Purification and characterization of the NADH:ferredoxinBPH oxidoreductase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.
NADH:ferredoxinBPH oxidoreductase (reductaseBPH) 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. ReductaseBPH transfers reducing equivalents from NADH to the catalytic oxygenase component (ISPBPH) via a ferredoxin (ferredoxinBPH) during the oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol. ReductaseBPH 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 reductaseBPH. 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. ReductaseBPH contained 0.89 mol FAD/mol protein. ReductaseBPH was required for oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol by ISPBPH and ferredoxinBPH. 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 ferredoxinBPH. The fastest rate of DCPIP reduction occurred at pH 7.2 and 32 degrees C. The apparent Km for NADH and NADPH in the DCPIP assay were 58 microM and 156 microM, respectively. Vmax 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.
Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA.
R M
RM
Broadus
J D
JD
Haddock
D001713:Biphenyl Compounds / Q000378:metabolism; D004579:Electron Transport; D004787:Environmental Pollution; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry*; D011078:Polychlorinated Biphenyls / Q000378:metabolism / Q000633:toxicity; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry
D001713:Biphenyl Compounds; D005182:Flavin-Adenine Dinucleotide; D007506:Iron-Sulfur Proteins; D009243:NAD; D010088:Oxidoreductases; D010105:Oxygenases; D011078:Polychlorinated Biphenyls; C010574:biphenyl; C074161:biphenyl-2,3-dioxygenase; C028878:ferredoxin-NAD+ reductase