We previously found that the soxABCD operon encodes a quinol oxidase complex in Sulfolobus acidocaldarius and this enzyme was purified and characterized. In this study, we have used a cloning procedure based on the conservation of oxidase sequences and the polymerase chain reaction to isolate a new gene (soxM) encoding a subunit of another terminal oxidase. This terminal oxidase is a fusion between two central components of cytochrome oxidases, subunits I and III. soxM forms a transcriptional unit which is expressed under heterotrophic growth conditions. The corresponding protein was detected by direct protein sequencing in a preparation enriched with a cytochrome absorbing light at 562 nm. This preparation contains a terminal oxidase which is able to oxidize the artificial substrate N,N,N′,N′ ‐tetramethyl‐p ‐phenylenediamine. This preparation also contains SoxC, a protein homologous to the mitochondrial cytochrome b, and a Rieske iron‐sulphur center. We suggest that SoxM is the core component of a second terminal oxidase complex and that this complex may share a subunit (SoxC) with the SoxABCD complex.

Mitochondrial cytochrome-c oxidase as well as several bacterial oxidases are known to reduce dioxygen to water. For the first time, a hemecontaining oxidase, the terminal enzyme of the aerobic respiratory system in the marine bacterium Pseudomonas nautica 617, is shown to reduce molecular oxygen only to hydrogen peroxide. Whereas the cell content is well protected from H,O, by catalase, the possible efflux of HZOZ into seawater could play an important role in the environment.Bacterial oxidase; Oxygen reduction; Hydrogen peroxide; (Pseudomonas nautica 617)

The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 61 7 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, 475-4791. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDSjPAGE showed the presence of four subunits. The pZ was found to be 4.45 and a M , 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 CI range. The prosthetic group is made of two hemes b, one high-spin ( S = 512, g , = 5.9, gii z 2.0), the other low-spin ( S = 1/2, g, = 2.94, g, = 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-' . mg protein-' and commercially available coenzyme (101.74 nmol O2 . min-' . mg protein-'). Exposure of the reduced enzyme to CO induced the collapse of a and 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.Of the terminal enzymes of aerobic respiratory systems, cytochrome-c oxidase of mammalian origin (aa3 type) is the most thoroughly investigated (see reviews in [l -31). This complex transmembrane protein, present in all eukaryotic cells, catalyzes the reduction of molecular oxygen to water in a reaction involving the transfer of four electrons. Coupled to this reaction is a proton pump function which contributes to converting the free energy of O2 reduction by building across the membrane a difference in the proton electrochemical potential. Oxidases of the same aa3-type have been purified from different genera of bacteria. They are far simpler than the enzyme of mammalian origin, being composed of 1 -3 subunits instead of 13 [2, 4, 51. There has, therefore, been a great deal of interest in studying their structures and the mechanisms of the reactions which they catalyze, under far simpler conditions. This is not the only peculiarity of prokaryotic cells, which, in the last few years, provided a number of new insights into electron-transfer mechanisms [6, 71. As far as terminal oxidases are concerned, a great diversity has emerged from investigations of an extending number of bacterial genCorrespondence to M. Denis, Centre d'ocednologie de Marseille,

When exposed IO CO, the aerobic respiratory system of the marine bacterium Pse~rdurrtortas twrrricu strain 617, previously reduced with dithionite. undergoes reoxidation. When dealing with the purified oxidasc (dithionite reduced) cxposurc of the enzyme to CO induces its reoxidation (collapse of its a band). Under our experimental conditions, this form of the oxidase could not be reduced again by dithionitc. Addition of formaldehyde to the native oxidized enzyme resulted in full inhibition of the oxidasc reduction by dithionitc. presumably due to complex formation. We hypothcsizcd a reduction ofC0 into formaldchydc and a locking of the active site by the reaction product. By using flash photolysis. it was possible to turn over the enzyme, accumulutc the reaction product and identify it as formaldehyde. When using the membrane-bound enzyme, formaldehyde accumulated without the help of Sash photolysis. This unusual reduction of CO to formaldehyde could bc related to the previously rcponcd uncommon kiturcs of the P. rwwicrr oxidasc. in particular 0. reduction into HzOz as end product [(1989) FEBS Lett. 247, 4754791. Brrctcrial oxidase, Carbon monoxide; CO reduction; P.se~crlorrrarras rmriru strain 617