DMSOR Structure: Controversy #2 The first Mo enzyme X-ray structure: DMSO Reductase Group Meeting Bryn Mawr College, October 2010 Doug Rees, 1996 Doug Rees,
Download ReportTranscript DMSOR Structure: Controversy #2 The first Mo enzyme X-ray structure: DMSO Reductase Group Meeting Bryn Mawr College, October 2010 Doug Rees, 1996 Doug Rees,
DMSOR Structure: Controversy #2 The first Mo enzyme X-ray structure: DMSO Reductase Group Meeting Bryn Mawr College, October 2010 Doug Rees, 1996 Doug Rees, Cal Tech Protein crystallographer SURPRISE!!!! • 2 molydopterin ligands! • nucleoside termini on pterin • very long Mo-S bonds DMSOR Structure: Controversy #2 The first look at molybdopterin was on a tungsten enzyme! Hyperthermophilic TungstonEnzyme, Aldehyde Ferredoxin Oxidoreductase Group Meeting Bryn Mawr College, October 2010 Doug Rees et al., Science,1995 SURPRISE!!!! • not the molydopterin ligand! • is that pyran ring actually right??? DMSOR Structure: Controversy #2 Group Meeting Bryn Mawr College, October 2010 Will the real active site structure in DMSO Reductase please stand up? (S J N Burgmayer, in Progress in Inorganic Chemistry, 2004) And the answer was: Hermann Schindelin, Würzburg, Germany Protein crystallographer DMSOR Structure: Controversy #2 Group Meeting Bryn Mawr College, October 2010 1.3 Å X-ray Structure in DMSO Reductase (Schindelin) Active form What does it mean? There are 2 superimposed structures. (only one is inactive!) Inactive form Group Meeting Bryn Mawr College, October 2010 Introduction Early view of Mo site Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 The Essential Moco Ralf Mendel: Prof., Dr. Ralf Mendel, Institut für Pflanzenbiologie Technische Universität Braunschweig Germany John Enemark: Prof. John Enemark, Regent’s Professor of Chemistry University of Arizona Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Moco Degradation K. V. Rajagopalan, James B. Duke Professor of Biochemistry, Duke Medicine Moco Degradation Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 We know some about its degradation Moco Rajagopalan Group Meeting Bryn Mawr College, October 2010 Moco Identity: Guess #1 Moco Identity: Controversy #2 Molybdopterin Ligand is full of mysteries What is true, functional oxidation state? (Rajagopalan, 1980) Group Meeting Bryn Mawr College, October 2010 + 2 eq [Fe(CN)6]3- 2 eq [Fe(CN)6]4- + 1 eq DCIP A 2 e- process; NOT a tetrahydropterin Oxidized pterin (fluorescent) A catalytic cycle for how Mo oxidizes SO32-: no role of pterin required!! Group Meeting Bryn Mawr College, October 2010 Moco Identity: Controversy #1 Moco Biosynthesis Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 We know a lot about its biosynthesis Prof., Dr. Ralf Mendel, Institut für Pflanzenbiologie Technische Universität Braunschweig Germany Introduction Repairing the Molybdenum Cofactor Baby Z Cured of Rare Disease in 3 Days (Southern Health/AFP/Getty Images) Orphan Drug Treatment Used Only on Mice to Get Hearing Before FDA By SUSAN DONALDSON JAMES, Nov. 9, 2009 University of Arizona, Tucson, October 2010 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. 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." MRI of brain of deceased baby with Sulfite Oxidase Deficiency MRI of healthy brain Introduction Biosynthetic Pathway for the Mo cofactor Repairing the Molybdenum Cofactor University of Arizona, Tucson, October 2010 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 6 day old mice WT w/o Moco w/ precursor Z injections X-ray structure of Sulfite Oxidase Caroline Kisker 1997 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 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 Structural Models (RH Holm, Harvard) Mo=O(mono-dithiolene) models for SO family Mo=O(di-dithiolene) models for DMSO family Differences with Moco? Different geometry, missing pterin A Functional Model OAT system Tp*Mo=X(S—S) Models Tp* = tris(pyrazolylborate) M.Kirk, J. Enemark, C. Young, Burgmayer lab Understanding Electronic Structure: Marty Kirk the redox active orbital, d2 as Mo(4+) Mo 4d orbitals Mo=O orbitals O 2p orbitals Mo=O bonds 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 Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Making Pterin Dithiolene Ligands on Molybdenum It’s all about the pterin. Ralf: Sharon J. Nieter Burgmayer John: BRYN MAWR COLLEGE Pennsylvania, USA University of Arizona, Tucson, October 2010 Introduction Pterin Redox: the essentials Introduction University of Arizona, Tucson, October 2010 Pterin Redox: the complications Introduction Pterin Redox: the essentials University of Arizona, Tucson, October 2010 PyranoPterin Redox: the peculiar