Oxidation of biphenyl and nine chlorinated biphenyls (CBs) by the biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400 was examined. The purified terminal oxygenase required the addition of partially purified electron transport components, NAD(P)H, and ferrous iron to oxidize biphenyl and CBs. cis-Biphenyl 2,3-dihydrodiol was produced with biphenyl as the substrate. Dihydrodiols were produced from all CBs, and more than one compound was produced with most substrates. Catechols were produced when the dioxygenasecatalyzed reaction occurred at the 2,3 position of a 2-chlorophenyl ring, resulting in dechlorination of the substrate. Oxidation at the 3,4 position of a 2,5-dichlorophenyl ring produced a 3,4-dihydrodiol. Compounds resulting from both types of reaction were produced during oxidation of 2,5,2-trichlorobiphenyl. The broad substrate specificity and the ability to oxidize at different ring positions suggest that the biphenyl 2,3-dioxygenase is responsible for the wide range of CBs oxidized by Pseudomonas sp. strain LB400.

Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.

The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISP BPH ) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (␣) subunit with an estimated molecular weight of 53,300 and a small (␤) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an ␣ 3 ␤ 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of ␣␤ heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxin BPH and ferredoxin BPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at g x ‫؍‬ 1.74, g y ‫؍‬ 1.92, and g z ‫؍‬ 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISP BPH in the presence of ferredoxin BPH , ferredoxin BPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISP BPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.

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