Molybdenum 1900 – discovered in plant ashes 1930 - azotobacter (N2 fixer) growth requirement for Mo 1940 – established as essential micronutrient for.
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Molybdenum 1900 – discovered in plant ashes 1930 - azotobacter (N2 fixer) growth requirement for Mo 1940 – established as essential micronutrient for plants 1950 - [Mo] affected rat XO activity; [Mo]~ [flavin] Mo associated with aldehyde oxidase in rabbit livers 1960 - Mo crucial to N-cycle Mo The only 4d metal essential to Life S Table 1. The Oxomolybdenum Enzymes The Xanthine Oxidase Family (LMoOS-Possessing Enzymes) enzyme source subunits cofactora xanthine oxidase cow’s milkb R2 MPT xanthine dehydrogenase chicken liverc R2 MPT rat liverd R2 MPT Micrococcus lactyliticuse Drosophila melanogasterf R2 MPT Chlamydomonas reinhardtiig humanh R2 (MPT) aldehyde oxidase rabbit liveri R2 (MPT) humanj R2 (MPT) cowk R2 (MPT) aldehyde oxidoreductase (dehydrogenase) Desulfovibrio gigasl R2 MCD Acetobacter polyoxogenesm formate dehydrogenase Alcaligenes eutrophusn Râçä Methylosinus trichosporumo R2â2ç2ä2 CO dehydrogenase (oxidoreductase) Pseudomonas carboxydovoransp R2â2ç2 Pseudomonas carboxydoflavaq R2â2ç2 MCD Oligotropha carboxidovoransr R2â2ç2 MCD quinoline-2-oxidoreductase Pseudomonas putida s R2â2ç2 MCD Rhodococcus sp. B1t R2â2ç2 MCD Comamonas testosteroni 63u R2â2ç2 MCD isoquinoline 1-oxidoreductase Pseudomonas diminutav Râ MCD quinoline-4-carboxylate-2-oxidoreductase Agrobacterium sp. 1Bw R2â2ç2 MCD quinaldine-4-oxidoreductase Arthrobacter sp.x R2â2ç2 MCD quinaldic acid 4-oxidoreductase Serratia marcescensy Pseudomonas sp. AK-2z Râ nicotinic acid hydroxylase (dehydrogenase) Clostridium barkeriaa,ab R2 Bacillus niaciniac R2â2ç2 Arthrobacter oxidansad Râç 6-hydroxynicotinate hydroxylase Bacillus niaciniac Râç nicotine dehydrogenase Arthrobacter oxidansad Râç Arthrobacter nicotinovoransae picolinate hydroxylase Arthrobacter picolinophilusaf R2â2ç2 MCD (2R)-hydroxycarboxylate oxidoreductase Proteus vulgarisag (R. Hille, Chem. Rev. 1996) The Sulfite Oxidase Family (LMoO2-Possessing Enzymes) sulfite oxidase bovine liverah R2 MPT chicken liverai R2 MPT rat liveraj R2 MPT humanak R2 MPT Thiobacillus novellusal R nitrate reductase (assimilatory) Neurospora crassaam R2 MPT spinachan R2 MPT Chlorella vulgarisao R4 MPT The DMSO Reductase Family (L2MoX-Possessing Enzymes) DMSO reductase Rhodobacter sphaeroidesap R MGD Rhodobacter capsulatus aq R MGD Escherichia coli ar Râç MGD biotin-S-oxide reductase Escherichia colias trimethylamine-N-oxide reductase Escherichia coliat R2 nitrate reductase (dissimilatory) Escherichia coli (NarGHI)au Râç MGD Escherichia coli (NarZYV)av Râç MGD Escherichia coli (FdoGHI)aw Râç MGD Paracoccus denitrificans (NapABCD)ax Râçä Haloferax volcaniiay formate dehydrogenase Escherichia coli (FdhF)az R MGD Escherichia coli (FdnGHI)ba Râç MGD Escherichia coli (FdoGHI)bb Râç MGD Methanobacterium formicicumbc (XdS) Râç MGD Wollinella succinogenesbd Râç polysulfide reductase Wolinella succinogenesbe Râç MGD arsenite oxidase Alcaligenes faecalisbf R MCD formylmethanofuran dehydrogenase) Râçä(ú) MGD, MAD, MHD Methanosarcina barkeribh MGD Unclassified Molybdenum-Containing Enzymes pyridoxal oxidase Drosophila melanogaster chlorate reductase Proteus mirabilis tetrathionite reductase Proteus mirabilis xanthine dehydrogenase Clostridium sp pyruvate:ferredoxin oxidoreductase Archaeoglobus fulgidus pyrogallol transhydroxylase Pelobacter acidigallici 2-furoyl-CoA dehydrogenase Pseudomonas putida NO2- Nitrate reductase N2 Nitrogen- cycle NO3- NH3 polysulfide reductase SH2 CH4 CODH FDH nitrogenase Sx Carbon- cycle Sulfur- cycle CO2 CH3CO2- (CH3)2S (CH3)2SO DMSO reductase Table 1. The Oxomolybdenum Enzymes (R. Hille, Chem. Rev. 1996) The