When Good Crystallography Goes Bad: The Active Site Structure of Mo Enzymes

Wednesday, March 17, 2004 Graham N. George

When Good Crystallography Goes Bad: When Good Crystallography Goes Bad: The The Active Site Structure of Mo EnzymesActive Site Structure of Mo Enzymes

Graham N. GeorgeDepartment of Geological Sciences,University of Saskatchewan.


OverviewOverview

• Introduction to X-ray Absorption SpectroscopyIntroduction to X-ray Absorption Spectroscopy

• Introduction to Molybdenum EnzymesIntroduction to Molybdenum Enzymes

• The Active Site Structure of DMSO ReductaseThe Active Site Structure of DMSO Reductase

• Recent workRecent work


E

Absorbance = loge(I0/I1)

Ion chamber detector Ion chamber detector

sample

I0 I1

Experiment: Scan the X-ray energy while monitoring the X-ray absorption.This can only be done using a synchrotron X-ray source.

What is X-ray Absorption Spectroscopy?What is X-ray Absorption Spectroscopy?


h

continuum

Emitted photo-electron

1s

Auger electron

hfluorescent photon

X-ray Absorption Spectroscopy – Basic PhysicsX-ray Absorption Spectroscopy – Basic Physics

2p

2s


K-edge XAS of some first transition elements.

X-ray absorption spectroscopy is element-specificX-ray absorption spectroscopy is element-specific


Setup includes a liquid He cryostat and 30 element Ge detector array.

Protein sample showing image of beam (X-ray induced color centers).

XAS – Experimental Setup.XAS – Experimental Setup.


E

Insufficient energy to eject core-electron

Just enough energy to eject core-electron(low energy photo-electron results).

core-electron easily ejected(high energy photo-electron)

transitions to bound-statesnear-edge

EXAFS oscillations

What is X-ray Absorption Spectroscopy?What is X-ray Absorption Spectroscopy?

h

continuum


1s


Near-edge spectrum

EXAFS oscillations (k3-weighted)

X-ray Absorption SpectroscopyX-ray Absorption Spectroscopy


h

continuum


1s

Auger electron

hfluorescent photon

X-ray Absorption Spectroscopy – Basic PhysicsX-ray Absorption Spectroscopy – Basic Physics

2p

2s


E

E

Bromine atom Br2 molecule

Schematics diagrams of final state wave functions.

Photo-electron DeBroglie wave.

EXAFS – Basic PhysicsEXAFS – Basic Physics


EXAFS oscillations (k3-weighted) Fourier transform

The EXAFS Fourier Transform



Phase-corrected Fourier transformEXAFS oscillations (k3-weighted)






MoS

Mo-S

[MoS4]2-




MoS

Mo-S

Fe

Mo····Fe

Cl

[MoS4FeCl2]2-

[MoS4]2-


Near-edge Spectra – Excitation to bound statesNear-edge Spectra – Excitation to bound states

Se-methionine

elemental Se

selenate

2-

selenite

2-



• Examines all of a particular element in a sample

• No sample pre-treatment required (don’t need crystals etc.)

• Near edge spectrum – gives information on electronic structure (oxidation state etc.)

• EXAFS (Extended X-ray Absorption Spectroscopy) oscillations in X-ray absorption Gives a Radial Structure.


Strengths and Limitations of EXAFSStrengths and Limitations of EXAFS

Structural parameters that are available from EXAFS analysis:

• Average bond-lengths, R

• Coordination Numbers, N

• Debye-Waller factors, σ2

σ2 is the mean-square displacement of the bond-length from the average value R. It has components from atomic vibration and disorder, and can be thought of as being similar to a crystallographic temperature factor. It differs from the temperature factor in that it is due to relative displacement of atoms.

• Geometric information is generally unavailable, although multiple scattering sometimes allows bond-angle determination.

• Data analysis is not always a routine matter.


Strengths and Limitations of EXAFSStrengths and Limitations of EXAFS

The Debye-Waller factor is not a total unknown

The Debye-Waller is a sum of static disorder and vibrational components.

σ2 = σ2stat. + σ2

vib.

σ2vib. – can be computed accurately for a given bond-length (using force

constants derived from vibrational spectroscopy or density functional theory).

σ2stat. – upper and lower limits for this can be computed from the k-range of

the data and the coordination number.

2 Mo-S at ~ 2.4 Å 0.0063 Å2 > σ2 > 0.0020 Å2

2 Mo-O at ~ 2.0 Å 0.0068 Å2 > σ2 > 0.0025 Å2


EXAFS EXAFS vs.vs. Crystallography CrystallographyComparison with small molecule X-ray crystal structures –• Use crystallographic bond-lengths and coordination numbers• Refine Debye-Waller factors σ2 within reasonable bounds

Expt.

