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

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???

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

And the answer was:

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

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

The Essential Moco Ralf Mendel: John Enemark: Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007 Prof. John Enemark, Regent’s Professor of Chemistry University of Arizona Prof., Dr. Ralf Mendel, Institut für Pflanzenbiologie Technische Universität Braunschweig Germany

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

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

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

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

A catalytic cycle for how Mo oxidizes SO 3 2- : no role of pterin required!!

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

We know a lot about its biosynthesis Prof., Dr. Ralf Mendel, Institut für Pflanzenbiologie Technische Universität Braunschweig Germany Moco Biosynthesis Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007

Repairing the Molybdenum Cofactor Introduction University of Arizona, Tucson, October 2010 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 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." (Southern Health/AFP/Getty Images)

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

Introduction 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 Repairing the Molybdenum Cofactor WT w/o Moco w/ precursor Z injections 6 day old mice Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli Günter Schwarz et al, Human Molecular Genetics, 2004 Biosynthetic Pathway for the Mo cofactor

X-ray structure of Sulfite Oxidase Caroline Kisker 1997

12.4 Å 5.4 Å 8.7 Å CO Dehydrogenase Aldehyde Oxidoreductase 3.5 Å 3.1 Å W Fe 4 S 4 cluster Fe 2 S 2 clusters Fe 2 S 2 clusters FAD MCD Mo MCD MPT molybdopterin Mo Aldehyde Ferredoxin Oxidoreductase Why use a pterin? One answer from X-ray Crystallography: Electron Transfer Conduit

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

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

Mo=O  bonds

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

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

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 Introduction University of Arizona, Tucson, October 2010 PyranoPterin Redox: the peculiar Burgmayer et al, J. Biol. Inorg. Chem A Pyranopterin behaves as a Dihydropterin

Molybdoterin Redox: the possible Introduction University of Arizona, Tucson, October 2010

2H + MQH 2 MQ 2e - NarG NarH NarI Cytoplasm Periplasm NO H + NO H 2 O [4Fe-4S] [3Fe-4S] [4Fe-4S] Mo-bisPGD bLbL bHbH Q [4Fe-4S] 2e - Group Meeting Bryn Mawr College, October 2010 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).”

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

NarGHI: A Complex Iron-Sulfur Molybdoenzyme (CISM) Heterotrimeric membrane- bound complex 224kDa: –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. 2H + MQH 2 MQ 2e - NarG NarH NarI Cytoplasm Periplasm NO H + NO H 2 O [4Fe-4S] [3Fe-4S] [4Fe-4S] Mo-bisPGD bLbL bHbH Q [4Fe-4S] 2e - Now, Dr. B. challenges you to explain this diagram!

Heme b L Heme b P FS4 FS3 FS2 FS1 FS Å (7.0) 14.35Å (11.2) 12.43Å (9.7) 12.95Å (9.6) 12.70Å (9.4) 14.38Å (8.9) 16.5Å (5.4) Electron transfer tunneling limit = 14Å NarG NarH NarI ETR: ~97.4Å Mo-bisPGD E m = mV E m = -55 mV E m = +130 mV E m = -420 mV E m = -55 mV E m = +180 mV E m = +125 mV E m = +25 mV

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

Is pyran ring scission/fusion part of active site mechanism P-pterin (Pyranopterin) Q-pterin (Molybdopterin)

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

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.

3.2Å 2.6Å 2.8Å 3.0Å 2.4Å 3.2Å 2.6Å S719 A719 H1163 S719 A1163 A719 A1163 WTS719A H1163A S719A/H1163A The mutations do not close the Q-pterin pyran ring. But, let’s look a little closer

WT S719A H1163A The single mutants have subtle effects on the conformation of atoms of the Q-pterin including C 10. The double mutant shows bending of the Q pterin ring. S719A H1163A  =0.5

The Molybdenum Cofactor: the most Redox Rich Cofactor in Biology Introduction University of Arizona, Tucson, October 2010 Mo Redox Dithiolene Redox Pterin Redox

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

+ 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

Our studies of pterin-dithiolene Moco models can be categorized into two groups by types of R-groups: 1. aryl substituents 2.  -hydroxyalkyl substituents Making Pterin Dithiolenes Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009

Sampleg1g1 g2g2 g3g3 b A1A1 A2A2 A3A3 cc  c  c hpH SO lpH SO Tp*MoO(S 2 PEPP) Tp*MoO(S 2 DIFPEPP) Tp*MoO(bdt) Model Spectroscopy EPR parameters indicate similar Mo environments in Tp*MoO(S 2 DIFPEPP) and Tp*MoO(bdt) simulation experimental Gordon Research Conference on Mo & W Enzymes, New Hampshire 2007

reduction KBH 4 The Three-Ring Circus Of Pterin-Dithiolene reduction KBH 4 oxidation (PPh 3, O 2 ) reduction KBH 4 oxidation (O 2 ) oxidation (H 2 O 2, O 2 ) The Pyranopterin Circus Gordon Research Conference on Mo & W Enzymes Lucca, Italy 2009

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

Hot springs Deep sea vents Hyperthermophilic bacteria “some like it hot”: 212 F Mo & W enzymes keep our ancient ancestors alive: archaebacteria Other places to find Mo and W enzymes

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???