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

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 (ISPBPH) 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,,, contained 0.89 mol FAD/mol protein. Reductase(BPH) was required for oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol by ISPBPH and ferredoxin(BPH). Potassium ferricyanide, 2,6-dichlorophenolindophenol (DCPIP), nitrobluetetrazolium and cytochrome c served as artificial electron accepters. 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 degrees C. The apparent K-m for NADH and NADPH in the DCPIP assay were 58 mu M and 156 mu M, 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.