Determination of reduction potential of an engineered CuA azurin by cyclic voltammetry and spectrochemical titrations

The reduction potentials of an engineered CuA azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4ʹ-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured CuA azurin are the same within the experimental error (422(plus-minus)5 and 425(plus-minus)5 mV vs. NHE, respectively, in 50 mM ammonium acetate buffer, pH 5.1, 300 mM NaCl, 25 °C), indicating that the potential is that of a nonnative state. In contrast, using a didodecyldimethylammonium bromide (DDAB) film-pyrolytic graphite edge (PGE) electrode, the reduction potentials of native and thermally denatured CuA azurin have been determined to be 271(plus-minus)7 mV (50 mM ammonium Aacetate buffer, pH 5.1, 4 °C) and 420(plus-minus)1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 °AC), respectively. Spectroscopic redox titration using [Ru(NH3)5Py]2+ resulted in a reduction potential (254(plus-minus)4 mV) (50 mM ammonium acetate buffer, pH 5.1, 4 °C) similar to the value obtained using the DDAB film-PGE electrochemical method. Complete reoxidation of [Ru(NH3)5Py]^(2+)-reduced CuA azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH3)5Py]^(2+) measures the reduction potential of native CuA azurin.