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 peakto-peak separation of ⌬E p ‫؍‬ 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 ؋ 10 ؊6 cm 2 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, UVvisible, 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 ؊1 are shifted to higher energy (ϳ10 cm ؊1

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

“…PAz isolated from P. pantotrophus has a formal reduction potential of 230 ± 5 mV, at pH 7.0, and three pK values were identified, 6.5, 7.2 and 10.5, from the dependence of that parameter with the pH, as verified for other small blue copper proteins [22,33,34].…”

“…Some of the pK values can be assigned from spectroscopic evidence for this PAz [16], by comparison with the results obtained for PAz from Achromobacter cycloclastes [22,33,34], which has 67% homology with P. pantotrophus PAz. The pK values of 6.5 and 7.2 have been assigned to the protonation of the histidine residue 6 [33].…”

“…The pK values of 6.5 and 7.2 have been assigned to the protonation of the histidine residue 6 [33]. The pK O2 value of 10.5 may be attributed, but this is still in discussion, to a structural transition brought about by the acid dissociation of the NH 3 + e-amino group of Lys77 (pK a = 10.4) [22].…”

“…Several works have reported the direct electrochemistry of blue copper proteins, including PAz from Achromobacter cycloclastes [21,22] and from Alcaligenes faecalis [23]. Few works dealing with the electrochemistry of other peroxidases have been published, with monohaem CCP being the most studied [24,25].…”

Towards Experimental Tests of Quantum Effects in Cytoskeletal Proteins

“…For instance, rotaxanes have been considered as switches and/or fuses [38] and carbon nanotubes as active channels in field effect transistors [39]. Many of these are not suitable for placement on traditional chips [40] or for forming ordered networks while virtually all of these attempt to hybridize some kind of electrical wires to chemical substrates in order to obtain current flows. This requirement adds a level of complexity to the task because of the need for appropriate nanomanufactured wires and connections.…”