MEASUREMENT OF ELECTRON-TRANSFER RATES BETWEEN ADSORBED AZURIN AND A GOLD ELECTRODE MODIFIED WITH A HEXANETHIOL LAYER

In this work we report the application of electroreflectance (ER) spec troscopy for the study of the redox reaction of the nonheme protein az urin, Azurin was irreversibly adsorbed on a polycrystalline gold surfa ce modified with a hexanethiol layer. Surface-enhanced resonant Raman spectroscopy (SERRS) demonstrated that the immobilized azurin's copper site remained intact upon adsorption and that it underwent reversible reduction-oxidation, Cyclic voltammetry (CV) data showed that the pea k current of the oxidation (or reduction) wave depended linearly on th e sweep rate as appropriate for an immobilized species. The formal pot ential of the adsorbed azurin was 95 +/- 8 mV vs Ag/AgCl electrode, ne arly identical to the earlier observed value of the native azurin in s olution. All measurements were analyzed in the context of Marcus theor y of electron transfer, The separation of the reduction-oxidation peak s in the CV data gave an estimate of the electron transfer (ET) rate i n the range from 4 to 12 s(-1). ER, with light of wavelength of 640 nm , yielded a strong signal at the same potential as the midpotential de termined in CV, The frequency dependence of the ER response was consis tent with an ET rate of 150 to 200 s(-1). Electrochemical impedance me asurements indicated an ET rate of the order of 300 s(-1). The dispari ty between the ET rates measured with CV and ER suggests that ET may n ot be a single-step process. The results also point to the importance of hydrophobic interactions in adsorption and redox transformations of azurin. (C) 1997 Academic Press.