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DMSOR Structure: Controversy #2

DMSOR Structure: Controversy #2. The first Mo enzyme X-ray structure: DMSO Reductase Doug Rees, 1996. Doug Rees, Cal Tech Protein crystallographer. Group Meeting Bryn Mawr College, October 2010. SURPRISE!!!! 2 molydopterin ligands! nucleoside termini on pterin very long Mo-S bonds.

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DMSOR Structure: Controversy #2

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  1. DMSOR Structure: Controversy #2 The first Mo enzyme X-ray structure: DMSO Reductase Doug Rees, 1996 Doug Rees, Cal Tech Protein crystallographer Group Meeting Bryn Mawr College, October 2010 • SURPRISE!!!! • 2 molydopterin ligands! • nucleoside termini on pterin • very long Mo-S bonds

  2. DMSOR Structure: Controversy #2 The first look at molybdopterin was on a tungsten enzyme! Hyperthermophilic TungstonEnzyme, Aldehyde Ferredoxin Oxidoreductase Doug Rees et al., Science,1995 Group Meeting Bryn Mawr College, October 2010 • SURPRISE!!!! • not the molydopterin ligand! • is that pyran ring actually right???

  3. DMSOR Structure: Controversy #2 Will the real active site structure in DMSO Reductase please stand up? Group Meeting Bryn Mawr College, October 2010 (S J N Burgmayer, in Progress in Inorganic Chemistry, 2004)

  4. And the answer was:

  5. Hermann Schindelin, Würzburg, Germany Protein crystallographer DMSOR Structure: Controversy #2 1.3 Å X-ray Structure in DMSO Reductase (Schindelin) Group Meeting Bryn Mawr College, October 2010 Inactive form Active form What does it mean? There are 2 superimposed structures. (only one is inactive!)

  6. Introduction Early view of Mo site Group Meeting Bryn Mawr College, October 2010

  7. Ralf Mendel: John Enemark: The Essential Moco Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Prof., Dr. Ralf Mendel, InstitutfürPflanzenbiologie TechnischeUniversitätBraunschweig Germany Prof. John Enemark, Regent’s Professor of Chemistry University of Arizona

  8. Moco Degradation K. V. Rajagopalan, James B. Duke Professor of Biochemistry, Duke Medicine Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007

  9. Moco Degradation We know some about its degradation Moco Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Rajagopalan

  10. Moco Identity: Guess #1 Group Meeting Bryn Mawr College, October 2010

  11. Moco Identity: Controversy #2 MolybdopterinLigand is full of mysteries What is true, functional oxidation state? (Rajagopalan, 1980) + 2 eq [Fe(CN)6]3- Oxidized pterin (fluorescent) - 2 eq [Fe(CN)6]4- + 1 eq DCIP Group Meeting Bryn Mawr College, October 2010 A 2 e- process; NOT a tetrahydropterin

  12. A catalytic cycle for how Mo oxidizes SO32-: no role of pterin required!!

  13. Moco Identity: Controversy #1 Group Meeting Bryn Mawr College, October 2010

  14. Moco Biosynthesis We know a lot about its biosynthesis Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Prof., Dr. Ralf Mendel, InstitutfürPflanzenbiologie TechnischeUniversitätBraunschweig Germany

  15. Introduction Repairing the Molybdenum Cofactor Baby Z Cured of Rare Disease in 3 Days Orphan Drug Treatment Used Only on Mice to Get Hearing Before FDA By SUSAN DONALDSON JAMES, Nov. 9, 2009 (Southern Health/AFP/Getty Images) Baby Z had a one in a million chance of developing a rare metabolic disorder called molybdenum cofactor deficiency and zero chance of avoiding the inevitable death sentence that comes with it. The Australian girl had a seemingly normal birth in May 2008 but, within hours, she began having multiple seizures -- as many as 10 an hour -- as sulfite build-up began to poison her brain. With the clock ticking, doctors who treated Baby Z gained approval from the hospital's ethics board and a family court to use the experimental treatment. The drug -- cPMP, a precurser molecule made from E. coli bacteria -- was airlifted on ice from the lab of German professor Guenter Schwarz and, within three days, it worked. University of Arizona, Tucson, October 2010 Worldwide, there are only about 50 cases of molybdenum cofactor, or sulfite oxidase deficiency, mostly in Europe and in the United States, according to the National Institutes of Health. Molybdenum, like other organic metals, is essential for the human body. Its cofactor is a small, complicated molecule that acts as a carrier to help the metal interact with proteins and enzymes so they can function properly. When the cofactor is missing, toxic sulfite builds and begins to cause degeneration of neurons on the brain and eventually death. "This was the first time I ever saw this," said Dr. Alex Veldman, the Monash neonatologist who headed up Baby Z's treatment. "It's very funny, now I am regarded a world specialist but I can tell you that before last May, I couldn't even spell it."

