Dibenzofuran 4,4a- dioxygenase ( DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i. e., terminal oxygenase ( DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21( DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine ( His)-tagged form. The reconstructed DFDO system with three purified components, i. e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 ( which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol ( specific activity: 14.4 nmol min(-1) mg(-1)) and convert dibenzofuran to 2,2', 3- trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV- visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.