S

S

O

M oN

N

NN

N

N

HB

H3C

H3C

H3C

C H3

C H3C H3

(Tp*)MoO(bdt)

Active Site of Sulfite Oxidase

Applications of Resonance Enhanced Raman Spectroscopy: Electronic Structure Probe of Metal-Sulfur Interactions in Oxo-Molybdenum

Ene-1,2-Dithiolate Systems

Frank E. InscoreThe University of Arizona

The Raman Spectroscopic TechniqueGeneral Considerations

Problematic: Inherent Weak Effect

Problematic: Fluorescence Complications

Problematic: Instrumental Limitations

Utility of Raman Spectroscopy

Research and Industrial Applications

Structure/ Solid State/ Biological Chemistry

Development of New Techniques

FT-RS/ SERS/ RERS

Resonance Raman Spectroscopy

• Catalyst Structure and Reactivity: Surface and In Situ Studies Heterogeneous processes: Supported metal oxides (MoO/ WO) used as catalyst. Hydrodesulfurization catalyst: Removal of sulfur from petroleum feedstocks.

Characterizing Structure/ Monitoring Reactivity in Catalytic Systems

• Structure/ Function In Situ Studies Protonation in Biomolecules: S-H/ S-S conversion. Mechanistic insight into Carcinogenesis: Blue/green particle in tumors; Cu-S bonding.

Structural Insight in Metalloproteins.

Biological Systems

Chemical and Petroleum/ Energy Production Industries

Overview of Presentation

• What we are doing?

• Why we are studying?

• How we will probe?

• Raman Applications• Background• Electronic Structure• Raman Instrumentation• Resonance Raman Studies• Implications for Mo Enzymes

S

S

O

M oN

N

NN

N

N

HB

H3C

H3C

H3C

C H3

C H3C H3

The Importance of Metal 1,2-Dithiolene ComplexesGeneral Considerations

Industrial Applications/ Commercial Uses:

Potential Biological Activity:

What Is an Ene-1,2-Dithiolate Ligand ?

Vulcanization Accelerators for Rubber Wear Additive Inhibitors in LubricantsCatalytic Inhibitors /Oxidation Catalyst Mode-Locking Additives in Nd Lasers

Correlations with Biological Systems containing Metal to Sulfur Bonds.

Relevance to structure, bonding and function of Metalloenzyme active site centers

Four Prototypical Ene-1,2-Dithiolate Systems:

S

S-S

-S

-S

-S

H

H N

N

-S

-S-S

-S- S

- SM

S

S =

Why the Interest in Transition Metal-Sulfur Complexes?

# X-ray crystallography reveals a common structural unit: Pyranopterin cofactor

HN

N N

N

OH2N OPO32-

S-S-

O H

H

Pyranopterin Molybdenum and Tungsten EnzymesBackground and Significance

2 W Families similar to DMSO reductase family

MoS

S

O

MoS

S

S

MoS

S

O

S

SO

S - C ys

OS er-O

O H2

Sulfite Oxidase Xanthine Oxidase DMSO Reductase

3 Mo Families based on structure and reactivity

The Resonance Raman Spectroscopic Probe Structure/ Bonding in the Active Site of DMSO Reductase

• XAS [Mo(VI,V,IV)]• MCD/ EPR [Mo(V)]• Electronic Absorption• Resonance Raman

Single Metal Redox Center

MSS

O

SS

RO(VI)

(Mo=O)

(Mo-S)

x

Observe enhanced isotopic sensitive Mo=O and Mo-S vibrations.

Parallel model studies on both relevant and simpler systems needed.

Outstanding Issues in Pyranopterin Mo Enzyme Catalysis

Research ObjectivesUtilize available physical characterization methods to determine the geometric and electronic structure of small synthetic active site analogs.

Derive key factors that define geometric/electronic structure relationships and correlate to the unique enzymatic spectroscopic features and their electronic contributions to structure-bonding/ function.

Primary IssueWhat is Structural and Functional Role of the Pyranopterin Ene-1,2-

Dithiolate Unit During Course of Catalysis?

