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

Recently Viewed Presentations

  • Strategy in the Global Environment - Cengage

    Strategy in the Global Environment - Cengage

    Strategy in the Global Environment ... Joint Venture Separate corporations come together to form a new corporate entity Two or more companies have an ownership stake, but combine resources for mutual benefit Sharing knowledge can be dangerous for the companies...
  • What is Water Potential? - Strongsville City Schools

    What is Water Potential? - Strongsville City Schools

    A flaccid cell is placed in the above beaker with a solute potential of -0.9 bars. a) What is the pressure potential of the flaccid cell before it was placed in the beaker? b) What is the water potential of...
  • Warm Up - Loudoun County Public Schools

    Warm Up - Loudoun County Public Schools

    Native American Crafts: Choose a tribe that you would like to learn more about. Each tribal station has a video about how to make a craft that is common in that tribe's culture. Watch the video and look at the...
  • MATSE 259 Properties and Processing of Engineering Materials

    MATSE 259 Properties and Processing of Engineering Materials

    Specimen geometry Experimental setup ASTM Standards E8 and E9 Callister, Materials Science and Engineering: An Introduction (2003) Ashby, Materials Selection in Mechanical Design (1999) A material property chart of Young's modulus and density. 0.30 207 Steel 0.34 107 Ti 0.33...
  • How Much Can Policy Affect How Much People Drive?

    How Much Can Policy Affect How Much People Drive?

    Adding in a recent RFF estimate of "externalities" would add a few cents per mile to the cost of autos Adding in the most extreme and implausible automobile costs proposed by smart growth activists would add about $0.23 per passenger-mile...
  • Essentials of GENETICS Eighth Edition 16 The Genetics

    Essentials of GENETICS Eighth Edition 16 The Genetics

    G1/S, G2/M, and M checkpoints Section 16.3 Cell-Cycle Control and Checkpoints G1/S checkpoints monitor cell size and determine whether DNA has been damaged. G2/M is where physiological conditions are checked (once G1/S are passed) prior to mitosis.
  • Disd Energy Management

    Disd Energy Management

    Attend more training provided by Michael Dorn, Executive Director of Safe Havens International, who is a recognized leader in school safety to increase the physical security of our campuses. ... Governor Abbot recommends the DPS "iWatch Texas" App. We will...
  • Diapositiva 1 - UCF Department of EECS

    Diapositiva 1 - UCF Department of EECS

    Digital Integral controller to regulate the stimulation current. Using the formula above will allow us to control the stimulators output. Shocking Device. Power Supply. Voltage buck/boost regulator dual output 3.7 volts supplies processor & 5 volts supplies DAC.