The multicopper enzyme nitrous oxide reductase reduces the greenhouse gas N2O to uncritical N2as the final step of bacterial denitrification. Its two metal centers require an elaborate assembly machinery that so far has precluded heterologous production as a prerequisite for bioremediatory applications in agriculture and wastewater treatment. Here, we report on the production of active holoenzyme inEscherichia coliusing a two-plasmid system to produce the entire biosynthetic machinery as well as the structural gene for the enzyme. Using this recombinant system to probe the role of individual maturation factors, we find that the ABC transporter NosFY and the accessory NosD protein are essential for the formation of the [4Cu:2S] site CuZ, but not the electron transfer site CuA. Depending on source organism, the heterologous hostE. colican, in some cases, compensate for the lack of the Cu chaperone NosL, while in others this protein is strictly required, underlining the case for designing a recombinant system to be entirely self-contained.
The tetranuclear Cu Z cluster is the unique active site of nitrous oxide reductase, the enzyme that catalyzes the reduction of nitrous oxide to dinitrogen as the fi nal reaction in bacterial denitrifi cation. Three-dimensional structures of orthologs of the enzyme from a variety of different bacterial species were essential steps in the elucidation of the properties of this center. However, while structural data fi rst revealed and later confi rmed the presence of four copper ions in spectroscopically distinct forms of Cu Z , the exact structure and stoichiometry of the cluster showed signifi cant variations. A ligand bridging ions Cu Z1 and Cu Z2 was initially assigned as a water or hydroxo species in the structures from Pseudomonas nautica (now Marinobacter hydrocarbonoclasticus ) and Paracoccus denitrifi cans . This ligand was absent in a structure from ' Achromobacter cycloclastes ' , and could be reconstituted by iodide that acted as an inhibitor of catalysis. A recent structure of anoxically isolated nitrous oxide reductase from Pseudomonas stutzeri revealed the bridging ligand to be sulfi de, S 2-, and showed an unprecedented side-on mode of nitrous oxide binding to this form of Cu Z .
The gas nitrous oxide (N₂O) is generated in a variety of abiotic, biotic, and anthropogenic processes and it has recently been under scrutiny for its role as a greenhouse gas. A single enzyme, nitrous oxide reductase, is known to reduce N₂O to uncritical N₂, in a two-electron reduction process that is catalyzed at two unusual metal centers containing copper. Nitrous oxide reductase is a bacterial metalloprotein from the metabolic pathway of denitrification, and it forms a 130 kDa homodimer in which the two metal sites CuA and CuZ from opposing monomers are brought into close contact to form the active site of the enzyme. CuA is a binuclear, valence-delocalized cluster that accepts and transfers a single electron. The CuA site of nitrous oxide reductase is highly similar to that of respiratory heme-copper oxidases, but in the denitrification enzyme the site additionally undergoes a conformational change on a ligand that is suggested to function as a gate for electron transfer from an external donor protein. CuZ, the tetranuclear active center of nitrous oxide reductase, is isolated under mild and anoxic conditions as a unique [4Cu:2S] cluster. It is easily desulfurylated to yield a [4Cu:S] state termed CuZ (*) that is functionally distinct. The CuZ form of the cluster is catalytically active, while CuZ (*) is inactive as isolated in the [3Cu(1+):1Cu(2+)] state. However, only CuZ (*) can be reduced to an all-cuprous state by sodium dithionite, yielding a form that shows higher activities than CuZ. As the possibility of a similar reductive activation in the periplasm is unconfirmed, the mechanism and the actual functional state of the enzyme remain under debate. Using enzyme from anoxic preparations with CuZ in the [4Cu:2S] state, N2O was shown to bind between the CuA and CuZ sites, suggesting direct electron transfer from CuA to the substrate after its activation by CuZ.
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