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

The reduction potentials of an engineered CU_A azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4′-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured CU_A azurin are the same within the experimental error (422 ± 5 and 425 ± 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 ± 7 mV (50 mM ammonium acetate buffer, pH 5.1, 4 °C) and 420 ± 1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 °C), respectively. Spectroscopic redox titration using [Ru(NH ₃)₅Py]²⁺ resulted in a reduction potential (254 ± 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 CU _A azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH₃)₅Py] ²⁺ measures the reduction potential of native CU_A azurin.