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A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and
T Alves1, S Besson, L C Duarte
1Departamento de Química, Centro de Tecnologia Química e Biologica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114, Caparica, Portugal.
Abstract:
Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.