Nitrous Oxide Focus Group launch event Friday February
Nitrous Oxide Focus Group launch event Friday February 22nd, 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 Nitrous Oxide Focus Group INCREASE IN ATMOSPHERIC N2O Estimated that today 76% arises from agricultural soils Haber-Bosch process for fixation of atmospheric Nitrogen Centre for Metalloprotein Spectroscopy & Biology
DJ Richardson Paracoccus denitrificans Oxygen Concentration How Do Soil Bacteria Respond to an Unlimited Supply of Fixed Nitrogen? NO2- NO3- Nitrate Reductas e NO N2O
Nitrite N 2O Reducta Reductase se NO Reductase Typical denitrifying bacterium Centre for Metalloprotein Spectroscopy & Biology N2 NO3- NO2- Nitrate Reductas
e Molybdenu m Centre for Metalloprotein Spectroscopy & Biology Nitrite Reduct ase Iron or Copper NO N 2O N2 N2O
NO ReductaseReductase Coppe r Iron The Enzyme that Produces N2O is Nitric Oxide Reductase Nitrate Reductase NO3- Nitrite Reductase NO2- Nitric Oxide Reductase NO
2NO + 2e- + 2H+ Nitrous Oxide Reductase N2O N2 N 2 O + H2 O (E`0 (pH 7.0) = +1.177V; G = -306.3 kJ mol-1) Centre for Metalloprotein Spectroscopy & Biology Km[NO] = <1 M Steady-state [NO] = <0.1 M Heme Iron Assists Nitric Oxide Reductase Transform NO
to N2O by Transferring electrons e- Binding substrate molecules? NO Centre for Metalloprotein Spectroscopy & Biology NO Our work involves the isolation and characterisation of Bacterial NOR Cell Culture Protein Purification Biochemistry and Spectroscopy Centre for
Metalloprotein Spectroscopy & Biology Three Hemes form a Nanowire that Conduct Electrons to the Site of N2O Formation 2NO + 2e- + 2H+ ELECTRONS N2O + H2O I NorC Centre for Metalloprotein Spectroscopy & Biology NorB Insulating
Bacterial Membrane We Have Identified a Channel that Moves Protons to the Site of N2O Formation Nitrite Reductase Nitrate Reductase NO3- NO2E122 Nitric Oxide Reductase NO Nitrous Oxide Reductase
N2O N2 PERIPLASM 2NO + 2e- + 2H+ E125 D185 T243 E198 E267 E2 02 CYTOPLASM Model of NorB based on the coordinates of R.sphaeroides cytochrome aa3 courtesy of
Joachim Reiman University of Stockholm N2O + H2O N2O Formation is Controlled by the Rate at Which Protons are Delivered?? e122d anpassad till kmax=160, pK=9.1 k0=0 1000 kobs (s-1) E122D wildtype: km=246 100 pk=6.589
k0=3.0000(L) WT 10 1 5 6 7 8 9 10 pH oton concentration (and the rate of N2O production?) will be highest in acidic soi
Centre for Metalloprotein Spectroscopy & Biology Why do we Need to Understand the Rate of N2O Production? E122 For PERIPLASM E125 Microbial Physiologists Metabolic Modellers Atmospheric Scientists D185 T243
E198 E267 E202 and others. CYTOPLASM Centre for Metalloprotein Spectroscopy & Biology Model of NorB based on the coordinates of R.sphaeroides cytochrome aa3 courtesy of Joachim Reiman University of Stockholm Nitrous Oxide Reductase An Enzyme that Destroys Laughing Gas Centre for
Metalloprotein Spectroscopy & Biology N2O + 2 e- + 2 H+ Free Energy N2O Reductase G = -338 kJ.M-1 N2 + H 2O Reaction pathway Kinetic pathways selected by catalysts leading to linked reactions and conservation of free
energy Centre for Metalloprotein Spectroscopy & Biology Different colours of N2OR Anaerobic purification: purple, high copper Caused byactivity, changehigh of the content oxidation state Aerobic purification: And state of the catalytic pink,
lowCu activity, low copper centre Z (multiple forms) 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 COPPER Centre for Metalloprotein Spectroscopy & Biology SULPHUR
R Eady, S Hasnain et al . JMB 2006, 362 55 Centre for Metalloprotein Spectroscopy & Biology Nitrous oxide reductase CuA Brown et al. (2000) Nature Struct. Biol. 7, 191-195; Brown et al. (2000) J. Biol. Chem. 275, 41133-41136 CuZ Catalytic redox-states of CuZ His
His CuI2CuII2S Cu Cu -1efast S Cu His His His Cu His +1eslow (2h) CuI3CuII1S
-1e- +1e- CuI4S His CuZ is a two electron storage catalytic centre Centre for Metalloprotein Spectroscopy & Biology N N O N N O
His Cu His Cu S Cu His His Cu His Centre for Metalloprotein Spectroscopy & Biology His
His Where next? To describe the catalytic process in molecular terms To understand the interaction between NOR and N 2OR 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 N20 to N2 as an autonomous respiratory process Reduce Nitrate to N2O Reduce Nitrate to N2 producing N2O to varying degrees
Centre for Metalloprotein Spectroscopy & Biology Regulation of Denitrification NO 3- (+5) Nitrate Reductase NO 2- (+3) Nitrite Reductase NO (+2) NO Reductase FNR Fumarate-Nitrate Regulator O2 NNR Nitric oxide Regulator -NO N 2O (+1)
N2O Reductase N 2 FUR Ferrous Uptake Regulator - Fe Gene regulators are DNA binding proteins that respond to small molecules Centre for Metalloprotein Spectroscopy & Biology (0) NO3- (+5) Nitrate Reductase Denitrificatio n
NO2- (+3) Nitrite Reductase NO (+2) Nitric Oxide Reductase N2O (+1) Centre for Metalloprotein Spectroscopy & Biology N2O Reductase N2 (0) REGULATION of CuZ BIOSYNTHESIS Copper deficiency in soils AMELIORATION STRATEGIES?
Centre for Metalloprotein Spectroscopy & Biology NO Reductase Biological Sciences Adam Baker Gareth Butland Sarah Field Karin Grnberg Verity Lyall David Richardson Lola Roldan Faye Thorndycroft Chemical Sciences and Pharmacy Myles Cheesman Harriet Seward Andrew Thomson Jess van Wonderen Centre for
Metalloprotein Spectroscopy & Biology EMBL Janneke Hendriks Matti Saraste University of Stockholm Pia Adelroth Ulrika Flock Joachim Reiman N2O Reductase T Rasmussen T Clarke J Farrar V Oganesyan NJ Watmough B Berks (U of Oxford)
T Brittain (U of Auckland, NZ) W Zumft (U of Karlsruhe, Germany) R Eady (JIC) BBSRC Nitrous Oxide Focus Group launch event Friday February 22nd, 2008 www.nitrousoxide.org Nitrous Oxide Focus Group
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