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 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.
Alves
T
T
Departamento de Química, Centro de Tecnologia Química e Biologica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114, Caparica, Portugal.
Besson
S
S
Duarte
L C
LC
Pettigrew
G W
GW
Girio
F M
FM
Devreese
B
B
Vandenberghe
I
I
Van Beeumen
J
J
Fauque
G
G
Moura
I
I
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003574:Cytochrome c Group / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D001426:Bacterial Proteins; D003574:Cytochrome c Group; D003578:Cytochrome-c Peroxidase