Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISP), a ferredoxin FER, and a ferredoxin reductase RED. RED and FER transfer electrons from NADH to an Fe-S active center of ISP which activates molecular oxygen for insertion into the substrate. In this work B-356 ISP complex and its and subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISP construction carrying a single His tail on the N-terminal portion of the subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an heteromer, with each unit containing a [2Fe-2S] Rieske center ( = 8,300 M cm) and a mononuclear Fe. Although purified His-tagged subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged subunit to reconstitute active ISP was weak. However, when His-tagged and subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISP could be purified on Ni-nitrilotriacetic acid resin.

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