2 Equipment for film voltammetry potentiostatinsulatorelectrodematerialreferenceElectroactive filmN2inletcounterworking electrodeE-t waveformCyclicvoltammetryE, VElectrochemical celltime
3 Ideal, reversible thin layer cyclic voltammogram Example cobalt complex: LCoIII + e-LCoIIQ = nFAGT GT = total surface concentration of electroactive speciesA = electrode area, F = Faraday’s constantEpIpreversible peak current Ip increases linearly as scan rate () is increased;And DEp = 0. Rate constants can be obtained by increasing to drivethe CV into a kinetically limited situation where DEp > 0. Q = area underreduction curve
4 Many types of electroactive films Ferrocene SAMElectroactive polymerProtein SAMSAM = self assembled monolayer
5 Real CVs, includeCharging currentAnd 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 proteinThin Film Electrochemistry of ProteinsProtein (monolayer)electrodeMeasure currentApply voltageInformation obtained:Redox potentials, free energies, re-organization energies2. Redox mechanism: protonation/deprotonation and chemical reaction stepsKinetics and thermodynamics of catalytic reactions4. Biosensors
7 One way to make a stable protein film A lipid-protein filmenzymeElectrode• Many other types of films possible - polyions,Adsorbed, crosslinked, etc.
8 Peak shapes, sizes, and Ep reveal details of redox chemistry Forward peakReversiblePeaks forDirect electronTransfer;Peak shapes, sizes, and Ep reveal details of redox chemistryNearly idealReversible ETReductionOf FeIIIOxidationOf FeIIReverse peak
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.
12 Human Metabolic Enzymes: Prof. John Schenkman, Pharmacology,Cell Biology, Uconn Health CenterCytP450s 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 nmSadagopan 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 PFeIIIPFeIIIPFeII-COUV-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 modelBackground subtracted cyclic voltammograms of LbL films on PG electrodes in anaerobic 50 buffer M NaCl, pH 7.0P450 2E1P450 camRate 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 modelLines were from digital simulation using
17 Conclusions for cyt P450 ET from thin Film voltammetry: • low spin cyt P450cam, ks = 95 s-1mixed 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 usede.g. Cu Cu2+ + 2e
19 Divided Electrolysis Cell for synthetic use Counter electrodeLarge working electrode + ref