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

Original Article

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 °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(NH3)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 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.

Keywords

Copper Electron transfer Cytochrome c oxidase 

Abbreviations

CcO

cytochrome c oxidase

N2OR

nitrous oxide reductase

ET

electron transfer

CV

cyclic voltammetry

NHE

normal hydrogen electrode

DDAB

didodecyldimethylammonium bromide

PGE

pyrolytic graphite edge

Notes

Acknowledgements

This material is based upon work supported by the National Science Foundation (CHE-0139203). We thank Drs. Kevin R. Hoke and Christophe Léger, and Professors Fraser A. Armstrong, Michael G. Hill and Sean J. Elliott for advice on electrochemistry.

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Copyright information

© SBIC 2004

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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