DMSOR Structure: Controversy #2 Group Meeting Bryn Mawr College, October 2010 SURPRISE!!!! 2...
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DMSOR Structure: Controversy #2 Group Meeting Bryn Mawr College, October 2010 SURPRISE!!!! 2 molydopterin ligands! nucleoside termini on pterin very long
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
Slide 2
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???
Slide 3
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?
Slide 4
And the answer was:
Slide 5
DMSOR Structure: Controversy #2 Group Meeting Bryn Mawr
College, October 2010 1.3 X-ray Structure in DMSO Reductase
(Schindelin) Active form What does it mean? There are 2
superimposed structures. (only one is inactive!) Hermann
Schindelin, Wrzburg, Germany Protein crystallographer Inactive
form
Slide 6
Early view of Mo site Introduction Group Meeting Bryn Mawr
College, October 2010
Slide 7
The Essential Moco Ralf Mendel: John Enemark: Gordon Research
Conference on Mo & W Enzymes, New Hampshire 2007 Prof. John
Enemark, Regents Professor of Chemistry University of Arizona
Prof., Dr. Ralf Mendel, Institut fr Pflanzenbiologie Technische
Universitt Braunschweig Germany
Slide 8
Moco Degradation K. V. Rajagopalan, James B. Duke Professor of
Biochemistry, Duke Medicine Gordon Research Conference on Mo &
W Enzymes, New Hampshire 2007
Slide 9
We know some about its degradation Moco Degradation Rajagopalan
Gordon Research Conference on Mo & W Enzymes, New Hampshire
2007 Moco
Slide 10
Moco Identity: Guess #1 Group Meeting Bryn Mawr College,
October 2010
Slide 11
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 ] 3- - 2 eq [Fe(CN) 6 ] 4- Oxidized pterin (fluorescent)
+ 1 eq DCIP A 2 e- process; NOT a tetrahydropterin
Slide 12
A catalytic cycle for how Mo oxidizes SO 3 2- : no role of
pterin required!!
Slide 13
Moco Identity: Controversy #1 Group Meeting Bryn Mawr College,
October 2010
Slide 14
We know a lot about its biosynthesis Prof., Dr. Ralf Mendel,
Institut fr Pflanzenbiologie Technische Universitt Braunschweig
Germany Moco Biosynthesis Gordon Research Conference on Mo & W
Enzymes, New Hampshire 2007
Slide 15
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)
Slide 16
MRI of brain of deceased baby with Sulfite Oxidase Deficiency
MRI of healthy brain
Slide 17
Introduction University of Arizona, Tucson, October 2010 Prof.
Gnter 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 Gnter
Schwarz et al, Human Molecular Genetics, 2004 Biosynthetic Pathway
for the Mo cofactor
Slide 18
X-ray structure of Sulfite Oxidase Caroline Kisker 1997
Slide 19
12.4 5.4 8.7 CO Dehydrogenase Aldehyde Oxidoreductase 3.5 3.1 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
Slide 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
Slide 21
Structural Models (RH Holm, Harvard) Mo=O(mono-dithiolene)
models for SO family
Slide 22
Mo=O(di-dithiolene) models for DMSO family
Slide 23
Differences with Moco? Different geometry, missing pterin
Slide 24
A Functional Model OAT system Tp*Mo=X(SS) Models Tp* =
tris(pyrazolylborate) M.Kirk, J. Enemark, C. Young, Burgmayer
lab
Slide 25
Slide 26
O 2p orbitals Mo=O orbitals Mo 4d orbitals the redox active
orbital, d2 as Mo(4+) Understanding Electronic Structure: Marty
Kirk
Slide 27
Mo=O bonds
Slide 28
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
Slide 29
Making Pterin Dithiolene Ligands on Molybdenum Sharon J. Nieter
Burgmayer BRYN MAWR COLLEGE Pennsylvania, USA Ralf:John: Its all
about the pterin. Gordon Research Conference on Mo & W Enzymes,
New Hampshire 2007
Slide 30
Pterin Redox: the essentials Introduction University of
Arizona, Tucson, October 2010
Slide 31
Pterin Redox: the complications Introduction University of
Arizona, Tucson, October 2010
Slide 32
Pterin Redox: the essentials Introduction University of
Arizona, Tucson, October 2010 PyranoPterin Redox: the peculiar
Burgmayer et al, J. Biol. Inorg. Chem. 2004 A Pyranopterin behaves
as a Dihydropterin
Slide 33
Molybdoterin Redox: the possible Introduction University of
Arizona, Tucson, October 2010
Slide 34
2H + MQH 2 MQ 2e - NarG NarH NarI Cytoplasm Periplasm NO 3 - +
2H + NO 2 - + 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).
Slide 35
Prof.. Joel Weiner, Prof of Biochemistry, U. Alberta
Slide 36
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 3 - + 2H + NO 2 - + 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!
Slide 37
Heme b L Heme b P FS4 FS3 FS2 FS1 FS0 13.80 (7.0) 14.35 (11.2)
12.43 (9.7) 12.95 (9.6) 12.70 (9.4) 14.38 (8.9) 16.5 (5.4) Electron
transfer tunneling limit = 14 NarG NarH NarI ETR: ~97.4 Mo-bisPGD E
m = +95 +190 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
Slide 38
Chemistry? But were 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
Slide 39
Is pyran ring scission/fusion part of active site mechanism
P-pterin (Pyranopterin) Q-pterin (Molybdopterin)
Slide 40
3.2 2.6 2.8 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
Slide 41
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.
Slide 42
3.2 2.6 2.8 3.0 2.4 3.2 2.6 S719 A719 H1163 S719 A1163 A719
A1163 WTS719A H1163A S719A/H1163A The mutations do not close the
Q-pterin pyran ring. But, lets look a little closer
Slide 43
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
Slide 44
The Molybdenum Cofactor: the most Redox Rich Cofactor in
Biology Introduction University of Arizona, Tucson, October 2010 Mo
Redox Dithiolene Redox Pterin Redox
Slide 45
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
Slide 46
+ Synthetic Strategy We dont want this hydrolysis to happen: *
No reaction with Mo=O Gordon Research Conference on Mo & W
Enzymes Lucca, Italy 2009
Slide 47
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
Slide 48
Sampleg1g1 g2g2 g3g3 b A1A1 A2A2 A3A3 cc c c hpH
SO1.9901.9661.9541.97054.421.011.328.901422 lpH
SO2.0071.9741.9681.98356.725.016.732.80180 Tp*MoO(S 2
PEPP)2.0061.9761.9361.97346.73.350.433.5000 Tp*MoO(S 2
DIFPEPP)2.0061.9761.9361.97347.33.351.033.9000
Tp*MoO(bdt)2.0041.9721.9341.97150.011.449.737.0000 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
Slide 49
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
Slide 50
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
Slide 51
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
Slide 52
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???