PURIFICATION AND PROPERTIES OF FERREDOXIN(BPH), A COMPONENT OF BIPHENYL 2,3-DIOXYGENASE OF PSEUDOMONAS SP STRAIN LB400

The ferredoxin component (ferredoxin(BPH)) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filt ration column chromatography. The protein was a monomer with a molecul ar weight of 15000 and contained 2 gram-atoms each of iron and acid-la bile sulfur, Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm, The spectrum was partially bleached in the visible region upon reduction by reduct ase(BPH) with NADPH as the source of electrons, Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein, Upo n reduction with sodium dithionite, signals with g(x) = 1.82, g(y) = 1 .92 and g(z) = 2.02 were detected. These results indicate that the pro tein contains a Rieske-type (2Fe-2S) iron-sulfur center, Ferredoxin(BP H) was required for the oxidation of biphenyl by the terminal oxygenas e component of the enzyme and is probably involved in the transfer of reducing equivalents from reductase, to the terminal oxygenase during catalysis.