1. Applications of Resonance Enhanced Raman
Spectroscopy: Electronic Structure Probe of
Metal-Sulfur Interactions in Oxo-Molybdenum
Ene-1,2-Dithiolate Systems
Frank E. Inscore
The University of Arizona
C H3 (Tp*)MoO(bdt)
O
H3C
N S
N
HB Mo
N
H3C N S
C H3 C H
3
N
N
H3C Active Site of Sulfite Oxidase

2. The Raman Spectroscopic Technique
General Considerations
Research and Industrial Applications
Structure/ Solid State/ Biological Chemistry
Utility of Raman Spectroscopy
Problematic: Inherent Weak Effect
Problematic: Fluorescence Complications
Problematic: Instrumental Limitations
Development of New Techniques
FT-RS/ SERS/ RERS

3. Resonance Raman Spectroscopy
Characterizing Structure/ Monitoring Reactivity in Catalytic Systems
Chemical and Petroleum/ Energy Production Industries
• 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.
Biological 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.

4. Overview of Presentation
• Raman Applications • What we are doing?
• Background
• Electronic Structure • Why we are studying?
• Raman Instrumentation
• Resonance Raman Studies • How we will probe?
• Implications for Mo Enzymes
C H3
O
H3C
N S
N
HB Mo
N
H3C N S
C H3 C H
3
N
N
H3C

5. The Importance of Metal 1,2-Dithiolene Complexes
General Considerations
Why the Interest in Transition Metal-Sulfur Complexes?
Industrial Applications/ Commercial Uses:
Vulcanization Accelerators for Rubber Wear Additive Inhibitors in Lubricants
Catalytic Inhibitors /Oxidation Catalyst Mode-Locking Additives in Nd Lasers
Potential Biological Activity:
Correlations with Biological Systems containing Metal to Sulfur Bonds.
What Is an Ene-1,2-Dithiolate Ligand ?
Four Prototypical Ene-1,2-Dithiolate Systems:
-S H -S -S N -S S
-S
M
S = -S -S -S N -S
S -S H S
Relevance to structure, bonding and function of Metalloenzyme active site centers

6. Pyranopterin Molybdenum and Tungsten Enzymes
Background and Significance
3 Mo Families based on structure and reactivity
2 W Families similar to DMSO reductase family
O S O
S er -O
S O O
Mo S S S
Mo Mo
S S - C ys S O H2 S S
Sulfite Oxidase Xanthine Oxidase DMSO Reductase
# X-ray crystallography reveals
a common structural unit:
Pyranopterin cofactor
O S-
H
N S-
HN
H2N N N O OPO 2-
H 3

7. The Resonance Raman Spectroscopic Probe
Structure/ Bonding in the Active Site of DMSO Reductase
Single Metal Redox Center RO O
(VI)
• XAS [Mo(VI,V,IV)] S S
M
• MCD/ EPR [Mo(V)] S S
(Mo-S)
• Electronic Absorption (Mo=O) x
• Resonance Raman
Observe enhanced isotopic sensitive Mo=O and Mo-S vibrations.
Parallel model studies on both relevant and simpler systems needed.

8. Outstanding Issues in Pyranopterin Mo Enzyme Catalysis
Primary Issue
What is Structural and Functional Role of the Pyranopterin Ene-1,2-
Dithiolate Unit During Course of Catalysis?
Research Goal
Derive fundamental understanding at molecular level, into how the unique
geometric and electronic structure of these enzyme active sites contribute
to their reactivity.
Research Objectives
Utilize 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.

9. Chemical Evolution of Mo and W Dithiolene Systems
The Reductionist Approach
CH3
H3C O O -1, -2 O -1
N S RO
HB N S S S S
N N M M M
H3C S S S S S
CH3 -1
N CH3 OR
N S S
-1,0 M
H3C S S
S S
CH3 M
S S
H3C S -2
N N Mo S OO
HB S S
H3C N N S M
S S
CH 0, +1
N 3CH3
N S
M
S
-2
H3C OO
CH3 S SR
O M
H3C 0, -1 S Y
N N
S
HB N Mo O
H3C N N S SS 0, -1, -2 -1
S SR
S S M
CH3 M S Y
N CH3 S S
N
H3C

10. Minimal Structural Models/ Effective Spectroscopic Models
Simple model; Mo coordinated by Ene-1,2-Dithiolate and terminal Oxo.
Isolated Oxo-Mo-Dithiolate Center; Controlled six coordinate environment.
Possess Mo(V) paramagnetic centers; Amenable to EPR/ MCD probes.
C H3
E
H3C
N
N S (S-S)
HB M
N S
H3 C N Cl
-S -S C H3 - S -S N
CH C H
3
N 3
-S -S -S -S N
N
(Tp*)ME(S-S) Cl
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.

