THE EFFECT OF FERREDOXIN(BED) OVEREXPRESSION ON BENZENE DIOXYGENASE ACTIVITY IN PSEUDOMONAS-PUTIDA ML2

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponen t complex comprising a flavoprotein reductase, a ferredoxin, and a ter minal iron-sulfur protein (ISP). The catalytic activity of the isolate d complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being f erredoxin(BED). The relative levels of the three components were analy zed by using I-125-labelled antibodies, and the functional molar ratio of ISPBED, ferredoxin(BED), and reductase(BED) was shown to be 1:0.9: 4.8, respectively. The concentration of ferredoxin,,, was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe- 2S centers in ferredoxin(BED) and ISPBED of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor i n dioxygenase activity in vitro. To determine if it is a limiting fact or in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was intr oduced into P. putida ML2. The benzene dioxygenase activity of this st rain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated l evel of ferredoxin(BED) protein and active redox centers in the recomb inant strain. However, in these cells, the increased level of ferredox in(BED) had no effect on the overall rate of benzene oxidation by whol e cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.