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 Fe 2 S 2 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 Å. Steady-state kinetics assays demonstrated that BPDO had an apparent k cat /K m for biphenyl of (1. The microbial catabolic activities responsible for the degradation of aromatic compounds constitute an essential link in the global carbon cycle. These activities are of considerable practical interest due to their potential to destroy toxic, persistent pollutants, a strategy known as bioremediation (1). In the case of highly chlorinated, structurally diverse xenobiotics such as PCBs, 1 the development of a practical bioremediation technology has been limited in part by the failure of existing microbial catabolic activities to effectively degrade these compounds (1, 2). This failure may arise because these activities have not yet evolved to degrade compounds that have only recently been introduced into the biosphere. An important aspect of the adaptation of catabolic activities for bioremediation is the study of the structure and function of key catabolic enzymes. Such studies provide insight into the molecular basis of an important biological process, thereby facilitating the modification of enzyme specificity and the design of novel metabolic pathways.2BPDO catalyzes the initial reaction in the aerobic degradation of biphenyl and some PCBs. BPDO is a typical aromatic ring-hydroxylating dioxygenase, utilizing O 2 and electrons originating from NADH to transform biphenyl to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene ( Fig. 1) (3, 4). This dihydroxylation prepares the ring for subsequent degradation by ring cleavage enzymes. The enzyme comprises an FAD-containing reductase (BphG), a Rieske-type ferredoxin (BphF), and a two-subunit oxygenase of ␣ 3 ␤ 3 constitution that contains a Rieske-type Fe 2 S 2 cluster and a mononuclear iron center. Accordingly, BPDO has been classified as a group IIB aromatic ring-hydroxylating dioxygenase together with benzene and toluene dioxygenases (5). Structural and spectroscopic studies of related dioxygenases indicate that the mononuclear iron center orchestrates substrate transformation (reviewed in Ref. 6). BphG, BphF, and the oxygenase Fe 2 S 2 cluster function to transfer electrons from NADH to this center.BPDO is a major determinant of the PCB-catabolizing capabilities of biphenyl-degrading strains, and the enzymes from different strains possess significantly different congener-transforming abilities. For example, BPDO LB400 from Burkholderia cepacia LB400 transforms a much broader range of congeners than BPDO KF707 from Pseudomonas ps...