Calc.

Mo=O

Mo-S

• There is excellent agreement between the two techniques• σ2 values match well with ab-initio calculated values (e.g. for Mo=O

0.0018(2) vs. 0.0017 Å2).


Molybdenum EnzymesMolybdenum Enzymes

• All molybdenum enzymes contain an organic cofactor.• This is called “molybdopterin”.

ONH

NH

NH

N

O

NH2

S

S

OPO3

Mo

• Almost all catalyze two-electron redox reactions involving oxygen transfer between Mo and substrate.

• Either one or two molybdopterin cofactors can be coordinated to the metal via the dithiolene linkage.


S

O

CH3 CH3

2H+

SCH3 CH3

H2O

Catalyses the two-electron reduction of dimethylsulfoxide (DMSO) to dimethylsulfide (DMS).

Mo is oxidized from Mo4+ to Mo6+ formal oxidation state in the process.

The Prototypical member of the DMSO reductase family of Mo enzymes.

The best studied DMSO reductases are those of Rhodobacter capsulatus and Rhodobacter sphaeroides. These have nearly identical sequences and properties.

DMSO reductase DMSO reductase

DMSO DMS

Mo4+ Mo6++ +


Structural studies of DMSO reductase active siteStructural studies of DMSO reductase active site

7 February 1996 - first EXAFS14 June 1996 - first crystallography18 October 1996 - more crystallography15 September 1997 - some more crystallography1 December 1997 - still more crystallography30 January 1998 - even more crystallography30 January 1998 - more EXAFS (a different group)27 November 1998 - crystallography of a closely related enzyme 17 February 1999 - more EXAFS16 August 2000 - yet more crystallography

Prior to the 1999 EXAFS study there was a lot of confusion and debate about the active site structure. To some extent the debate still continues.


First EXAFS of DMSO reductaseFirst EXAFS of DMSO reductase

“X-ray Absorption Spectroscopy of Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides” G. N. George, J. Hilton and K. V. RajagopalanJ. Am. Chem. Soc. 1996, 118(5), 1113-1117

7 February 1996


First EXAFS of DMSO reductaseFirst EXAFS of DMSO reductase

“X-ray Absorption Spectroscopy of Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides” G. N. George, J. Hilton and K. V. RajagopalanJ. Am. Chem. Soc. 1996, 118(5), 1113-1117

7 February 1996

• Mono-oxo Mo6+ and des-oxo Mo4+

• Four Mo-S indicated two cofactors

• Postulated oxo-transfer mechanism

• Suggested that one cofactor might dissociate.

• “It seems likely that the active site can adopt at least two different structures”


First Crystallography of DMSO reductaseFirst Crystallography of DMSO reductase

“Crystal Structure of DMSO Reductase: Redox-Linked Changes in Molybdopterin Coordination” H. Schindelin, C. Kisker, J. Hilton, K. V. Rajagopalan and D. C. ReesScience, 1996, 272 1615-1621

14 June 1996

Gave us our first look at the 3-dimensional structure of the protein.


First Crystallography of DMSO reductaseFirst Crystallography of DMSO reductase

“Crystal Structure of DMSO Reductase: Redox-Linked Changes in Molybdopterin Coordination” H. Schindelin, C. Kisker, J. Hilton, K. V. Rajagopalan and D. C. ReesScience, 1996, 272 1615-1621

14 June 1996

Big changes in Mo coordination on changing metal oxidation state!

Mo6+

Mo4+

mono-oxo Mo6+ des-oxo Mo4+

Ser147 Ser147


Proposed Catalytic MechanismProposed Catalytic Mechanism


More crystallographic results followed…More crystallographic results followed…

“Crystal Structure of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus at 1.88 Å Resolution” F. Schneider, J. Löwe, R. Huber, H. Schindelin, C. Kisker and J. KnäbleinJ. Mol. Biol. 1996 263, 53-69

18 October 1996

The structure was different! – a dioxo Mo6+ site with only one of two cofactors bound.


