Electrochemical activation of proteins at electrode surfaces through electrostatic promotion and discrimination

Scope and method of study. The purpose of this research is to develop surface modifiers and electrode surfaces that will electrostatically promote the electrochemistry of redox-active proteins. The selective electrostatic promotion and discrimination of proteins is utilized to selectively reduce lower potential proteins (OM cytochrome b5, spinach ferredoxin) in the presence of higher potential proteins (cytochrome c, cytochrome P450cam). This selective reduction is followed by a second order homogeneous electron transfer reaction between the proteins, which gives rise to a characteristic cyclic voltammetric response. These electrochemical responses are simulated by various mechanisms in order to obtain rate constants for the reactions occurring between the proteins in solution.;Findings and conclusion. In lipid layers composed of didodecyldimethylammonium bromide (DDAB) or a modifier consisting of 0.02% nafion and 0.02% poly(2-methacryloxyethyltrimethyl ammonium bromide (PMAT), the direct electrochemical response of outer mitochondrial (OM) cyt b 5 was obtained. The homogeneous electron transfer reaction between OM cyt b5 and cytochrome c was hindered in the lipid modified electrodes due to the low diffusion coefficient of OM cyt b5 inside the lipid films. OM cyt b5 was strongly adsorbed at the 0.02% nafion and 0.02% PMAT electrodes, thereby preventing the homogeneous electron transfer between OM cyt b5 and cytochrome c from occurring. The second order homogeneous electron transfer rate constant for the reaction of OM b 5 and cytochrome c was measured at ultrasound activated glassy carbon electrodes and at indium tin oxide (ITO) electrodes. The sonochemically activated glassy carbon electrodes were also found to promote the electrochemistry of spinach ferredoxin. Cytochrome P450cam, on the other hand, was irreversibly adsorbed at this electrode surface. This is in contrast to ITO electrodes where spinach ferredoxin was selectively reduced in the presence of cytochrome P450cam. At the ITO electrodes, in the presence of substrate (i.e hexachloroethane), a catalytic current is obtained. From the simulation of this catalytic electrochemical response it has been postulated that the rate limiting step for reductive dehalogenation carried out by the enzyme is the egress of product from the active site.