Enzymes that make and consume

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• Enzymes that make and consume
• Nitrous Oxide

Nicholas Watmough and Andrew J Thomson Centre for
Metalloprotein Spectroscopy and BiologySchool of
Biological Sciences, School of Chemical Sciences
and Pharmacy University of East Anglia
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INCREASE IN ATMOSPHERIC N2O Estimated that today
76 arises from agricultural soils
Haber-Bosch process for fixation of atmospheric
Nitrogen
DJ Richardson
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How Do Soil Bacteria Respond to an Unlimited
Supply of Fixed Nitrogen?
Paracoccus denitrificans
Oxygen Concentration
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N2O
NO
NO2-
NO3-
N2
Nitrite Reductase
N2O Reductase
Nitrate Reductase
NO Reductase
Typical denitrifying bacterium
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NO
NO2-
N2O
NO3-
N2
Nitrite Reductase
N2O Reductase
Nitrate Reductase
NO Reductase
Iron or Copper
Copper
Molybdenum
Iron
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The Enzyme that Produces N2O is Nitric Oxide
Reductase
KmNO lt1 µM Steady-state NO lt0.1 µM
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Heme Iron Assists Nitric Oxide Reductase
Transform NO to N2O by
Transferring electrons
e-
Binding substrate molecules?
NO
NO
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Our work involves the isolation and
characterisation of Bacterial NOR

• Cell Culture
• Protein Purification
• Biochemistry and Spectroscopy

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Three Hemes form a Nanowire that Conduct
Electrons to the Site of N2O Formation
Insulating Bacterial Membrane
NorB
NorC
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We Have Identified a Channel that Moves Protons
to the Site of N2O Formation
2NO 2e- 2H
N2O H2O
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N2O Formation is Controlled by the Rate at Which
Protons are Delivered??
Proton concentration (and the rate of N2O
production?) will be highest in acidic soils
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Why do we Need to Understand the Rate of N2O
Production?

• For
• Microbial Physiologists
• Metabolic Modellers
• Atmospheric Scientists
• and others.

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Nitrous Oxide Reductase An Enzyme that Destroys
Laughing Gas

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N2O Reductase
N2O 2 e- 2 H
Free Energy
?G -338 kJ.M-1
N2 H2O
Reaction pathway
Kinetic pathways selected by catalysts leading to
linked reactions and conservation of free energy
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Different colours of N2OR
Anaerobic purification purple, high activity,
high copper content
Aerobic purification pink, low activity, low
copper content
Semi-aerobic purification Ink-blue, high
activity, high copper content
Dithionite-reduced anaerobic form Mutant-form
without CuZ (similar aerobic form) Oxidised
anaerobic form
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COPPER
SULPHUR
R Eady, S Hasnain et al . JMB 2006, 362 55
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Nitrous oxide reductase
Brown et al. (2000) Nature Struct. Biol. 7,
191-195 Brown et al. (2000) J. Biol. Chem. 275,
41133-41136
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Catalytic redox-states of CuZ
CuI2CuII2S
1e- slow (2h)
-1e- fast
CuI3CuII1S
CuI4S
CuZ is a two electron storage catalytic centre
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Where next?

• To describe the catalytic process in molecular
  terms
• To understand the interaction between NOR and
  N2OR
• To understand biosynthesis of the catalyst CuZ
• To understand the genetic regulation of the
  enzyme - e.g. dependence on copper

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Bacterial species that can

• Reduce N20 to N2 as an autonomous respiratory
  process
• Reduce Nitrate to N2O
• Reduce Nitrate to N2 producing N2O to varying
  degrees

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Regulation of Denitrification
Nitrate Reductase
Nitrite Reductase
NO (2)
NO Reductase
FNR Fumarate-Nitrate Regulator O2
N2O Reductase
NNR Nitric oxide Regulator -NO
FUR Ferrous Uptake Regulator - Fe
Gene regulators are DNA binding proteins that
respond to small molecules
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Denitrification
Nitrate Reductase
Nitrite Reductase
NO (2)
Nitric Oxide Reductase
REGULATION of CuZ BIOSYNTHESIS Copper deficiency
in soils
N2O Reductase
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AMELIORATION STRATEGIES?
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NO Reductase

• Biological Sciences
• Adam Baker
• Gareth Butland
• Sarah Field
• Karin Grönberg
• Verity Lyall
• David Richardson
• Lola Roldan
• Faye Thorndycroft

• EMBL
• Janneke Hendriks
• Matti Saraste

• University of Stockholm
• Pia Adelroth
• Ulrika Flock
• Joachim Reiman

• Chemical Sciences and Pharmacy
• Myles Cheesman
• Harriet Seward
• Andrew Thomson
• Jess van Wonderen

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N2O Reductase
T Rasmussen T Clarke J Farrar V Oganesyan NJ
Watmough R Eady (JIC)
B Berks (U of Oxford) T Brittain (U of
Auckland, NZ) W Zumft (U of Karlsruhe,
Germany)
BBSRC
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