Protein Film Voltammetry: Cyt P450s James F. Rusling, Univ. of CT, Dept. of Chemistry Dept. of Surgery, UConn Health Course materals:  http://rusling.research.uconn.edu/teaching/.

Thin Film Electrochemistry of Proteins Electrochemistry of proteins in solution • electrode fouling, proteins denature • large D, tiny signals • need lots of protein Thin Film Electrochemistry of Proteins Protein (monolayer) tiny amt. of protein 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

Equipment for protein film voltammetry potentiostat insulator electrode material reference Protein film N2 inlet counter working electrode E-t waveform Cyclic voltammetry E, V Electrochemical cell time Figure1

Thin Films of proteins: Differences from Solution Voltammetry • Electrochemical reactions occur in films • If enzymes, reatant usually delivered from solution to films; then must enter film • Diffusion in film much slower than in solutions

Ideal, reversible thin layer protein cyclic voltammogram Example: Iron heme proteins: PFeIII + e- PFeII Q = nFAGT  GT = total surface concentration of protein A = electrode area, F = Faraday’s constant 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.

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

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

Kinetically limited CV at 0 Kinetically limited CV at 0.1 V s-1 for 40 mm myoglobin-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. Value of ks (s-1) obtained by fitting data to theoretical curves of DEp vs. log scan rate or by fitting with best fit digital simulations of the CVs.

Additional methods used to make protein films Nanostructured electrodes, SWCNTs, AuNPs

LbL

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 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

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

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.

Voltammetry and rate constant estimates assuming reversible electron transfer 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 (Ep) corrected for scan rate independent non-kinetic contribution. Lines for Butler-Volmer theory for the rate constant (ks) shown and a= 0.5.

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

Conclusions for cyt P450 ET: • 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

Thanks to NIEHS/NIH and USDA for funding! Thanks again to all coworkers and collaborators http://web.uconn.edu/rusling/ Thanks to YOU for listening! Thanks to intangible creative factors Joe Cooley, Co. Galway playing a Paolo Soprani Accordion