Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. Strain LB400

Abstract

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 (α) subunit with an estimated molecular weight of 53,300 and a small (β) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an α3β3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of αβ 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.