DMSOR Structure: Controversy #2G

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SURPRISE!!!!• 2 molydopterin ligands!• nucleoside termini on pterin• very long Mo-S bonds

The first Mo enzyme X-ray structure: DMSO ReductaseDoug Rees, 1996

Doug Rees, Cal TechProtein crystallographer

DMSOR Structure: Controversy #2G

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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 #2G

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(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 #2G

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1.3 Å X-ray Structure in DMSO Reductase (Schindelin)

Active formWhat does it mean?

There are 2 superimposed structures. (only one is inactive!)

Hermann Schindelin, Würzburg, GermanyProtein crystallographer

Inactiveform

Early view of Mo site

IntroductionG

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The Essential Moco

Ralf Mendel: John Enemark:

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Prof. John Enemark, Regent’s Professor of ChemistryUniversity of Arizona

Prof., Dr. Ralf Mendel,Institut für Pflanzenbiologie

Technische Universität BraunschweigGermany

Moco Degradation

K. V. Rajagopalan,James B. Duke Professor of Biochemistry,Duke Medicine

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We know some about its degradation

Moco Degradation

Rajagopalan

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Moco

Moco Identity: Guess #1G

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Moco Identity: Controversy #2G

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0 Molybdopterin Ligand is full of mysteriesWhat is true, functional oxidation state? (Rajagopalan, 1980)

+ 2 eq [Fe(CN)6]3-

- 2 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 SO32-: no role of pterin required!!

Moco Identity: Controversy #1G

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We know a lot about its biosynthesis

Prof., Dr. Ralf Mendel,Institut für Pflanzenbiologie

Technische Universität BraunschweigGermany

Moco BiosynthesisGo

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Repairing the Molybdenum CofactorIntroduction

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0 Baby Z Cured of Rare Disease in 3 DaysOrphan 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 babywith Sulfite Oxidase Deficiency MRI of healthy brain

IntroductionU

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Professor and Chair in BiochemistryInstitute of Biochemistry and Center for Molecular Medicine Cologne University

Repairing the Molybdenum Cofactor

WT w/oMoco

w/ precursor Z injections

6 day old mice

Rescue of lethal molybdenum cofactor deficiencyby a biosynthetic precursor from Escherichia coliGünter Schwarz et al, Human Molecular Genetics, 2004

Biosynthetic Pathway for the Mo cofactor

X-ray structure of Sulfite OxidaseCaroline Kisker 1997

12.4 Å

5.4 Å

8.7 Å

CO Dehydrogenase

Aldehyde Oxidoreductase

3.5 Å

3.1 Å

W

Fe4S4

cluster

Fe2S2

clusters Fe2S2

clusters

FAD

MCD

Mo

MCD

MPT

molybdopterin

Mo

Aldehyde Ferredoxin Oxidoreductase

Why use a pterin?One answer fromX-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 orbitalsMo=O orbitals

Mo 4d orbitals

the redox activeorbital, d2 as Mo(4+)

Understanding Electronic Structure: Marty Kirk

Mo=O bonds

Why a Dithiolene not a Dithiolate?

This orbital is especiallyimportant: it shows how the redox active d(xy) orbital is directly influenced by a dithiolene interaction

DithioleneDithiolate

Making Pterin Dithiolene Ligands on Molybdenum

Sharon J. Nieter Burgmayer BRYN MAWR COLLEGE Pennsylvania, USA

Ralf: John:

It’s all about the pterin.

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Pterin Redox: the essentials

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Pterin Redox: the complications

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Pterin Redox: the essentialsIntroduction

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PyranoPterin Redox: the peculiar Burgmayer et al, J. Biol. Inorg. Chem. 2004

A Pyranopterin behaves as a Dihydropterin

Molybdoterin Redox: the possibleIntroduction

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2H+

MQH2

MQ

2e-

NarGNarH

NarI

Cytoplasm

Periplasm

NO3- + 2H+

NO2- + H2O

[4Fe-4S]

[3Fe-4S]

[4Fe-4S]

[4Fe-4S]

Mo-

bisP

GD

bL

bH

Q

[4Fe-4S]2e-

Gro

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Still more structural controversy surrounds molybdopterinin 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+

MQH2

MQ

2e-

NarGNarH

NarI

Cytoplasm

Periplasm

NO3- + 2H+

NO2- + H2O

[4Fe-4S]

[3Fe-4S]

[4Fe-4S]

[4Fe-4S]M

o-bi

sPGD

bL

bH

Q

[4Fe-4S]2e-

Now, Dr. B. challenges you to explain this diagram!

Heme bL

Heme bP

FS4

FS3

FS2

FS1

FS013.80Å (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 Em = +95 +190 mV

Em = -55 mV

Em = +130 mV

Em = -420 mV

Em = -55 mV

Em = +180 mV

Em = +125 mV

Em = +25 mV

Chemistry?

But we’re suspicious…

Nitrate Reductase J. Weiner 2003

“open” MPTNo pyrano ringWhat is theoxidation state of MPT?

Pyranopterin of MPTDihydro-oxidation state

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Is pyran ring scission/fusion part of active site mechanism

N

NHNH

N

O

NH2OH

S

S

OPO3

H

H1

2

3

4

4a55a67

8

9

9a

10

10a

Mo

GN

NHNH

N

O

NH2OH

S

S

OPO3

H

H1

2

3

4

4a55a67

8

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9a

10

10a

Mo

G

N

NHNH

NH

O

NH2 O

S

SH

H1

2

3

4

4a5

5a67

8

9

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Mo

OPO3 GN

NHNH

NH

O

NH2 O

S

SH

H1

2

3

4

4a5

5a67

8

9

9a 1010a

Mo

OPO3 G

P-pterin(Pyranopterin)

Q-pterin(Molybdopterin)

3.2Å

2.6Å

2.8Å

FS0

MoD222

Q-pterin

Guanine

P-pterin

S719H1163

Residues Surrounding the Open Pyran Ring of the Q-pterinM

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. J. B

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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

2.4Å3.2Å

2.6Å

2.6Å

S719 A719H1163 H1163 S719

A1163

A719

A1163

WT S719A 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 C10.

• The double mutant shows bending of the Q pterin ring.

S719A H1163A

=0.5

The Molybdenum Cofactor: the most Redox Rich Cofactor in Biology

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Mo Redox Dithiolene Redox

Pterin Redox

Introduction

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• 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

oxidativering opening

no reduction

+

Synthetic Strategy

We don’t want this hydrolysis to happen:

* No reaction with Mo=O

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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

Making Pterin DithiolenesGo

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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

Model Spectroscopy

EPR parameters indicate similar Mo environments in Tp*MoO(S2DIFPEPP) and Tp*MoO(bdt)

simulation

experimental

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reductionKBH4

The Three-Ring CircusOfPterin-Dithiolene

reductionKBH4

oxidation(PPh3, O2)

reductionKBH4

oxidation (O2)

oxidation(H2O2, O2)

The Pyranopterin CircusGo

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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 springsDeep sea vents

Hyperthermophilic bacteria“some like it hot”: 212 F

Mo & W enzymes keepour ancient ancestors alive:archaebacteria

Other placesto find Mo andW enzymes

DMSOR Structure: Controversy #2G

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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???