Determination of reduction potential of an engineered Cu(A) azurin by cyclic voltammetry and spectrochemical titrations

• Published in: Journal of biological inorganic chemistry Vol. 9; no. 4; pp. 489 - 494
• Main Authors: Hwang, Hee Jung, Ang, Majorie, Lu, Yi
• Format: Journal Article
• Language: English
• Published: Germany 01.06.2004
• Subjects: Azurin - chemistry; Copper - chemistry; Electrochemistry; Electron Transport; Oxidation-Reduction; Protein Engineering; Spectrum Analysis; Titrimetry
• ISSN: 0949-8257
• DOI: 10.1007/s00775-004-0547-y

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 4,4'-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured CuA 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 degrees 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 degrees C) and 420 +/- 1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 degrees C), respectively. Spectroscopic redox titration using [Ru(NH3)5Py]2+ resulted in a reduction potential (254+/-4 mV) (50 mM ammonium acetate buffer, pH 5.1, 4 degrees 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.