A Pyranopterin behaves as a Dihydropterin Burgmayer et al, J. Biol. Inorg. Chem. 2004 Introduction University of Arizona, Tucson, October 2010 Molybdoterin Redox: the possible Still more structural controversy surrounds molybdopterin in E. coli dissimilatory Nitrate Reductase “Escherichia coli, when grown anaerobically with nitrate as respiratory oxidant, develops a respiratory chain terminated by a membrane-bound quinol:nitrate oxidoreductase (NarGHI).” NarH - NarG [4Fe-4S] NO2 + H2O [4Fe-4S] 2e- Mo-bisPGD Group Meeting Bryn Mawr College, October 2010 Cytoplasm [4Fe-4S] [4Fe-4S] [3Fe-4S] MQ MQH2 Periplasm bH Q 2H+ NarI 2e- bL NO3- + 2H+ Prof.. Joel Weiner, Prof of Biochemistry, U. Alberta NarGHI: A Complex Iron-Sulfur Molybdoenzyme (CISM) Now, Dr. B. challenges you to explain this diagram! NarG NarH [4Fe-4S] [4Fe-4S] Mo-bisPGD Cytoplasm 2e- [4Fe-4S] [4Fe-4S] [3Fe-4S] MQ MQH2 Periplasm bH Q 2H+ NarI 2e- bL • Heterotrimeric membranebound complex 224kDa: NO2- + H2O NO3- + 2H+ – NarG (1246 AA, 140.4kDa), catalytic subunit; – 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. Mo-bisPGD NarG 13.80Å (7.0) FS0 14.35Å (11.2) FS1 Em = +95 +190 mV Em = -55 mV Em = +130 mV 12.43Å (9.7) NarH Em = -420 mV FS2 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 Chemistry? Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 But we’re suspicious… Pyranopterin of MPT Dihydro-oxidation state “open” MPT No pyrano ring What is the oxidation state of MPT? Nitrate Reductase J. Weiner 2003 Is pyran ring scission/fusion part of active site mechanism P-pterin HN (Pyranopterin) 8 7 6 N H2N 5a 9a 9 H H N 5 10 4 HN Q-pterin (Molybdopterin) 8 H2N 6 S 4a 3 10a 2 N O H H 5a H H N 5 9 9a N 10 G Mo S 4 S 4a 3 10a N OPO3 1 O 7 Mo S O 2 OPO3 H OH 1 G Moura et al. (2004). J. Biol. Inorg. Chem. 9, 791 Residues Surrounding the Open Pyran Ring of the Q-pterin P-pterin D222 Mo FS0 Guanine 3.2Å 2.8Å 2.6Å S719 Q-pterin H1163 Part I Hypothesis: 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. WT 3.2Å 2.8Å 2.4Å 3.2Å 3.0Å 2.6Å S719 H1163A S719A H1163 A719 A1163 H1163 S719 S719A/H1163A • The mutations do not close the Q-pterin pyran ring. • But, let’s look a little closer 2.6Å 2.6Å A1163 A719 WT S719A H1163A S719A =0.5 H1163A • 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. Introduction The Molybdenum Cofactor: the most Redox Rich Cofactor in Biology University of Arizona, Tucson, October 2010 Mo Redox Pterin Redox Dithiolene Redox Introduction Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 Why are we doing this work? • The two main components of Moco are the dithiolene chelate and the pterin • Much about the dithiolene chelate on Mo is fairly well understood Fold Angle Oxo Gate Electronic Buffer • Pterin chemistry is not understood, especially when part of a dithiolene oxidative ring opening Burgmayer JBIC 2004 no reduction Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 Synthetic Strategy + We don’t want this hydrolysis to happen: * No reaction with Mo=O Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 Making Pterin Dithiolenes Our studies of pterin-dithiolene Moco models can be categorized into two groups by types of R-groups: 1. aryl substituents 2. a-hydroxyalkyl substituents Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Model Spectroscopy EPR parameters indicate similar Mo environments in Tp*MoO(S2DIFPEPP) and Tp*MoO(bdt) simulation experimental Sample g1 g2 g3 <g>b A1 A2 A3 <A>b c c c hpH SO 1.990 1.966 1.954 1.970 54.4 21.0 11.3 28.9 0 14 22 lpH SO 2.007 1.974 1.968 1.983 56.7 25.0 16.7 32.8 0 18 0 Tp*MoO(S2PEPP) 2.006 1.976 1.936 1.973 46.7 3.3 50.4 33.5 0 0 0 Tp*MoO(S2DIFPEPP) 2.006 1.976 1.936 1.973 47.3 3.3 51.0 33.9 0 0 0 Tp*MoO(bdt) 2.004 1.972 1.934 1.971 50.0 11.4 49.7 37.0 0 0 0 Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009 The Pyranopterin Circus The Three-Ring Circus Of Pterin-Dithiolene oxidation (O2) oxidation (PPh3, O2) reduction KBH4 reduction KBH4 oxidation (H2O2, O2) reduction KBH4 Molybdopterin, the “special ligand” for Mo (and W) in several views Molybdopterin (MPT) (a) (b) Molybdopterin guanine dinucleotide (MGD) (d) (c) Flavin adenine dinucleotide (FAD) (e) Other places to find Mo and W enzymes Hot springs Deep sea vents Hyperthermophilic bacteria “some like it hot”: 212 F Mo & W enzymes keep our ancient ancestors alive: archaebacteria DMSOR Structure: Controversy #2 The first look at molybdopterin was on a tungsten enzyme! Hyperthermophilic TungstonEnzyme, Aldehyde Ferredoxin Oxidoreductase Group Meeting Bryn Mawr College, October 2010 Doug Rees et al., Science,1995 SURPRISE!!!! • not the molydopterin ligand! • is that pyran ring actually right???