Pomowski A, Zumft WG, Kroneck PM, Einsle O. (2011)
N2O binding at a
[4Cu:2S] copper-sulphur cluster in nitrous oxide reductase
Nature 477: 234-237.
Nitrous oxide (N2O)
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 CO2 300-fold. In bacterial denitrification, N2O is reduced to N2
by the copper-dependent nitrous oxide reductase (N2OR).
This enzyme carries the mixed-valent CuA
centre and the unique, tetranuclear CuZ
site. Previous structural data were obtained with enzyme isolated in the
presence of air that is catalytically inactive without prior reduction. Its CuZ
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 N2OR 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
N2O bound side-on at CuZ, in close
proximity to CuA. With the substrate located between the two clusters,
electrons are transferred directly from CuA to N2O,
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 N2OR 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 N2O 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.