And more crystallography followed those …And more crystallography followed those …

“Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus: crystal structure of the oxidised enzyme at 1.82-Å resolution and the dithionite-reduced enzyme at 2.8-Å resolution” A. S. McAlpine, A. G. McEwan, A. L. Shaw and S. BaileyJBIC, 1997, 2, 690-701

1 December 1997

The structures were different again!

di-oxo Mo6+ mono-oxo Mo4+


Even more crystallography followed …Even more crystallography followed …

“The High Resolution Crystal Structure of DMSO Reductase in Complex with DMSO”A. S. McAlpine, A. G. McEwan and S. Bailey J. Mol. Biol. 1998, 275, 613-623

30 January 1998

This work found that if the product (DMS) is added to oxidized enzyme then a pink-purple species was formed which had DMSO bound to the active site.

Oxidized Mo6+ + DMS (gray-green) DMSO-bound form (pink purple)

This DMSO-bound form was studied by X-ray crystallography.


Even more crystallography followed …Even more crystallography followed …

“The High Resolution Crystal Structure of DMSO Reductase in Complex with DMSO”A. S. McAlpine, A. G. McEwan and S. Bailey J. Mol. Biol. 1998, 275, 613-623

30 January 1998

A seven-coordinate mono-oxo molybdenum site, with DMSO covalently bound with an unusually long S=O bond length (1.7 Å).


Lots of different structures for the same active site...Lots of different structures for the same active site...

Mo6+ Mo6+

Mo4+

Mo6+Mo4+ Mo4+

DMSO

All protein folds were essentially identical


And then more EXAFS followed …And then more EXAFS followed …

The conclusions of this study totally supported the crystal structures of Bailey and co-workers…

“X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus” P. E. Baugh, C. D. Garner, J. M. Charnock, D. Collison, E. S. Davies, A. S. McAlpine, S. Bailey, I. Lane, G. R. Hanson and A. G. McEwanJBIC, 1998, 2, 634-643.

30 January 1998

And another mechanism was postulated…


Mo4+

DMSOExptl.

Calc.

EXAFS EXAFS vs.vs. protein crystallography protein crystallography

The crystal structure does not agree with our EXAFS data.

Comparison with protein X-ray crystal structures –• Use crystallographic bond-lengths and coordination numbers• Refine Debye-Waller factors σ2 within reasonable bounds


EXAFS EXAFS vs.vs. small molecule crystallography small molecule crystallographyComparison with small molecule X-ray crystal structures –• Use crystallographic bond-lengths and coordination numbers• Refine Debye-Waller factors σ2 within reasonable bounds

Expt.

Calc.

Mo=O

Mo-S

• There is excellent agreement between the two techniques• σ2 values match well with ab-initio calculated values (e.g. for Mo=O

0.0018(2) vs. 0.0017 Å2).


EXAFS EXAFS vs.vs. protein crystallography protein crystallography

Mo6+ Mo6+

Mo4+

Mo6+Mo4+ Mo4+

DMSO

None of the crystal structures fitted our EXAFS data…


Resonance Raman Resonance Raman doesdoes agree with our EXAFS data agree with our EXAFS data

“Active Site Structures and Catalytic Mechanism of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase as Revealed by Resonance Raman Spectroscopy”S. D. Garton, J. Hilton, H. Oku, B. R. Crouse, K. V. Rajagopalan and M. K. JohnsonJ. Am. Chem. Soc. 1997, 119, 12906-12916

31 December 1997

• Resonance Raman spectroscopy indicated a mono-oxo Mo6+ species for the oxidized enzyme

• Both Mo=O and Mo-S frequencies agreed quantitatively with our EXAFS-derived bond-lengths


The first structure was unusual…The first structure was unusual…

More than one half of the metal coordination sphere is empty!

Caltech Rees group, oxidized form


• A 3-coordinate Mo4+ site – a totally unknown coordination• Mo-O-C bond angle close to 180º• More than ½ the metal coordination sphere totally empty

The other crystal structures were odd too…The other crystal structures were odd too…Caltech Rees group, reduced form


The other crystal structures were odd too…The other crystal structures were odd too…

The O=Mo=O bond angle is impossibly small.

Martinsried Huber group, oxidized form


The other crystal structures were odd too…The other crystal structures were odd too…

• Many supposedly non-bonded atoms with overlapping Van der Waals radii• O=Mo=O bond-angle impossibly small (70º)

Daresbury Bailey group, oxidized form


The other crystal structures were odd too…The other crystal structures were odd too…Daresbury Bailey group, oxidized form

• Overlapping Van der Waals radii with non-bonded atoms – the Mo6+ structure showed six of these.

• All the other Bailey structures had the same problem.• So did crystal structures of related enzymes (TMAO reductase).

• Too many atoms, not enough room.


Were all of the structures wrong?Were all of the structures wrong?

On close inspection ALL of the structures had some chemically implausible or impossible features.

