Nitrous Oxide Focus Group Nitrous Oxide Focus Group launch event Friday February 22 nd, 2008 Enzymes that make and consume Nitrous Oxide Nicholas Watmough and Andrew J Thomson Centre for Metalloprotein Spectroscopy and Biology School of Biological Sciences, School of Chemical Sciences and Pharmacy University of East Anglia

DJ Richardson INCREASE IN ATMOSPHERIC N 2 O Estimated that today 76% arises from agricultural soils Haber-Bosch process for fixation of atmospheric Nitrogen Centre for Metalloprotein Spectroscopy & Biology

How Do Soil Bacteria Respond to an Unlimited Supply of Fixed Nitrogen? Paracoccus denitrificans Oxygen Concentration

NO Reductase N 2 O Reductase Nitrate Reductase Nitrite Reductase NO 3 - NO 2 - NO N2ON2O N2N2 Typical denitrifying bacterium Centre for Metalloprotein Spectroscopy & Biology

NO Reductase N 2 O Reductase Nitrate Reductase Nitrite Reductas e NO 3 - NO 2 - NO N2ON2O N2N2 Molybdenum Iron or Copper Iron Copper Centre for Metalloprotein Spectroscopy & Biology

The Enzyme that Produces N 2 O is Nitric Oxide Reductase NO 3 - NO 2 - NON2N2 N2ON2O Nitrate Reductase Nitrite Reductase Nitric Oxide Reductase Nitrous Oxide Reductase 2NO + 2e - + 2H + N 2 O + H 2 O (E` 0 (pH 7.0) = V; ΔG° = kJ mol -1 ) K m [NO] = <1 µM Steady-state [NO] = <0.1 µM Centre for Metalloprotein Spectroscopy & Biology

NO e-e- Transferring electrons Binding substrate molecules? Heme Iron Assists Nitric Oxide Reductase Transform NO to N 2 O by…… Centre for Metalloprotein Spectroscopy & Biology

Our work involves the isolation and characterisation of Bacterial NOR Cell Culture Protein Purification Biochemistry and Spectroscopy Centre for Metalloprotein Spectroscopy & Biology

I ELECTRONS NorC NorB 2NO + 2e - + 2H + N 2 O + H 2 O Insulating Bacterial Membrane Three Hemes form a Nanowire that Conduct Electrons to the Site of N 2 O Formation Centre for Metalloprotein Spectroscopy & Biology

We Have Identified a Channel that Moves Protons to the Site of N 2 O Formation NO 3 - NO 2 - NON2N2 N2ON2O Nitrate Reductase Nitrite Reductase Nitric Oxide Reductase Nitrous Oxide Reductase 2NO + 2e - + 2H + N 2 O + H 2 O E122 E125 D185 T243 E267 E198 E20 2 PERIPLASM CYTOPLASM Model of NorB based on the coordinates of R.sphaeroides cytochrome aa 3 courtesy of Joachim Reiman University of Stockholm

Proton concentration (and the rate of N 2 O production?) will be highest in acidic soils N 2 O Formation is Controlled by the Rate at Which Protons are Delivered?? Centre for Metalloprotein Spectroscopy & Biology

Why do we Need to Understand the Rate of N 2 O Production? E122 E125 D185 T243 E267 E198 E202 PERIPLASM CYTOPLASM Model of NorB based on the coordinates of R.sphaeroides cytochrome aa 3 courtesy of Joachim Reiman University of Stockholm For…… Microbial Physiologists Metabolic Modellers Atmospheric Scientists and others…. Centre for Metalloprotein Spectroscopy & Biology

Nitrous Oxide Reductase An Enzyme that Destroys Laughing Gas Centre for Metalloprotein Spectroscopy & Biology

Free Energy Reaction pathway Kinetic pathways selected by catalysts leading to linked reactions and conservation of free energy N 2 O + 2 e- + 2 H+ N 2 + H 2 O ΔG = -338 kJ.M -1 N 2 O Reductase Centre for Metalloprotein Spectroscopy & Biology

Aerobic purification: pink, low activity, low copper content Anaerobic purification: purple, high activity, high copper content Semi-aerobic purification: Ink-blue, high activity, high copper content Caused by change of the oxidation state And state of the catalytic centre Cu Z (multiple forms) Different colours of N 2 OR Dithionite-reduced anaerobic form Mutant-form without Cu Z (similar: aerobic form) Oxidised anaerobic form

R Eady, S Hasnain et al. JMB 2006, COPPER SULPHUR Centre for Metalloprotein Spectroscopy & Biology

Centre for Metalloprotein Spectroscopy & Biology

Nitrous oxide reductase Cu A Cu Z Brown et al. (2000) Nature Struct. Biol. 7, ; Brown et al. (2000) J. Biol. Chem. 275,

Cu I 4 S Cu I 3 Cu II 1 S Cu I 2 Cu II 2 S +1e - slow (2h) -1e - fast +1e - -1e - Cu Z is a two electron storage catalytic centre Cu S His Catalytic redox-states of CuZ Centre for Metalloprotein Spectroscopy & Biology

Cu S His N NO N N O Centre for Metalloprotein Spectroscopy & Biology

Centre for Metalloprotein Spectroscopy & Biology Where next? To describe the catalytic process in molecular terms To understand the interaction between NOR and N 2 OR To understand biosynthesis of the catalyst CuZ To understand the genetic regulation of the enzyme - e.g. dependence on copper

Centre for Metalloprotein Spectroscopy & Biology Bacterial species that can Reduce N 2 0 to N 2 as an autonomous respiratory process Reduce Nitrate to N 2 O Reduce Nitrate to N 2 producing N 2 O to varying degrees

Gene regulators are DNA binding proteins that respond to small molecules NO (+5) 3 - NO (+3) 2 - NO (+2) N O (+1) 2 N (0) 2 NO Reductase N 2 O Reductase Nitrate Reductase Nitrite Reductase FNR Fumarate-Nitrate Regulator – O 2 FUR Ferrous Uptake Regulator - Fe NNR Nitric oxide Regulator -NO Regulation of Denitrification Centre for Metalloprotein Spectroscopy & Biology

NO (+5) 3 - NO (+3) 2 - NO (+2) N 2 O (+1) N (0) 2 Denitrification Nitric Oxide Reductase N 2 O Reductase Nitrate Reductase Nitrite Reductase REGULATION of Cu Z BIOSYNTHESIS Copper deficiency in soils Centre for Metalloprotein Spectroscopy & Biology

AMELIORATION STRATEGIES? Centre for Metalloprotein Spectroscopy & Biology

Biological Sciences  Adam Baker  Gareth Butland  Sarah Field  Karin Grönberg  Verity Lyall  David Richardson  Lola Roldan  Faye Thorndycroft Chemical Sciences and Pharmacy  Myles Cheesman  Harriet Seward  Andrew Thomson  Jess van Wonderen University of Stockholm  Pia Adelroth  Ulrika Flock  Joachim Reiman EMBL  Janneke Hendriks  Matti Saraste NO Reductase Centre for Metalloprotein Spectroscopy & Biology

T Rasmussen T Clarke J Farrar V Oganesyan NJ Watmough R Eady (JIC) BBSRC B Berks (U of Oxford) T Brittain (U of Auckland, NZ) W Zumft (U of Karlsruhe, Germany) N 2 O Reductase

Nitrous Oxide Focus Group Nitrous Oxide Focus Group launch event Friday February 22 nd,