Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617.
The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-479]. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130,000 was determined by gel filtration. The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560-nm band for the reduced form in the alpha range. The prosthetic group is made of two hemes b, one high-spin (S = 5/2, gl = 5.9, g parallel approximately 2.0), the other low-spin (S = 1/2, gz = 2.94, gy = 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one-electron, single redox couple, with Em = 87 +/- 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2.min-1.mg protein-1 and commercially available coenzyme (101.74 nmol O2.min-1.mg protein-1). Exposure of the reduced enzyme to CO induced the collapse of alpha and beta bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.
Arnaud
S
S
Centre d'Océanologie de Marseille, CNRS URA 41, Parc Scientifique et Technologique de Luminy, Marseille, France.
Malatesta
F
F
Guigliarelli
B
B
Gayda
J P
JP
Bertrand
P
P
Miraglio
R
R
Denis
M
M
D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D012623:Seawater; D013053:Spectrophotometry
D000596:Amino Acids; D006418:Heme; D010088:Oxidoreductases; C020094:duroquinol oxidase