Significant contribution of arginine-112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin.
Cytochrome P-450cam 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-450cam in which Arg-112 was replaced by Gln or Glu, expressed them in Escherichia coli and purified the mutant proteins. Their enzymic activities were analyzed in the reconstituted system to determine the function of Arg-112. Kd values for d-camphor of Arg112-Gln and Arg112-Glu were much the same as those of the wild-type enzyme, whereas Kd values for the oxidized form of putidaredoxin, which is an acidic protein and is the redox partner of P-450cam, were 240 and 530 microM, respectively. These values are 8 and 19 times larger than that of the wild-type enzyme (28 microM), thereby indicating lower affinities of the mutant enzymes for the oxidized putidaredoxin. Reaction rate constants for reduction by the reduced form of putidaredoxin, measured using the stopped flow method, were 45.5, 9.0 x 10(-3) and 9.0 x 10(-4) s-1 for the wild type, Arg112-Gln and Arg112-Glu, respectively. Thus, Arg-112 of P-450cam plays an important role in the interaction with putidaredoxin and in the high efficiency of the electron transfer; the positive charge of the residue seeming to contribute to the process. The yields in Escherichia coli, the heme contents in the purified fractions and heat stability of the mutant proteins were lower than those of the wild type enzyme, suggesting that Arg-112 of P-450cam is also important for stability of P-450cam.
Nakamura
K
K
Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Horiuchi
T
T
Yasukochi
T
T
Sekimizu
K
K
Hara
T
T
Sagara
Y
Y
D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D002164:Camphor / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004579:Electron Transport; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry*; D006418:Heme / Q000032:analysis; D008969:Molecular Sequence Data; D009154:Mutation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D005288:Ferredoxins; D006418:Heme; C100137:putidaredoxin; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine