Two azurins with unusual redox and spectroscopic properties isolated from the Pseudomonas chlororaphis strains DSM 50083^T and DSM 50135

Two azurins (Az624 and Az626) were isolated from the soluble extract of two strains of Pseudomonas chlororaphis, DSM 50083^T and DSM 50135, respectively, grown under microaerobic conditions with nitrate as final electron acceptor. The azurins, purified to electrophoretic homogeneity in three chromatographic steps, exhibit several peculiar properties. They have high reduction potentials and lower pI than most azurins described in the literature. As previously observed for Pseudomonas aeruginosa azurin, their reduction potentials are pH-dependent, but the pK values of their oxidized forms are lower, which suggests that deeper structural changes are associated with the oxidation process of these novel azurins. A hitherto undescribed pH-dependence of the diffusion coefficient was observed in Az624, that could be caused either by conformational changes, or by the formation of supramolecular aggregates associated with a protonation process. Both azurins exhibit axial X-band electron paramagnetic resonance spectra in frozen solution showing a typical hyperfine with the copper nucleus (I=3/2) and a well-resolved superhyperfine structure with two equivalent ¹⁴N nucleus (I=1), which is not usually observed for azurins from other species.