Xanthine Oxidase Family (LMoOS-type) - in mammalian milk but >50% de-Mo - in liver: purine metabolism (gout) - implicated in heart damage (ROS form’n) The Sulfite Oxidase Family (LMoO2-type) Nitrate Reductase - in all plants - assimilatory form (plants, fungi, algae) NO3- + 2H+ + 2e- NO2- + H2O The DMSO Reductase Family (L2MoX-type) - all bacterial or from archea - many in respiration using specific e- acceptors - recall DMSOR in cloud formation …. - simplest Mo-enzymes: w/o other cofactors Sulfite Oxidase - most critical for humans - detoxification of sulfite - also has role in S-metabolism, from Cys Met SO32- SO42SO32- + H2O SO42- + 2H+ + 2e- Locating the Molybdenum Cofactor All plants require the molybdenum enzyme All mammals require molybdenum enzymes Nitrate Reductase Sulfite Oxidase NO3- + 2H+ + 2e- SO32- + H2O NO2- + H2O SO42- + 2H+ + 2e- Mo(4+) + X-O + 2H+ Mo(6+) + X + H2O Tomato plants with and without molybdenum available. Tobacco plants (Arabidopsis thaliana) Nitrate Nitrite Proteins nitrate Healthy (wild type) All plants require Assimilatory Nitrate Reductase Sick (mutant) nitrite Environmental impact of DMSOR and the smell of the ocean DMS CH3SO3- cloud nucleation sites hn, photo-oxidation DMS (CH3)2S(CH2)2CO2from algae DMSO Reductase DMSO Why the correct oxidation state matters MRI of brain of deceased baby with Sulfite Oxidase Deficiency MRI of healthy brain The baby died because this reaction didn’t happen: SO32- + Sulfite S4+ H2O ---> SO42- + 2H+ + Sulfate S6+ 2e- The baby has a genetic defect in the enzyme that catalyzes this reaction. Babies with this genetic disease die within hours. The enzyme is Sulfite Oxidase. You have it in your liver. Table 1. The Oxomolybdenum Enzymes (R. Hille, Chem. Rev. 1996) The Xanthine Oxidase Family (LMoOS-type) The Sulfite Oxidase Family (LMoO2-type) Xanthine Oxidase/Dehydrogenase - in mammalian milk but >50% de-Mo - in liver: purine metabolism (gout) - implicated in heart damage (ROS form’n) Nitrate Reductase - in all plants - assimilatory form (plants, fungi, algae) The DMSO Reductase Family (L2MoX-type) - all bacterial or from archea - many in respiration using specific e- acceptors - recall DMSOR in cloud formation …. - simplest Mo-enzymes: w/o other cofactors Sulfite Oxidase - most critical for humans - detoxification of sulfite - also has role in S-metabolism, from Cys Met SO32- SO42- Table 1. The Oxomolybdenum Enzymes (R. Hille, Chem. Rev. 1996) The Xanthine Oxidase Family (LMoOS-type) The Sulfite Oxidase Family (LMoO2-type) The DMSO Reductase Family (L2MoX-type) Used to be classed with XO b/c structural similarity. Unique Mo enzyme with a 2nd metal, Cu The Generic Molybdenum Cofactor MAD MGD MCD MPT There’s not only one, but a family of Moco’s Why molybdopterin? dithiolene Mo pterin 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 Group Meeting Bryn Mawr College, October 2010 The first look at molybdopterin was on a tungsten enzyme! Hyperthermophilic TungstonEnzyme, Aldehyde Ferredoxin Oxidoreductase Doug Rees et al., Science,1995 SURPRISE!!!! • not the molydopterin ligand! • is that pyran ring actually right??? Caroline Kisker Würzburg, Germany Protein crystallographer X-ray structure of chicken liver Sulfite Oxidase Kisker, Enemark Moco locked into position by H-bonds to pterin A catalytic cycle for how Mo oxidizes SO32- Water mediated OAT Similar cycles can be devised for Sulfite Oxidase and Nitrate Reductase SO32- + H2O Sulfite S4+ NO3- + Nitrate N5+ ---> SO42- + 2H+ + Sulfate S6+ 2H+ + 2e- 2e- ---> NO2- + H2O Nitrite N3+ 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 Group Meeting Bryn Mawr College, October 2010 Moco Identity: Controversy #2 Molybdopterin Ligand is full of mysteries What is true, functional oxidation state? (Rajagopalan, 1980) + 