A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strai n 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 denitrif icans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activa tion of the enzyme through spin state transition of the peroxidatic haem. M onohaemic cytochrome c(552) from Ps. nautica was identified as the physiolo gical electron donor, with a half-saturating concentration of 122 mu M 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 streng th. There are indications that the interactions between the two redox partn ers are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All ri ghts reserved.