Nitrous oxide reductase (N2OR) is a multicopper enzyme catalyzing the reduction of nitrous oxide to dinitrogen and water. It consists of two identical subunits, each with four Cu atoms/65.8 kDa. N2OR as isolated under the exclusion of air is a purple enzyme with an intense absorption maximum at 540 nm (sigma 16 mM-1 cm-1), and a characteristic CW EPR spectrum (X-band) with an unusual seven-line hyperfine pattern in the g-parallel region. The current model for N2OR suggests that the protein possesses two identical subunits, each carrying four copper atoms arranged in two novel binuclear centers: (1) the EPR-detectable, mixed-valence [Cu(1.5+)...Cu(1.5+)], S=1/2, CuA site, and (2) an EPR-silent, antiferromagnetically coupled [Cu(2+)...Cu(2+)] CuZ site. CuA is the primary electron acceptor and cycles between the mixed-valence and the fully reduced [Cu(1+)...Cu(1+)] state. CuZ either binds the substrate N2O in its [Cu(2+)...Cu(2+)] form and then accepts two electrons followed by cleavage of N2O and release of the products N2 and H20. Alternatively, formation of the reduced CuZ site precedes the binding and cleavage of N2O. The electronic and magnetic structures of the two dinuclear copper centers will be described based on extensive biochemical (amino acid sequence, catalytic activity) and spectroscopic (UV/VIS, EPR/ENDOR, EXAFS, CD/MCD) studies.?
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