Impossibly crowded atomsImpossibly acute or unlikely bond-anglesUnusual bond-lengths (this is expected)

We concluded that ALL crystal structures were either wrong or had major problems.

We re-examined all the EXAFS (several times), including the DMSO bound form.

We examined the EXAFS of other closely related Mo enzymes (biotinsulfoxide reductase, trimethylamineoxide reductase). They looked just like DMSO reductase.


But what about the other EXAFS?But what about the other EXAFS?

The experimental data of Baugh et al. looks the same as ours – so why did they form such very different conclusions?

– Physically impossible Debye-Waller factors.

Some of the σ2 values used by Baugh et. al. were so big that they effectively removed the EXAFS.

Baugh et al. started from the crystal structure coordinates, then (probably) refined σ2 values with no boundaries imposed, and finally refined bond-lengths etc. to obtain their final result. This gave conclusions that were heavily biased towards the crystal structure.



Bond N R (Å) σ2 (Å2)

Mo=O 1 1.69 0.014

Mo-O 1 1.91 0.001

Mo-O 1 2.11 0.009

Mo-S 2 2.28 0.016

Mo-S 2 2.37 0.001

Mo-S 1 2.81 0.013

Mo-C 2 3.26 0.013

Mo-C 2 3.41 0.001

Mo-C 1 3.17 0.001



DMSO-bound form:

A total of 9 components with 18 variables were refined.



Mo=O 1 1.69 0.014

Mo-O 1 1.91 0.001

Mo-O 1 2.11 0.009

Mo-S 2 2.28 0.016

Mo-S 2 2.37 0.001

Mo-S 1 2.81 0.013

Mo-C 2 3.26 0.013

Mo-C 2 3.41 0.001

Mo-C 1 3.17 0.001

Too big

Too big

Too big

Too big

Too big



Some of the σ2 values were impossibly big.

This reduces the EXAFS intensity of these components



Mo=O 1 1.69 0.014

Mo-O 1 1.91 0.001

Mo-O 1 2.11 0.009

Mo-S 2 2.28 0.016

Mo-S 2 2.37 0.001

Mo-S 1 2.81 0.013

Mo-C 2 3.26 0.013

Mo-C 2 3.41 0.001

Mo-C 1 3.17 0.001

Too big

Too big

Too big

Too big

Too big

Too small

Too small

Too small

Too small



Other σ2 values were impossibly small.

This increases the EXAFS intensity of these components



Mo=O 1 1.69 0.014

Mo-O 1 1.91 0.001

Mo-O 1 2.11 0.009

Mo-S 2 2.28 0.016

Mo-S 2 2.37 0.001

Mo-S 1 2.81 0.013

Mo-C 2 3.26 0.013

Mo-C 2 3.41 0.001

Mo-C 1 3.17 0.001

Too big

Too big

Too big

Too big

Too big

Too small

Too small

Too small

Too small



Ignore the very low intensity components in the fit, and those which cancel



Mo=O 1 1.69 0.014

Mo-O 1 1.91 0.001

Mo-O 1 2.11 0.009

Mo-S 2 2.28 0.016

Mo-S 2 2.37 0.001

Mo-S 1 2.81 0.013

Mo-C 2 3.26 0.013

Mo-C 2 3.41 0.001

Mo-C 1 3.17 0.001

Too big

Too big

Too big

Too big

Too big

Too small

Too small

Too small

Too small




“Structure of the Molybdenum Site of Dimethyl Sulfoxide Reductase” G. N. George, J. Hilton, C. Temple, R. C. Prince and K. V. RajagopalanJ. Am. Chem. Soc. 1997, 121, 1256-1266.

17 February 1999

EXAFS and EPR spectroscopyEXAFS and EPR spectroscopy

• This paper suggested that the conclusions of all of the crystallographic studies and the Daresbury EXAFS study were wrong.

• The structural conclusions presented in this work were essentially the same as in our original 1996 paper.

• We suggested that multiple species might be present in the crystals and that this caused erroneous conclusions.


More crystallography…More crystallography…

“The Crystal Structure Of Rhodobacter Sphaeroides Dimethylsulfoxide Reductase Reveals Two Distinct Molybdenum Coordination Environments” H-K Li, C. Temple, K. V. Rajagopalan, and H. Schindelin.J. Am. Chem. Soc. 2000, 122, 7673-7680.

16 August 2000

• 1.3 Å structure resolved two different active site structures within the crystals• One of them looked quite like our solution structure!