  16. MRI of brain of deceased baby with Sulfite Oxidase Deficiency MRI of healthy brain

  17. Introduction Biosynthetic Pathway for the Mo cofactor Repairing the Molybdenum Cofactor Prof. Günter Schwarz, PhD Professor and Chair in Biochemistry Institute of Biochemistry and Center for Molecular Medicine Cologne University Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli Günter Schwarz et al, Human Molecular Genetics, 2004 University of Arizona, Tucson, October 2010 6 day old mice w/o Moco w/ precursor Z injections WT

  18. X-ray structure of Sulfite Oxidase Caroline Kisker1997

  19. CO Dehydrogenase 8.7 Å Mo 3.5 Å 12.4 Å FAD Fe2S2 clusters Mo 5.4 Å Fe2S2 clusters MCD Aldehyde Oxidoreductase MCD Aldehyde Ferredoxin Oxidoreductase molybdopterin Fe4S4 cluster Why use a pterin? One answer from X-ray Crystallography: Electron Transfer Conduit W 3.1 Å MPT

  20. Models of Moco Functional: display OAT reactions, proton-coupled redox Structural: display same inner sphere constituents display same secondary sphere constituents Electronic: display same spectroscopic signatures; presumed similar orbital description

  21. Structural Models (RH Holm, Harvard) Mo=O(mono-dithiolene) models for SO family

  22. Mo=O(di-dithiolene) models for DMSO family

  23. Differences with Moco? Different geometry, missing pterin

  24. A Functional Model OAT system Tp*Mo=X(S—S) Models Tp* = tris(pyrazolylborate) M.Kirk, J. Enemark, C. Young, Burgmayer lab

  25. Mo 4d orbitals Mo=O orbitals Understanding Electronic Structure: Marty Kirk the redox active orbital, d2 as Mo(4+) O 2p orbitals

  26. Mo=O  bonds

  27. Why a Dithiolene not a Dithiolate? Dithiolene Dithiolate This orbital is especially important: it shows how the redox active d(xy) orbital is directly influenced by a dithiolene interaction

  28. It’s all about the pterin. Ralf: John: Making Pterin Dithiolene Ligands on Molybdenum Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Sharon J. NieterBurgmayerBRYN MAWR COLLEGE Pennsylvania, USA

  29. Introduction PterinRedox: the essentials University of Arizona, Tucson, October 2010

  30. Introduction PterinRedox: the complications University of Arizona, Tucson, October 2010

  31. Introduction PterinRedox: the essentials PyranoPterinRedox: the peculiar Burgmayer et al, J. Biol. Inorg. Chem. 2004 University of Arizona, Tucson, October 2010 A Pyranopterin behaves as a Dihydropterin

  32. Introduction MolybdoterinRedox: the possible University of Arizona, Tucson, October 2010

  33. Still more structural controversy surrounds molybdopterin inE. coli dissimilatory Nitrate Reductase “Escherichia coli, when grown anaerobically with nitrate as respiratory oxidant, develops a respiratory chain terminated by a membrane-bound quinol:nitrateoxidoreductase (NarGHI).” Cytoplasm NarG NarH [4Fe-4S] NO2- + H2O [4Fe-4S] 2e- [4Fe-4S] Mo-bisPGD NO3- + 2H+ [4Fe-4S] Group Meeting Bryn Mawr College, October 2010 [3Fe-4S] NarI bH MQ 2e- Q MQH2 bL Periplasm 2H+

  34. Prof.. Joel Weiner, Prof of Biochemistry, U. Alberta

  35. NarGHI: A Complex Iron-Sulfur Molybdoenzyme (CISM) Now, Dr. B. challenges you to explain this diagram! • Heterotrimeric membrane-bound complex 224kDa: • NarG (1246 AA, 140.4kDa), catalyticsubunit; • NarH (512 AA, 58.1kDa), electron-transfer subunit or Four Cluster Protein (FCP); • NarI (225 AA, 25.5kDa) membrane-anchor subunit. • 8 prosthetic groups. • Enzyme turnover produces a proton electrochemical potential. Cytoplasm NarG NarH [4Fe-4S] NO2- + H2O [4Fe-4S] 2e- [4Fe-4S] Mo-bisPGD NO3- + 2H+ [4Fe-4S] [3Fe-4S] NarI bH MQ 2e- Q MQH2 bL Periplasm 2H+

