Nitrous Oxide Reductase

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Nitrous Oxide Reductase

• Structure and Mechanism
• 1st February 2007
• Rajiv Luthra

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Introduction

• During denitrification nitrate ? nitrite ? nitric
  oxide ? nitrous oxide ? dinitrogen gas
• N2O is generated by denitrifying bacteria as a
  product of NO reduction

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Introduction

• N2O 2e- 2H ? N2 H2O
• Highly exergonic (?G 340 kJ/mol)
• N2O is a very inert gas at room temperature
• Efficient catalyst is necessary
• N2O is metabolized further by the enzyme N2O
  reductase (N2OR)
• N2OR Periplasmic multi-copper protein
• converts N2O into dinitrogen and water

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The need for N2O Reductase

• Terminal respiratory oxidant under anaerobic
  conditions generating ATP
• Lower intracellular nitrous oxide concentrations
  in order to prevent inhibition of nitrogen
  fixation
• High solubility of nitrous oxide (25 mM at 25C)
  relative to dinitrogen (0.6 mM at 25C)
• Preferential reduction of nitrous oxide by
  nitrogenase

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Structure

• Two redoxactive copper centres
• binuclear electron input site called CuA
• catalytic site termed CuZ

CuA
CuZ
Source Biochem. J. (2003) 369 (7788) Crystal
structure of nitrous oxide reductase from
Paracoccus denitrificans at 1.6Å resolution
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CuA centre of N2OR

• Related to the electron entry site of cyt c
  oxidase
• Site developed first in N2OR and was then adopted
  by cytochrome c oxidase
• Accepts electron from a reducing substrate
  undergoing a one e- redox change
• Feeds them to the CuZ site
• No role in the proton pumping

From Formation of a cytochrome cnitrous oxide
reductase complex is obligatory for N2O reduction
by Paracoccus pantotrophus Tim Rasmussen, Thomas
Brittain, Ben C. Berks, Nicholas J. Watmougha and
Andrew J. Thomson
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CuZ

• CuZ site where N2O is activated and reduced
• Evolved from an NO reductase
• CuZ is a tetranuclear copper cluster residing in
  the middle of a sevenbladed ß-propeller domain
• Inorganic sulphur bridging the four coppers of
  CuZ
• Novel type of copper-sulphur cluster

From Formation of a cytochrome cnitrous oxide
reductase complex is obligatory for N2O reduction
by Paracoccus pantotrophus Tim Rasmussen, Thomas
Brittain, Ben C. Berks, Nicholas J. Watmougha and
Andrew J. Thomson
8
Overall Mechanism
From Formation of a cytochrome cnitrous oxide
reductase complex is obligatory for N2O reduction
by Paracoccus pantotrophus Tim Rasmussen, Thomas
Brittain, Ben C. Berks, Nicholas J. Watmougha and
Andrew J. Thomson
9
CuA and CuZ
CuZ
CuA
From Formation of a cytochrome cnitrous oxide
reductase complex is obligatory for N2O reduction
by Paracoccus pantotrophus Tim Rasmussen, Thomas
Brittain, Ben C. Berks, Nicholas J. Watmougha and
Andrew J. Thomson
10
Suggested Structural Mechanism
Adapted from Mechanism of N2O Reduction by the
u4-S Tetranuclear CuZ Cluster of Nitrous Oxide
Reductase Serge I. Gorelsky, Somdatta Ghosh,
and Edward I. Solomon
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Suggested Structural Mechanism
H
H
e-
e-
Adapted from Mechanism of N2O Reduction by the
u4-S Tetranuclear CuZ Cluster of Nitrous Oxide
Reductase Serge I. Gorelsky, Somdatta Ghosh,
and Edward I. Solomon
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Inhibitors

• Acetylene most specific inhibitor of nitrous
  oxide reductase
• Other inhibitors azide anion and thiocyanate
• All isoelectronic with nitrous oxide
• Cyanide, nitrite and fluoride
• general copper ligands
• Prolonged exposure of the oxidised enzyme to high
  concentrations of nitrous oxide