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 University of Arizona, Tucson, October 2010 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 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." Introduction Biosynthetic Pathway for the Mo cofactor University of Arizona, Tucson, October 2010 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 6 day old mice WT w/o Moco w/ precursor Z injections 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 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 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 Introduction University of Arizona, Tucson, October 2010 The Molybdenum Cofactor: the most Redox Rich Cofactor in Biology 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 University of Arizona, Tucson, October 2010 Introduction Pterin Redox: the essentials Introduction University of Arizona, Tucson, October 2010 Pterin Redox: the complications Introduction University of Arizona, Tucson, October 2010 Pterin Redox: the essentials 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 Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 What is the role of the pterin- dithiolene ligand of Moco? A model chemistry investigation Sharon J. Nieter Burgmayer Department of Chemistry, Bryn Mawr College Bryn Mawr, Pennsylvania, USA Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 ~ The Pterin-Dithiolene Model System ~ We have developed a general synthetic route to oxomolybdenum pterin-dithiolene complexes Tp*, occupies remaining coordination sites the pterin-dithiolene ligand Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 ~ The Pterin-Dithiolene Model Syntheses ~ A two-pronged synthetic pathway to pterin-dithiolene model complexes + Next step: Hydrolysis of S=Mo(4+) to O=Mo(4+) facilitated by PR3 + 3 eq PR3 + H2O ESI-MS [NEt4+][Tp*Mo(4+)(S)(S2BMOPP)—] [M-] m/Z 820 in ESI-MS- FT-IR Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 ~ The Pterin-Dithiolene Model Syntheses ~ n(Mo=S) 485 cm-1 [NEt4+][Tp*Mo(4+)(O)(S2BMOPP)—] [M-] m/Z 804 in ESI-MS- n(Mo=O) 924cm-1 Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 ~ The Pterin-Dithiolene Model Structure ~ The crystal gods smile upon the Burgmayer lab! Tp*Mo(5+)(=O)(S2BMOPP) Ben Williams A spontaneous cyclization of the side chain –OH across the pterin C7-N8 bond occurred providing the first synthetic example of a pyranopterin dithiolene. Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 ~ The Pterin-Dithiolene Model Structure ~ The crystal gods smile upon the Burgmayer lab . . . Twice! [Tp*Mo(4+)(=O)(S2BMOPP)] anion Yichun Fu The model complex in both Mo(5+) and Mo(4+) oxidation states exhibit cyclization of the side chain –OH across the pterin C7-N8 bond forming two examples of an oxo-Mo-pyranopterin dithiolene unit. Molybdenum and Tungsten Enzymes ~ Sintra, Portugal 2013 ~ The Pterin-Dithiolene Model System ~ So we speculate: 1) reversible pyran cyclization can occur in Mo enzym 2) it is controlled by protein environment Mo(4+)-open pterin (NMR) Mo(5+)-open pterin (??) Mo(4+)-pyranopterin (X-ray) Mo(5+)-pyranopterin (X-ray) 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 – NarG (1246 AA, 140.4kDa), catalytic subunit; – NarH (512 AA, 58.1kDa), NO3- + 2H+ 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 12.43Å (9.7) NarH FS2 Em = +95 +190 mV Em = -55 mV Em = +130 mV Em = -420 mV 12.95Å (9.6) Em = -55 mV FS3 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 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 Group Meeting Bryn Mawr College, October 2010 The first look at molybdopterin was on a tungsten enzyme! Hyperthermophilic TungstonEnzyme, Aldehyde Ferredoxin Oxidoreductase Doug Rees et al., Science,1995 SURPRISE!!!! • not the molydopterin ligand! • is that pyran ring actually right???