clade I and II, that differ in their assembly pathways and in the machinery that couples them to their cellular electron supply, the quinone pool in the cytoplasmic membrane [8-11]. Here the energetics of the reaction come into play, as the quinone pool should not be sufficiently reducing to convert N2O. The nos operon of clade I N2O reducers contains the nosRgene, encoding an 80 kDa membrane-integral iron-sulfur flavoprotein [12]. NosR shows few homologies to other known proteins, and we have consequently produced the protein and analyzes its spectroscopy, structure and dynamics. The highly unusual arrangement of cofactors in NosR suggests a novel mechanism of energy transduction that might address key mechanistic questions in microbial N2O reduction.