Research GoalDerive fundamental understanding at molecular level, into how the unique geometric and electronic structure of these enzyme active sites contribute to their reactivity.

Chemical Evolution of Mo and W Dithiolene Systems

The Reductionist Approach

S

SO

MN NN N

NN

S

SMoN N

N N

NN

S

S

SMoN N

N N

NN

NO

HB

H3C

H3C

H3C

CH3

CH3CH3

HB

H3C

H3C

H3C

CH3

CH3CH3

HB

H3C

H3C

H3C

CH3

CH3CH3

0, -1

MS

S

MS

SS

S

MS

SO

S

S

MS

SS

S

S

S

0, +1

-1, -2

-1,0

0, -1, -2

MS

SO

S

S

MS

SOR

S

S

MS

SO

S

S

MY

SRO

S

S

MY

SRO

S

S

RO

O

O

-1

-1

-2

-2

-1

Possess Mo(V) paramagnetic centers; Amenable to EPR/ MCD probes.

Minimal Structural Models/ Effective Spectroscopic Models

- S

- S N

N

- S

- S- S

- S

- S

- S

C H3

C l

C l

S

S

E

MN N

NN

N

N

HB

H3C

H 3 C

C H 3

C H 3CH 3

H3C

Probe fundamental properties of Oxo-Mo mono-ene1,2-dithiolate complexes:Metal (M = Mo, W), axial (E = O, S, NO) and dithiolate (S-S) coordination effects.

(Tp*)ME(S-S)

(S-S)

Isolated Oxo-Mo-Dithiolate Center; Controlled six coordinate environment.

Simple model; Mo coordinated by Ene-1,2-Dithiolate and terminal Oxo.

-4 10-6

-3 10-6

-2 10 -6

-1 10-6

0

1 10-6

2 10 -6

3 10-6

4 10-6

-1500.0-1000.0-500.000.0000500.001000.01500.0

(Tp*)MoO(bdt) (1)I

II

1 5.7 14.7 13.7 12.7 11.7 10.7 9.7 8.7 7.7 6.7 5.7

Ionization Energy (eV)

C27

C23

C24

C17C13

C14

C16

O

MOC15

C26N21B

N32

C37C33

C34

C36

N31

S1 C1 C6C5

C4

C3

C2S2

Resonance Raman Spectroscopy

Electrochemistry Magnetic Circular Dichroism

Electronic AbsorptionPhotoelectron Spectroscopy

Electron Paramagnetic Resonance

X-ray Crystallography

Density Functional Theory

Synthesis/ Purification

Characterization:NMR, IR, HR-MS, XAS

Minimal Structural/ Effective

Spectroscopic Active SiteModels

-1 105

-5 104

0

5 104

1 105

1.5 105

3100 3200 3300 3400 3500 3600 3700Field (Gauss)

n= 9.4510 GHz

(3)

Physical Characterization: The (Tp*)MoO(bdt) Benchmark

MCD and Electronic Absorption Spectroscopy

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

-1000

-500

0

500

1000

8000 12000 16000 20000 24000 28000 32000

Ab

sorb

ance

MC

D In

tensity (m

deg)

Energy (wavenumbers)

1 2 (3, 4) 5 6 7

Complimentary selection rules: resolve electronic transitions in spectra

MCD (5K /7T)

Low Temperature Solid-State (PDMSO Mull) Studies

Absorption (5K)

Band Assignments from Combined Spectroscopic Approach

0

800

1600

2400

3200

4000

4800

5600

6400

8000 12000 16000 20000 24000 28000 32000

Ep

silo

n (

M-1

cm

-1)

Energy (wavenumbers)

1 2 (3, 4)

5

6

7y

1 2

3

5

6 7

4

ya''op

ya''ip

ya'op

ya'ip

ya'z2

ya''x2-y2

a''x'z ya'

y'z

y a'xy

O

O

M= 90 0

M

a'xy + a'

ip

S

S

> 90 0

a'xy + a'

op a'yz + a'

op

Solution Electronic Absorption (DCE)

Resonance Raman Scattering

Enhancement of the Raman signal

RayleighNormal RamanResonance Raman

nn'

nono

IR

E0

E1

FC - A TermHT - B Term

E’

Intensitiy: selective enhancement associated with absorbing metal center.