12. MCD and Electronic Absorption Spectroscopy
Low Temperature Solid-State (PDMSO Mull) Studies
0.8 1000
Absorption (5K) MCD (5K /7T)
0.7
0.6 500
MCD Intensity (mdeg)
0.5
Absorbance
0.4 0
0.3
0.2 1 2 -500
(3, 4) 5 6 7
0.1
0 -1000
8000 12000 16000 20000 24000 28000 32000
Energy (wavenumbers)
Complimentary selection rules:
resolve electronic transitions in spectra

13. Band Assignments from Combined Spectroscopic Approach
y a'z2
Solution Electronic Absorption (DCE)
y a''x2-y2
4 6400
y x'z y a'y'z
a''
7
3 6 7
5600
4800
y a'xy
1 2 5
Epsilon (M cm )
-1
4000 6
y a''op
y a'op -1 3200
2400
y a'ip 5
1600
y a''ip
800 1 2 (3, 4)
a'xy + a'ip a'xy + a'op a'yz + a'op
O 0
M S
8000 12000 16000 20000 24000 28000 32000
= 90 0
Energy (wavenumbers)
O
M
S
> 90 0

14. Resonance Raman Scattering
Enhancement of the Raman signal
Sensitive and selective probe of structure/ bonding
Vibrational frequencies: sensitive to inner coordination environment.
Intensitiy: selective enhancement associated with absorbing metal center.
Resonance FC - A Term
Normal Raman Rayleigh
Raman O HT - B Term
E’
E1
o o M S
O S
IR
' M S

E0
S
Selectivity based on resonant electronic transition and excited state distortion.
Intensity depends on energy and intensity of electronic absorption band.
Enhancement result of coupling with electronic excited state.

15. Raman Experimental Instrumentation and Techniques
Design and Methodology
Goal: Obtain Low-frequency vibrational information regarding M-S bonding.
Computer System
Controller Interface
Titanium Sapphire Laser Argon Ion Laser
CCD
Sample
Krypton Ion Laser
Illumination/ Collection
Pre Monochromator Optics SPEX 1877
SPEX 1405 Triplemate

16. Collection Geometry
Computer System
Controller Interface
Argon Ion Laser
90 degree geometry
CCD
Sample
Illumination/ Collection
Optics SPEX 1877
135 degree back scattering geometry Triplemate

17. The Resonance Raman Experiment

18. Laser Enhanced Raman Spectroscopy

20. Sample Illumination and Collection Optics

21. Sample Handling, Detection and Dispersal System
Samples Problematic: Photo Decomposition/ Thermal Degradation?

22. Vibrational Raman Spectroscopy
C H3
(Tp*)MoO(bdt) in NaCl/ Na2SO4
O 8500
H 3C 140K 528.7 nm ~40 mW
N S 8000
N
HB Mo 7500 3
N
H3C N S
7000
1
C H3 C H 6500 6
Raman Intensity (cps)
N 3
6000
N
5500
300 400 500 600 700 800 900 1000 1100
(Tp*)MoO(bdt) in Benzene
H 3C 2000
293K 514.5 nm ~75 mW
1900
3 vibrational bands observed 1800
1700
1600
 6 = 362 cm-1 1 = 393 cm-1  3 = 932 cm-1 1500
3
6 1
1400
1300
300 400 500 600 700 800 900 1000 1100
Raman-shift (wavenumbers)
Identify normal modes coupled to electronic transitions

