N2O binding at a [4Cu:2S] copper–sulphur cluster in nitrous oxide reductase

Abstract

Nitrous oxide (N(2)O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO(2) 300-fold. In bacterial denitrification, N(2)O is reduced to N(2) by the copper-dependent nitrous oxide reductase (N(2)OR). This enzyme carries the mixed-valent Cu(A) centre and the unique, tetranuclear Cu(Z) site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its Cu(Z) site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N(2)OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N(2)O-pressurized crystals. The active Cu(Z) cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N(2)O bound side-on at Cu(Z), in close proximity to Cu(A). With the substrate located between the two clusters, electrons are transferred directly from Cu(A) to N(2)O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N(2)OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N(2)O bound to its reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase.

Copper control of bacterial nitrous oxide emission and its impact on vitamin B 12 -dependent metabolism

“…(for nitrite reductase), norCBQDEF (for nitric oxide reductase) and nosCRZDFYLX (for nitrous oxide reductase) loci, respectively. NosZ is the catalytic subunit of the N 2 O reductase and binds 12 Cu ions per functional homodimer in the copper-sulfide redox centers Cu A and Cu Z (8). To examine if Cu limitation has an impact on the denitrification genes at the level of transcription, global gene-expression analyses were performed using total RNA isolated from anaerobically grown cells under either Cu-H or Cu-L conditions.…”

“…Under certain conditions, the final step in denitrification is dispensed with and N 2 O is released into the atmosphere. One limiting factor in this process is copper (Cu) availability, the metal cofactor required by the N 2 O reductase (NosZ) that destroys N 2 O (5,7,8). During Cu-limitation the catalytic capacity of the Nos system may be exceeded by the rate of the preceding reactions that generate N 2 O (i.e., NO 3 − , NO 2 − , and NO reduction) and thus, N 2 O is emitted by denitrifying bacteria (7,9,10).…”

Nature’s way of handling a greenhouse gas: the copper-sulfur cluster of purple nitrous oxide reductase

“…A form II enzyme was investigated from ' Achromobacter cycloclastes ' (Paraskevopoulos et al , 2006 ), an isolate from a denitrifying community (Iwasaki and Matsubara , 1972 ) that is not a taxonomically valid species by current defi nition. Recently, the purple form I enzyme from Pseudomonas stutzeri was crystallized (Pomowski et al , 2010 ), yielding a structure that represents the physiologically active form of N 2 OR (Pomowski et al , 2011 ). The structures show slight differences in the metal centers, but to date the functional implications of this variability are scarcely understood (Table 1 ).…”

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

“…Catalysis of the head-to-tail homodimeric enzyme requires two copper centers (35,36). The periplasmic domain of the transmembrane iron-sulfur flavoprotein NosR is essential for NosZ function (37).…”