Selectivity based on resonant electronic transition and excited state distortion.

Vibrational frequencies: sensitive to inner coordination environment.

Sensitive and selective probe of structure/ bonding

Intensity depends on energy and intensity of electronic absorption band.

Enhancement result of coupling with electronic excited state.

M

O

S

S

M

S

O

S

Raman Experimental Instrumentation and Techniques Design and Methodology

CCD

Computer Controller

System Interface

Argon Ion LaserTitanium Sapphire Laser

Krypton Ion Laser

Pre Monochromator SPEX 1405

Sample

Illumination/ Collection Optics

Goal: Obtain Low-frequency vibrational information regarding M-S bonding.

SPEX 1877Triplemate

CCD

Computer Controller

System Interface

Argon Ion Laser

Sample

Illumination/ Collection Optics SPEX 1877

Triplemate135 degree back scattering geometry

90 degree geometry

Collection Geometry

The Resonance Raman Experiment

Laser Enhanced Raman Spectroscopy

Sample Illumination and Collection Optics

Sample Handling, Detection and Dispersal System

Samples Problematic: Photo Decomposition/ Thermal Degradation?

Vibrational Raman Spectroscopy

5500

6000

6500

7000

7500

8000

8500

300 400 500 600 700 800 900 1000 1100

Ra

ma

n I

nte

ns

ity

(c

ps

)

Raman-shift (wavenumbers)

1300

1400

1500

1600

1700

1800

1900

2000

300 400 500 600 700 800 900 1000 1100

n6

n1 n 3

n 6

n1

n1 n6

n3

n3

140K 528.7 nm ~40 mW

(Tp*)MoO(bdt) in NaCl/ Na2SO4

(Tp*)MoO(bdt) in Benzene

293K 514.5 nm ~75 mW

3 vibrational bands observed

= 932 cm-1= 393 cm-1= 362 cm-1

Identify normal modes coupled to electronic transitions

S

S

O

MoN

N

NN

N

N

HB

H3C

H3C

H3C

CH3

CH3CH3

Solution Raman Depolarization Studies

Ram

an I

nte

nsi

ty (

cps)

= 932 cm-1

= 393 cm-1

= 362 cm-1

1300

1400

1500

1600

1700

1800

1900

2000

300 400 500 600 700 800 900 1000 1100

n3 n1 n6

1300

1400

1500

1600

1700

1800

1900

2000

300 400 500 600 700 800 900 1000 1100

Raman-shift (wavenumbers)

n1 n6 n3

(Tp*)MoO(bdt) in Benzene293K 496.5 nm ~75 mW

n6 ( A' )

n3 ( A' )

n1 ( A' )

I

I

Parallel polarization

Perpendicular polarization

Depolarization Ratio

= I/ I 0 3/4

Totally symmetric (polarized)

¾

Non-totally symmetric (depolarized)

Ratio indicates 3 modes are totally symmetric

Vibrational Analysis

1300

1400

1500

1600

1700

1800

1900

2000

300 400 500 600 700 800 900 1000 1100

Ram

an I

nte

nsi

ty (

cps)

Raman-shift (cm-1

)

Key Points:3 bands observed – polarized (A’ symmetry)

Resonance Raman spectroscopy probes: Differences in bonding between ground and excited states via distortions along specific normal modes.