More crystallography…More crystallography…

“The Crystal Structure Of Rhodobacter Sphaeroides Dimethylsulfoxide Reductase Reveals Two Distinct Molybdenum Coordination Environments” H-K Li, C. Temple, K. V. Rajagopalan, and H. Schindelin.J. Am. Chem. Soc. 2000, 122, 7673-7680.

16 August 2000

• Most of this structure was in quantitative agreement with the EXAFS• But it still had non-bonded atoms that were too close!• Probably there are more than just two forms present.


A combined approach –A combined approach –

Use the information from EXAFS, crystallography and density functional calculations (DFT) to investigate nature of DMSO-bound form.

• DMSO-bound DMSO reductase has a highly characteristic electronic spectrum with absorption bands at 478 and 546 nm.

• Trimethylarsine [(CH3)3As] forms a similar Mo4+ complex with an essentially identical electronic spectrum in which trimethylarsineoxide [(CH3)3As=O] is bound covalently to Mo.

• We will attempt to combine the information from the Mo and As EXAFS of this complex to help understand the coordination of DMSO.


data fit

As

Mo

Mo-O

Mo-S

Mo····As

data

fitAs=O

As-C

As····Mo

• EXAFS shows (CH3)3As located at Mo site.• Both As=O and As-C interactions are clearly resolved.

Mo and As EXAFSMo and As EXAFS


• Arsenic is oxidized (As5+) and molybdenum is reduced (Mo4+)

• As=O bond-length is within normal range – no particular distortion is present.

• Cofactor and Ser147 coordinates from crystal structure

2.37 Å

3.44 Å

2.23 Å2.01 Å

1.70 Å

MoS

As

OSer147

1.91 Å

EXAFS of (CHEXAFS of (CH33))33As-bound DMSO reductaseAs-bound DMSO reductase


• (CH3)3As remains bound but with longer than observed Mo-O=As distance.

• DFT Mo-S 2.41, Mo-O(Ser) 1.95, Mo-O(AsMe3) 2.45, Mo-As 3.56

• EXAFS Mo-S 2.37, Mo-O(Ser) 2.01, Mo-O(AsMe3) 2.23, Mo-As 3.44

DFT of (CHDFT of (CH33))33As=O-bound DMSO reductaseAs=O-bound DMSO reductase


data

fit

Mo-S + Mo-O

Mo-O

EXAFS indicates4 Mo-S at 2.37 Å1 Mo-O at 2.23 Å1 Mo-O at 1.98 Å

• Estimate geometry of DMSO-bound enzyme from crystal structure and EXAFS with adjustments from (CH3)3As=O experiment.

EXAFS of (CHEXAFS of (CH33))22S=O bound DMSO reductaseS=O bound DMSO reductase


• DMSO dissociates – rather than DMS – tendency to go in reverse.• Active site pocket must be important in stabilization and direction.• Future calculations will use hybrid approach to include protein.

DFT Calculation – (CHDFT Calculation – (CH33))22S=O leaves active site…S=O leaves active site…


Future work and directionsFuture work and directions

Protein crystal structure solution is traditionally aided by structural restraints of the amino acids derived from small molecule crystallography.

• For metalloproteins the information from crystallography should be supplemented by careful and independent spectroscopic studies.

• Modern computational techniques could aid in providing restraints.

• Crystallographers – don’t ignore the spectroscopy!

• Some sort of check for overlapping Van der Waals radii ought to be routinely applied to non-bonded “hetero-atoms”


K.V. Rajagopalan Duke University Medical CenterJim Hilton Duke University Medical CenterCarrie Temple Duke University Medical CenterKimberly Johnson Duke University Medical Center

Eileen Yu Sneeden Stanford Synchrotron Radiation Lab.Hugh H. Harris SSRL (now University of Sydney)Roger C. PrinceExxonMobil Research & Eng. Co.

Christian J. Doonan University of Saskatchewan

Ingrid J. Pickering University of Saskatchewan

AcknowledgementsAcknowledgements


AcknowledgementsAcknowledgements

Canada Research Chairs ProgramCanada Research Chairs ProgramUniversity of SaskatchewanUniversity of SaskatchewanProvincial GovernmentProvincial GovernmentCanada Foundation for InnovationCanada Foundation for Innovation

Canadian Light SourceCanadian Light Sourceand its many sponsorsand its many sponsors

www.lightsource.cawww.lightsource.ca

Stanford Synchrotron Radiation Stanford Synchrotron Radiation Laboratory, Laboratory, U.S. DOE and NIHU.S. DOE and NIH

Documents

When Good Crystallography Goes Bad: The Active Site Structure of Mo Enzymes