A presentation of my work on peptidyl-hydroxylating monooxygenase conducted at Oregon Health and Science University.

1. Production and Mechanistic Characterization of Peptidylglycine Hydroxylating Monooxygenase (PHM) Andrew Bauman Senior Research Associate @ OHSU

2. Function of PHM and its partner PAL Vederas, J. C. et.al . J. Chem. Soc., Chem. Commun. , (1991) 571-572. Eipper, B. A. et. al ., Biochemistry, 41 (2002) 12384-12394.

3. Structure of PHMcc (aa 42 – 356)

4. PHM, A Copper Monooxygenase Cu H H172 H108 H107 H244 H242 Di-I-YG Substrate Cu M Y318 R240 N316 D1 D2 Q170 Amzel, L. M. et. al ., Science, 278 (1997) 1300-1305. Substrate C  is in close-proximity to Cu M Cu M is the site of dioxygen binding and catalysis. S = C-terminal D-aminoacid

5. Active Oxidized State of PHM

6. General PHM Mechanism

9. Substrate-Mediated Electron Transfer Amzel, L. M. et. al ., Science, 278 (1997) 1300-1305.

12. Bauman, Andrew, T.; Blackburn, Ninian, J.; Ralle, Martina. Large Scale Production of the Copper Enzyme Peptidylglycine Monooxygenase Using an Automated Bioreactor. Protein Expr. Purif. (2007), 51(1), 34-8. Bauman, Andrew, T.; Jaron, Shula; Yukl, Eric, T.; Burchfiel, Joel, R.; Blackburn, Ninian, J. pH Dependence of Peptidylglycine Monooxygenase. Mechanistic Implications of Cu-Methionine Binding Dynamics. Biochemistry. (2006), 45(37), 11140-50. Bauman, Andrew, T.; Yukl, Erik, T.; Alkevich, Katsiaryna; McCormack, Ashley; Blackburn, Ninian, J. The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate. J. Biol. Chem. (2006), 281(7), 4190-8. Bauman, Andrew, T.; Boers, Brenda.; Blackburn, Ninian, J.; Characterization of the Peptidylglycine Monooxygenase M314H Mutant. New Insights Into Methionine Coordination, Oxygen Binding, and Electron Transfer. In preparation. Publications

13. Experiments Stopped-Flow Spectrokinetic Analyzer

14. Experiments Freeze Quench Spectrokinetic Analyzer

15. Experiments Dissolved Oxygen Electrode

16. Electron Paramagnetic Resonance Experiments

17. Experiments EXAFS Shell R( Å ) 2 σ 2 ( Å -1 ) 2.5 N(im) 1.97 0.009 1.5 O/N 1.97 0.009 Cu N1 C2 C5 N4 C3 Cu N1 C2 C5 N4 C3

19. Production of PHM Harvest media Ammonium Sulfate Gel Filtration Anion Exchange Reconstitution Experiments

22. B2 Schematic of B2 (Accusyst Minimax)

27. Quality Comparison of B1 and B2

28. Quality Comparison of B1 and B2

29. MALDI-MS of PHM from B2 ESI-MS of reduced/alkylated PHM provided evidence of an intact N-terminus 35,625 daltons was observed ~ (35,048 Da + (10*58 Da)) Quality Control of PHM from B2

30. Visible spectrum of PHM pH 8.0 Quality Control of PHM from B2

32. Substrate: Dansyl-Y-V-G Mix: Buffer pH 5.5, 5uM Cu++, 5uM PHM, Catalase PHM Reaction Mix ± Reductant TFA Quench every 30s Initiate Reaction RPHPLC Equipped with Fluorescence Detector Monitor Oxygen Consumption Quench entire reaction with TFA Peroxide Concentration Assay

33. Dissolved Oxygen Electrode Standard Hydrogen Peroxide Reaction + Oxygen Consumption

34. Standard Reaction Using Ascorbate as Reductant Substrate: Dansyl-Y-V-G Buffer pH 5.5, 5uM Cu++, 5uM PHM, 1mM ascorbate TFA Quench every 30s Add substrate to 300 uM RPHPLC equipped with Fluorescence Detector

35. Substrate: Dansyl-Y-V-G Standard Hydrogen Peroxide Reaction + HPLC Buffer pH 5.5, 5uM Cu++, 300uM substrate, 5uM PHM TFA Quench every 30s Add H 2 O 2 to 1mM RPHPLC equipped with Fluorescence Detector Peroxide Concentration Assay

36. Substrate: Dansyl-Y-V-G Standard Hydrogen Peroxide Reaction + Oxygen Consumption Quench entire reaction with TFA Buffer pH 5.5, 5uM Cu++, 300uM substrate, 5uM PHM Monitor Oxygen Consumption Add H 2 O 2 to 1mM Peroxide Concentration Assay

39. oxygen evolution from peroxide measured in the O2-electrode under different conditions. Initial trace , 100 mM MES pH 5.5, 5 μM Cu2+ and 5 μM PHM; A , addition of 1 mM H2O2; B , addition of 200 μM dansyl-YVG substrate. Evolution of Oxygen From Peroxide and PHM

41. Substrate: Dansyl-Y-V-G Peroxide Generation by Glucose/Glucose Oxidase (GO) Buffer pH 5.5, 50mM Glucose, 300uM substrate, 5uM PHM Quench entire reaction with TFA RPHPLC equipped with Fluorescence Detector Peroxide Concentration Assay GO addition 45µg/mL Monitor Oxygen Consumption

42. Peroxide Generation by Glucose/Glucose Oxidase (GO)

44. PHM Kinetics and Thermodynamics

45. PHM Kinetics and Thermodynamics

48. Proposed Mechanism

52. XAS Edge Results from Core Ionization Energies (keV)

53. EXAFS – Photoelectron Scattering a s E 0 absorption coefficient Energy (eV) 1 E a s 2 E

55. Essential Information from EXAFS How many of what type of ligands are at what distance from metal? Observable Frequency Phase Shift Amplitude Information Distance Type of Atom # of Atoms

56. EXAFS of Oxidized PHM Shell R( Å ) 2 σ 2 ( Å -1 ) 2.5 N(im) 1.97 0.009 1.5 O/N 1.97 0.009 Peaks at ~2 Ǻ (Cu-N/O) ~ 3 Ǻ (C2/C5 imidazole) ~ 4 Ǻ (C3/N4 imidazole) Cu N1 C2 C5 N4 C3 Cu N1 C2 C5 N4 C3

57. EXAFS of the reduced PHM shows major changes in coordination First shell is split into two peaks at ~1.90 Ǻ (Cu-N) and ~2.3 Ǻ (Cu-S) Outer shell signatures of histidine are still present Histidine shell splits if copper sites are refined separately Shell R(Å) 2 σ 2 (Å -1 ) 1.0 N(im) 1.98 0.007 0.5 S(met) 2.26 0.003 1.0 N(im) 1.88 0.007

71. Acknowledgements NIH DOE Stanford Synchotron Radiation Laboratory Staff Ninian Blackburn, Ph.D. Pierre Moienne Loccoez, Ph.D. Caitlin Grammer Gnana Sutha, Ph.D. Martina Ralle, Ph.D. Luisa Andruzzi, Ph.D. Joel Burchfiel