Purification and characterization of the NADH:ferredoxinBPH oxidoreductase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400

Abstract

NADH:ferredoxin_BPH oxidoreductase (reductase_BPH) of biphenyl 2,3-dioxygenase was purified over 47-fold to homogeneity with a yield of 41% from cell extract of Pseudomonas sp. strain LB400. Reductase_BPH transfers reducing equivalents from NADH to the catalytic oxygenase component (ISP_BPH) via a ferredoxin (ferredoxin_BPH) during the oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol. Reductase_BPH was a monomer with a molecular weight of 43,600 as determined by electrophoresis under denaturing conditions. Gel filtration column chromatography gave a molecular weight of 41,500 for native reductase_BPH. The absorbance spectrum of the protein in its oxidized state had maxima at 271 nm, 376 nm and 448 nm with shoulders at 422 nm and 476 nm. The peak around 448 nm was partially bleached upon reduction with NADH under anoxic conditions. Reductase_BPH contained 0.89 mol FAD/mol protein. Reductase_BPH was required for oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol by ISP_BPH and ferredoxin_BPH. Potassium ferricyanide, 2,6-dichlorophenolindophenol (DCPIP), nitrobluetetrazolium and cytochrome c served as artificial electron acceptors. Reduction of cytochrome c was dependent upon the presence of ferredoxin_BPH. The fastest rate of DCPIP reduction occurred at pH 7.2 and 32° C. The apparent K _m for NADH and NADPH in the DCPIP assay were 58 μM and 156 μM, respectively. V _max was 3,120 U mg^–1 for NADH and 1,140 U mg^–1 for NADPH. NADH is most likely the physiological electron donor for oxidation of biphenyl and polychlorinated biphenyls.