No Laughing Matter: The Unmaking of the Greenhouse Gas Dinitrogen Monoxide by Nitrous Oxide Reductase

Abstract

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.

Functional assembly of nitrous oxide reductase provides insights into copper site maturation

“…The 7-line hyperfine pattern of the X-band electron paramagnetic resonance (EPR) spectrum ( Fig. 2D) confirmed the mixed-valent [Cu 1.5+ :Cu 1.5+ ] state of oxidized Cu A (7,11). These are the exact spectral features defining the native form I of the enzyme (5,23).…”

“…This transition was direct, as evidenced by an isosbestic point at 615 nm ( Fig. 2C) (11). The 7-line hyperfine pattern of the X-band electron paramagnetic resonance (EPR) spectrum ( Fig.…”

“…Complete oxidation with potassium ferricyanide led to a color change to purple, with absorption maxima at 538 nm and 780 nm and a shoulder at 485 nm ( Fig. 2 A, purple), the defining spectral features of form I N 2 O reductase, with Cu Z as a [4Cu:2S] cluster (4,5,11). From here, the addition of ascorbate resulted in the exclusive reduction of Cu A , retaining a Cu Z spectrum with two distinct bands at 562 nm and 625 nm (Fig.…”

“…Initial 3D structures of NosZ from Marinobacter hydrocarbonoclasticus (8), Paracoccus denitrificans (9), and Achromobacter cycloclastes (10) consistently contained Cu Z as a [4Cu:μ 4 -S] center, i.e., four Cu ions with a central, interstitial sulfide, while in Pseudomonas stutzeri ZoBell, the tetranuclear cluster contained an additional sulfide, revealing the complete, native [4Cu:2S] configuration (4). This form, Cu Z , is readily desulfurylated during protein isolation to yield a [4Cu:S] form termed Cu Z * (11). Beside its two copper centers, NosZ also binds one ion each of Ca 2+ , K + , and Cl − per monomer (4,12).…”

Country, Cover or Protection: What Shapes the Distribution of Red Deer and Roe Deer in the Bohemian Forest Ecosystem?

“…The only large predator is Eurasian lynx ( Lynx lynx ), which was reintroduced in the 1970s and 1980s [32]. At present, this lynx population is stagnant [33]. Lynx prey mainly on roe deer and to a much lesser extent on red deer [34].…”