Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe2S2 center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 Angstrom. Steady-state kinetics assays demonstrated that BPDO had an apparent K-cat/K-m for biphenyl of (1.2 +/- 0.1) x 10(6) M-1 s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4,4'-diClB. Strikingly, the ability of BPDO to utilize O-2 depended strongly on the biphenyl substrate; k(cat)/K-m(O2) = (3.6 +/- 0.3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M-1 S-1 the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O-2 consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O-2 was detected as H2O2. Thus, PCB congeners such as 2,2' diClB exact a high energetic cost, produce a cytotoxic compound (H2O2), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.