Spectroscopic and Electrochemical Studies on Active-site Transitions of the Type 1 Copper Protein Pseudoazurin from Achromobacter cycloclastes

Abstract

The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode. Measurements carried out at 25.0°C indicate a midpoint reduction potential of E1/2 = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of ΔEp = 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 × 10-6 cm2 s-1 and 3.7 × 10-2 cm s-1, respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 × 10-3 cm s-1, consistent with a slower process at the positively charged electrode surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure. The trigonal form has elongated axial bonding and an axial EPR spectrum. At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm- are shifted to higher energy (˜10 cm-1), consistent with a stronger Cu-S bond. The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode. Measurements carried out at 25.0°C indicate a midpoint reduction potential of E1/2 = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of ΔEp = 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 × 10-6 cm2 s-1 and 3.7 × 10-2 cm s-1, respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 × 10-3 cm s-1, consistent with a slower process at the positively charged electrode surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure. The trigonal form has elongated axial bonding and an axial EPR spectrum. At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm- are shifted to higher energy (˜10 cm-1), consistent with a stronger Cu-S bond.