23. Solution Raman Depolarization Studies
2000
Depolarization Ratio (Tp*)MoO(bdt) in Benzene
1900
293K 496.5 nm ~75 mW
1800
 = I/ I  1700
Parallel polarization
0   3/4 1600 I 
  ¾
1500
3
1400 6 1
Raman Intensity (cps)
1300
300 400 500 600 700 800 900 1000 1100
Totally symmetric (polarized)
Non-totally symmetric (depolarized) 2000
1900
1800
Perpendicular polarization
Ratio indicates 3 modes are totally symmetric 1700
6(A = 362 cm-1
') 1600
I
1500
1(A = 393 cm-1
')
1400 6 1 3
3(A = 932 cm-1
') 1300
300 400 500 600 700 800 900 1000 1100
Raman-shift (wavenumbers)

24. Vibrational Analysis
(Tp*)MoO(bdt) in Benzene
2000 CH3
O
H3C O z z
S
NN S
1900
Mo
N N Mo S
HB
S
Raman Intensity (cps)
H3C
1800 O O
CH3 CH
N 3
1700 N M y  (zy) M y
B N
N S S
1600 S x S
362 cm-1 H C 3
932 cm-1
x N
-1
1500  ( A' ) 393 cm  ( A' )
6 3
 ( A' )
1
1400 O
O O
1300
300 400 500 600 700 800 900 1000 1100 M M M
-1 S S S S S S
Raman-shift (cm )
1 ( A' ) 2 ( A'' ) 3 ( A' )
Key Points:
3 bands observed – polarized (A’ symmetry) O O O
M M M
Intensity enhancement patterns consistent S S S S S S
With M-S/ M=O vibrational assignments 4 ( A' ) 5 ( A'' )
 ( A' )
6
Resonance Raman spectroscopy probes:
Differences in bonding between ground and excited states via distortions along specific normal modes.

25. Solid-State Excitation Profiles
Key Points:
Observe large differential enhancement of Mo=O
Transitions probed are orthogonal (in-plane vs out-of-plane)
O
(Tp*)MoO(bdt): 8K PDMSO mull EA; 100K RR NaCl/ Na2SO4 M S
S
3( A' )
Sop  Mo dxz,yz
O
Sip  Mo dxy S
M
S
1( A' )
O
M S
S
6( A' )
Conclusions:
Sip  Mo dxy CT probes covalent contributions to ground-state
S Mo CT probes electronic contributions to redox potentials

26. Implications for Catalytic Reactivity in Enzymes
Lowest energy (intense) CT must be Sip  Mo dxy
This CT transition probes covalency contributions to ET pathway.
M=O aligns redox orbital for facile ET via unique 3-center 2-electron bond.
O SO32- SO42- O
O OH 2
S S
yxya' Mo (VI) Mo (IV)
S S cys S S cys
xya' H+, e- H2O
S-Moxy
3-center H+, e-
pseudo- antibonding
O OH
S-Moxy
3-center S
pseudo- bonding Mo (V)
a'
ip S S cys
Criteria for efficient ET
yipa' Reason Nature has chosen ene-1,2-dithiolate and M=O groups
Good M-L overlap/ Minimize ROE

27. 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 RR
Especially when Combined with other Spectroscopic Techniques.
RR Spectroscopy Important Tool for Characterizing Enzyme Active Sites
when Interpreted within Context of Well-Defined Small Molecular Models.
Protocols Developed can be Applied to more Complicated Systems.

28. Acknowledgements and Funding
*
HeI
Prof. John H. Enemark HOMO
Pseudo anti-bonding
Mo dxy
h = 579 nm
Enemark Research Group HOMO -1&-2
HeII
Sip
University of Arizona
Pseudo bonding
* HOMO-3 &-4
* *
10.5 10 9.5 9 8.5 8 7.5 7 6.5 HOMO-5
Ionization Energy (eV)
Prof. Martin L. Kirk
National Institutes of Health National Science Foundation
Kirk Research Group
University of New Mexico
Petroleum Research Fund Sandia National Laboratories
C16
O
C14
C15 S2 C4
C2
C13 MO
C17 C3
C24 C26 S1 C5
N21 C1 C6
B N31
C23 N32 C36
(Tp*)M oO(qdt) in Benze ne at 514.5nm
C27 C33
C37 C34
4000
(Tp*)MoO(bdt) 3800
Raman Intensity (cps)
3600
3400
(Tp*)WO(bdt)
3200
3000
10 9.5 9 8.5 8 7.5 7 6.5 6
400 600 800 1000 1200 1400
Ionization Energy (eV) -1
Ram an S h ift (cm)