Intensity enhancement patterns consistentWith M-S/ M=O vibrational assignments

M

O

S SM

O

S S

M

O

S S

M

O

S SM

O

S SM

O

S S

SS

M

O

1 ( A' ) 2 ( A'' )

4 ( A' )

3 ( A' )

5 ( A'' )

z

y

x

(zy)SS

M

O

z

y

x N

NB N

n6 ( A' )

932 cm-1

393 cm-1

362 cm-1

n6 ( A' ) n3 ( A' )n1 ( A' )

S

SO

MoS

SO

MoN NN N

NN

HB

H3C

H3C

H3C

CH3

CH3CH3

(Tp*)MoO(bdt) in Benzene

Solid-State Excitation Profiles

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0

0.5

1

1.5

2

16000 18000 20000 22000 24000

Energy (wavenumbers)

S

S

M

O

n3 ( A' )

S

S

M

O

S

S

M

O

n1 ( A' )

n6 ( A' )

(Tp*)MoO(bdt): 8K PDMSO mull EA; 100K RR NaCl/ Na2SO4

Observe large differential enhancement of Mo=O

Key Points:

Transitions probed are orthogonal (in-plane vs out-of-plane)

Conclusions:

Sip Mo dxy CT probes covalent contributions to ground-state

S Mo CT probes electronic contributions to redox potentials

Sip Mo dxy

Sop Mo dxz,yz

Implications for Catalytic Reactivity in Enzymes

xya'

ipa'

xya'

ipa'

S-Moxy3-center

pseudo- antibonding

S-Moxy3-center

pseudo- bonding

S

S

S

O

c ys

Mo

OH2

(IV)

S

S

S

O

c ys

Mo

OH

(V)

S

S

S

O

c ys

Mo (VI)

OSO3

2- SO42-

H2O

H+, e-H+, e-

Lowest energy (intense) CT must be Sip Mo dxy

This CT transition probes covalency contributions to ET pathway.

Criteria for efficient ET

Good M-L overlap/ Minimize ROE

Reason Nature has chosen ene-1,2-dithiolate and M=O groups

M=O aligns redox orbital for facile ET via unique 3-center 2-electron bond.

Conclusions

Resonance Raman Important Probe of Ground and Excited State Structure

State of the Art Equipment Necessary for probing M-S Bonding.

Contributions of M-L Bonding to Electronic Structure Elucidated by RREspecially when Combined with other Spectroscopic Techniques.

RR Spectroscopy Important Tool for Characterizing Enzyme Active Siteswhen Interpreted within Context of Well-Defined Small Molecular Models.

Protocols Developed can be Applied to more Complicated Systems.

3000

3200

3400

3600

3800

4000

400 600 800 1000 1200 1400

Ram

an I

nte

nsi

ty (

cps)

Raman Shift (cm -1)

(Tp*)MoO(qdt) in Benzene at 514.5nm

Acknowledgements and Funding

Mo

E

S

S C

C

z

y

x

E = O, NO

= 42.1

(Tp*)MoO(bdt) (Tp*)MoO(bdtCl2)

(Tp*)Mo(NO)(bdt) (Tp*)Mo(NO)(bdtCl2)

= 6.9 = 21.3

= 44.4

14

2

21 37

C27

C23

C24

C17C13

C14

C16

O

MOC15

C26N21B

N32

C37C33

C34

C36

N31

S1 C1 C6C5

C4

C3

C2S2

Small change with changeof dithiolate ligand

Significantchangewithchange ofaxial ligand

10.5 10 9.5 9 8.5 8 7.5 7 6.5 Ionization Energy (eV)

HeI

HeII

HOMO

HOMO -1&-2

HOMO-3 &-4

HOMO-5

10 9.5 9 8.5 8 7.5 7 6.5 6

Ionization Energy (eV)

(Tp*)MoO(bdt)

(Tp*)WO(bdt)

C27

C23

C24

C17C13

C14

C16

O

MOC15

C26N21B

N32

C37C33

C34

C36

N31

S1 C1 C6C5

C4

C3

C2S2

Mo dxy

Pseudo anti-bonding

Pseudo bonding

Sip

**

*

*

h = 579 nmEnemark Research Group

University of Arizona

Kirk Research GroupUniversity of New Mexico

National Institutes of Health National Science Foundation

Petroleum Research Fund Sandia National Laboratories

Prof. John H. Enemark

Prof. Martin L. Kirk