1. Thin Film Cyclic Voltammetry

2. Equipment for film voltammetry
potentiostat
insulator
electrode
material
reference
Electroactive film N2
inlet
counter
working electrode
E-t waveform
Cyclic
voltammetry
E, V
Electrochemical cell
time

3. Ideal, reversible thin layer cyclic voltammogram
Example cobalt complex: LCoIII + e-
LCoII
Q = nFAGT  GT = total surface concentration of electroactive species
A = electrode area, F = Faraday’s constant Ep Ip
reversible peak current Ip increases linearly as scan rate () is increased;
And DEp = 0. Rate constants can be obtained by increasing  to drive
the CV into a kinetically limited situation where DEp > 0. Q = area under
reduction curve

4. Many types of electroactive films
Ferrocene SAM
Electroactive polymer
Protein SAM
SAM = self assembled monolayer

5. Real CVs, include
Charging current
And some non-ideality

6. Thin Film Electrochemistry of Proteins
Electrochemistry of proteins in solution
• electrode fouling, proteins denature
• large size means small D, tiny signals
• need lots of protein
Thin Film Electrochemistry of Proteins
Protein (monolayer)
electrode
Measure current
Apply voltage
Information obtained:
Redox potentials, free energies, re-organization energies
2. Redox mechanism: protonation/deprotonation and chemical reaction steps
Kinetics and thermodynamics of catalytic reactions
4. Biosensors

7. One way to make a stable protein film
A lipid-protein film
enzyme
Electrode
• Many other types of films possible - polyions,
Adsorbed, crosslinked, etc.

8. Peak shapes, sizes, and Ep reveal details of redox chemistry
Forward peak
Reversible
Peaks for
Direct electron
Transfer;
Peak shapes, sizes, and Ep reveal details of redox chemistry
Nearly ideal
Reversible ET
Reduction
Of FeIII
Oxidation
Of FeII
Reverse peak

9. LbL

10. Kinetically limited CV at 0
Kinetically limited CV at 0.1 V s-1 for 40 nm myoglobin (Mb)-polyion film on a PG electrode in pH 5.5 buffer at 35 oC. Example where rate constants can be obtained by increasing  to drive the CV into a kinetically limited situation; DEp >> 0. Mb is another iron heme protein, peaks are for redox reactions of iron.
Value of ks (s-1) cas be obtained by fitting data to theoretical curves of DEp vs. log scan rate or by fitting with best fit digital simulations of the CVs.

11. Cytochrome P450 Enzymes

12. Human Metabolic Enzymes:
Prof. John Schenkman, Pharmacology,
Cell Biology, Uconn Health Center
CytP450s in LbL polyion films:
• ET reduction rates from CV depend on spin state of cyt P450 iron heme (low spin fastest); conformational equilibria
• rates of oxidation by peroxide depend on spin state (high spin fastest) and secondary structure

13. Thin Film voltammetry of human cyt P450s
LbL films of cyt P450s and polyions on pyrolytic graphite electrodes. Polyions are purple strands and proteins are green/red ribbons . Thickness nm
Sadagopan Krishnan, Amila Abeykoon, John B. Schenkman, and James F. Rusling, Control of Electrochemical and Ferryloxy Formation Kinetics of Cyt P450s in Polyion Films by Heme Iron Spin State and Secondary Structure, J. Am. Chem. Soc. 2009, 131, 16215–16224.

14. Spectral characterization of cyt P450 films
PFeIII
PFeIII
PFeII-CO
UV-vis spectra of cyt P450 films on aminosilane-functionalized fused silica slides: (A) CO difference spectrum confirming native protein in PEI(/PSS/cyt P450 1A2)6 film after reducing to the ferrous form and purging the pH 7 buffer with CO; (B) ferric high spin form of enzyme in PEI(/PSS/cyt P450 1A2)6; and (C) ferric low spin form of enzyme in PSS(/PEI/cyt P450cam)6 film.

15. Cyclic Voltammetry and rate constant (ks) estimates
Assuming simple electron transfer model
Background subtracted cyclic voltammograms of LbL films on PG electrodes in anaerobic 50 buffer M NaCl, pH 7.0
P450 2E1
P450 cam
Rate const. estimation for cyt P450/polyion films experimental () peak separation (Ep) corrected for scan rate independent non-kinetic contribution. Lines for Butler-Volmer theory for the rate constant (ks) shown and a= 0.5.

16. The simple reversible theory did not fit peak potential vs
The simple reversible theory did not fit peak potential vs. scan rate data, so complex model
Lines were from digital simulation using

17. Conclusions for cyt P450 ET from thin Film voltammetry:
• low spin cyt P450cam, ks = 95 s-1
mixed spin cyt P450 1E2, ks = 18 s-1 (80% high spin)
high spin cyt P450 1A2, ks = 2.3 s-1
• ks for the reduction step correlates with spin state of the iron heme in the cyt P450, as found for solution reductions
• rates of oxidation by peroxide depend on spin state (high spin fastest) also

18. Divided cell – keep products apart
Undivided cell – sacrificial anode can be used
e.g. Cu  Cu2+ + 2e

19. Divided Electrolysis Cell for synthetic use
Counter electrode
Large working electrode + ref