Thin Film Cyclic Voltammetry

Equipment for film voltammetry

potentiostat

insulator electrode material reference N 2 inlet counter Electroactive film

working electrode E-t waveform

E, V

Cyclic voltammetry

Electrochemical cell time

Ideal, reversible thin layer cyclic voltammogram

Example cobalt complex: LCo III + e LCo II Q = nFA G T G T = total surface concentration of electroactive species A = electrode area, F = Faraday’s constant E p I p reversible peak current I p increases linearly as scan rate (  ) is increased; And D E p = 0. Rate constants can be obtained by increasing  the CV into a kinetically limited situation where D E p to drive > 0. Q = area under reduction curve

Many types of electroactive films

Ferrocene SAM Electroactive polymer Protein SAM SAM = self assembled monolayer

Real CVs, include Charging current And some non-ideality

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

Apply voltage Measure current Information obtained:

1. Redox potentials, free energies, re-organization energies

2. Redox mechanism: protonation/deprotonation and chemical reaction steps

3. Kinetics and thermodynamics of catalytic reactions 4. Biosensors

One way to make a stable protein film A lipid-protein film

enzyme Electrode

• Many other types of films possible - polyions, Adsorbed, crosslinked, etc.

Reversible Peaks for Direct electron Transfer; Peak shapes, sizes, and E p reveal details of redox chemistry Nearly ideal Reversible ET

Forward peak Reduction Of Fe III Reverse peak Oxidation Of Fe II

LbL

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 o C. Example where rate constants can be obtained by increasing  to drive the CV into a kinetically limited situation; D E p >> 0. Mb is another iron heme protein, peaks are for redox reactions of iron.

Value of k s (s -1 ) cas be obtained by fitting data to theoretical curves of D E p vs. log scan rate or by fitting with best fit digital simulations of the CVs.

Cytochrome P450 Enzymes

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 (low spin fastest); conformational equilibria of cyt P450 iron heme • rates of oxidation by peroxide depend on spin state and secondary structure (high spin fastest)

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 10-25 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.

PFe II -CO

Spectral characterization of cyt P450 films

PFe III PFe III 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.

Cyclic Voltammetry and rate constant (k s ) estimates Assuming simple electron transfer model

Background subtracted cyclic voltammograms of LbL films on PG electrodes in anaerobic 50 buffer + 0.1 M NaCl, pH 7.0 P450 2E1 P450 cam Rate const. estimation for cyt P450/polyion films experimental (  ) peak separation ( D E p ) corrected for scan rate independent non-kinetic contribution. Lines for Butler-Volmer theory for the rate constant (k

s

) shown and a = 0.5.

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

Conclusions for cyt P450 ET from thin Film voltammetry:

• low spin cyt P450cam, k s = 95 s -1 mixed spin cyt P450 1E2, k s = 18 s -1 (80% high spin) high spin cyt P450 1A2, k s = 2.3 s -1 • k s 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 fastest) also (high spin

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

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