Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617

Abstract

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 130000 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 α range. The prosthetic group is made of two hemes b, one high‐spin (S= 5/2, g⊥= 5.9, g∥∼ 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 α and β 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. Copyright © 1991, Wiley Blackwell. All rights reserved