title: Determination of reduction potential of an engineered Cu(A) azurin by cyclic voltammetry and spectrochemical titrations.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Hee Jung
authors.initials: HJ
authors.lastname: Hwang
authors.forename: Majorie
authors.initials: M
authors.lastname: Ang
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D001400:Azurin / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D004563:Electrochemistry; D004579:Electron Transport; D010084:Oxidation-Reduction; D015202:Protein Engineering; D013057:Spectrum Analysis; D017186:Titrimetry
chemical_list: D001400:Azurin; D003300:Copper