1. A soluble enzyme system which oxidizes benzene to cis-1,2-dihydroxycyclohexa-3,5-diene (cis-benzene glycol) was obtained from a species of Pseudomonas grown on benzene as the major carbon source. 2. The system was shown to consist of three protein components. Two of these were non-haem-iron proteins of molecular weight approx. 21,000 and approx. 186,000 and the other was a flavoprotein of molecular weight approx. 60,000. 3. Fe2+ and NADH were essential cofactors for benzene oxidation.

1. Protocatechuate 4,5-oxygenase, purified 21-fold from extracts of Pseudomonas testosteroni, was examined in the ultracentrifuge and assigned a mol.wt. of about 140000. 2. When diluted, the enzyme rapidly lost activity during catalysis. Inactivation was partially prevented by l-cysteine. 3. With a saturating concentration of protocatechuate (1.36mm), K(m) for oxygen was 0.303mm. This value is greater than the concentration of oxygen in water saturated with air at 20 degrees . 4. Cell extracts converted protocatechuate into gamma-carboxy-gamma-hydroxy-alpha-oxovalerate, which was isolated as its lactone. 5. gamma-Carboxy-gamma-hydroxy-alpha-oxovalerate pyruvate-lyase activity was stimulated by Mg(2+) ions and mercaptoethanol. Cells grown with p-hydroxybenzoate as carbon source contained higher concentrations of this enzyme than those grown with succinate.

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis.

1. cis-Benzene glycol dehydrogenase was purified to a homogeneous state from a species of Pseudomonas grown with benzene as the major carbon source. 2. The enzyme was specific for the cis-isomer of its substrate and required NAD(+) as hydrogen acceptor. 3. Partial inactivation of the enzyme, which was observed during purification, could be reversed by the addition of Fe(2+) and GSH. 4. A molecular weight of 440000 was calculated from data obtained by sedimentation-velocity and diffusion analysis in the ultracentrifuge. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis indicated a subunit of molecular weight 110000. 5. p-Chloromercuribenzoic acid and 1,10-phenanthroline were shown to inhibit the enzyme.