SIGNIFICANT CONTRIBUTION OF ARGININE-112 AND ITS POSITIVE CHARGE OF PSEUDOMONAS-PUTIDA CYTOCHROME P-450(CAM) IN THE ELECTRON-TRANSPORT FROMPUTIDAREDOXIN

Cytochrome P-450(cam) of Pseudomonas putida is a prototype of various eukaryotic cytochrome P-450 molecules. Arg-112 located on the surface of this protein is highly conserved among various other cytochromes P- 450. In this study, we constructed mutant genes for P-450(cam) in whic h Arg-112 was replaced by Gln or Glu, expressed them in Escherichia co li and purified the mutant proteins. Their P-450 enzymic activities we re analyzed in the reconstituted system to determine the function of A rg-112. K-d values for d-camphor of Arg(112)-Gln and Arg(112)-Glu were much the same as those of the wild-type enzyme, whereas K-d values fo r the oxidized form of putidaredoxin, which is an acidic protein and i s the redox partner of P-450(cam) were 240 and 530 mu M, respectively. These values are 8 and 19 times larger than that of the wild-type enz yme (28 mu M), thereby indicating lower affinities of the mutant enzym es for the oxidized putidaredoxin. Reaction rate constants for reducti on by the reduced form of putidaredoxin, measured using the stopped fl ow method, were 45.5, 9.0.10(-3) and 9.0.10(-4) s(-1) for the wild typ e, Arg(112)-Gln and Arg(112)-Glu, respectively. Thus, Arg-112 of P-450 (cam) plays an important role in the interaction with putidaredoxin an d in the high efficiency of the electron transfer; the positive charge of the residue seeming to contribute to the process. The yields in Es cherichia coli, the heme contents in the purified fractions and heat s tability of the mutant proteins were lower than those of the wild type enzyme, suggesting that Arg-112 of P-450(cam) is also important for s tability of P-450(cam).