  36. Em = +95 +190 mV Mo-bisPGD 13.80Å (7.0) NarG Em = -55 mV FS0 14.35Å (11.2) Em = +130 mV FS1 12.43Å (9.7) Em = -420 mV FS2 NarH 12.95Å (9.6) FS3 Em = -55 mV 12.70Å (9.4) Em = +180 mV FS4 14.38Å (8.9) Em = +125 mV Heme bP NarI 16.5Å (5.4) Em = +25 mV Heme bL ETR: ~97.4Å Electron transfer tunneling limit = 14Å

  37. Chemistry? But we’re suspicious… Pyranopterin of MPT Dihydro-oxidation state “open” MPT No pyrano ring What is the oxidation state of MPT? Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Nitrate Reductase J. Weiner 2003

  38. Mo Mo S S O O H H H H 4 4 S S N N 7 7 5a 5a 6 6 5 5 H H N N 4a 4a 3 3 10a 10a 2 2 8 8 G G OPO OPO 10 10 1 1 3 3 9a 9a N N N N O O H H N N 2 2 H H 9 9 H H Mo Mo S S O O H H H H S S 4 4 N N 7 7 5a 5a 6 6 5 5 H H N N 4a 4a 3 3 10a 10a 2 2 8 8 G G OPO OPO 3 3 9a 9a N N N N H H H H N N 2 2 9 9 10 10 OH OH 1 1 Is pyran ring scission/fusion part of active site mechanism P-pterin (Pyranopterin) Q-pterin (Molybdopterin)

  39. Residues Surrounding the Open Pyran Ring of the Q-pterin P-pterin D222 Mo FS0 Guanine Moura et al. (2004). J. Biol. Inorg. Chem.9, 791 2.8Å 3.2Å 2.6Å Q-pterin S719 H1163

  40. Part IHypothesis: Mutation of S719 and H1163 wll convert the Q-pterin from a molybdopterin to a pyranopterin • S719A, H1163A, and S719A/H1163A mutants were generated, enzymes purified and characterized and their structures solved by X-ray crystallography. • EPR was used to characterize the Mo electrochemistry.

  41. H1163A WT S719A 2.8Å 2.6Å 2.4Å 3.2Å 3.2Å 3.0Å 2.6Å A1163 H1163 H1163 S719 S719 A719 S719A/H1163A • The mutations do not close the Q-pterin pyran ring. • But, let’s look a little closer 2.6Å A1163 A719

  42. WT S719A H1163A S719A H1163A =0.5 • The single mutants have subtle effects on the conformation of atoms of the Q-pterin including C10. • The double mutant shows bending of the Q pterin ring.

  43. Introduction The Molybdenum Cofactor: the most Redox Rich Cofactor in Biology DithioleneRedox Mo Redox University of Arizona, Tucson, October 2010 PterinRedox

  44. Introduction Why are we doing this work? • The two main components of Moco are the dithiolene chelate and the pterin • Much about the dithiolenechelate on Mo is fairly well understood Fold Angle Oxo Gate Electronic Buffer Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 • Pterin chemistry is not understood, especially when part of a dithiolene oxidative ring opening no reduction Burgmayer JBIC 2004

  45. Synthetic Strategy + Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 We don’t want this hydrolysis to happen: * No reaction with Mo=O

  46. Making PterinDithiolenes Our studies of pterin-dithioleneMocomodels can be categorized into two groups by types of R-groups: 1. aryl substituents 2.a-hydroxyalkylsubstituents Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009

  47. Model Spectroscopy EPR parameters indicate similar Mo environments in Tp*MoO(S2DIFPEPP) and Tp*MoO(bdt) simulation experimental Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007

  48. The Pyranopterin Circus The Three-Ring Circus Of Pterin-Dithiolene oxidation (O2) oxidation (PPh3, O2) reduction KBH4 Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 reduction KBH4 oxidation (H2O2, O2) reduction KBH4

  49. Molybdopterin, the “special ligand” for Mo (and W) in several views Molybdopterin (MPT) (a) (b) (c) Molybdopterin guanine dinucleotide (MGD) Flavin adenine dinucleotide (FAD) (d) (e)

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