Determination of the Active Form of the Tetranuclear Copper Sulfur Cluster in Nitrous Oxide Reductase

N₂OR has been found to have two structural forms of its tetranuclear copper active site, the 4CuS Cu_Z* form and the 4Cu2S Cu_Z form. EPR, resonance Raman, and MCD spectroscopies have been used to determine the redox states of these sites under different reductant conditions, showing that the Cu_Z* site accesses the 1-hole and fully reduced redox states, while the Cu_Z site accesses the 2-hole and 1-hole redox states. Single-turnover reactions of N₂OR for Cu_Z and Cu_Z* poised in these redox states and steady-state turnover assays with different proportions of Cu_Z and Cu_Z* show that only fully reduced Cu_Z* is catalytically competent in rapid turnover with N₂O.