Essential role of His163 of cytochrome P450 1A2 in catalytic functions associated with cytochrome b5.
The effect of cytochrome b5 on the activity of cytochrome P450 1A2 (P450 1A2) and three site-directed mutants designed to alter surface charges has been investigated. Cytochrome b5 increased the rate of oxidation of methanol and/or 7-ethoxycoumarin 2- to 10-fold in both wild type and mutant P450. Cytochrome b5 also increased the coupling of electron transfer to substrate hydroxylation as opposed to the uncoupling reaction which leads to peroxide production. The effect of cytochrome b5 on both wild type and the Lys99Glu and Lys401Glu mutants are similar. In contrast, turnover numbers in the His163Glu mutant did not change in the presence of cytochrome b5. The reduction potential of the His163Glu mutant decreased approximately -40 mV while the Lys99Glu and Lys401Glu mutants exhibited little change. The rate of photoreduction decreased from 1.1 x 10(-1) min-1 to 8.3 x 10(-3) min-1 in the His163Glu mutant while the rate of electron transfer from ferrous P450 to ferric cytochrome b5 increased from 0.02 min-1 to > 5 min-1. Overall, the present study suggests that His163 is important to keep an appropriate redox potential of P450 1A2 for optimum electron transfer to occur from cytochrome b5. Based on the P450 101 crystal structure, His163 is not expected to directly contact the heme. Therefore, the resulting change in redox properties in the His163Glu mutant is probably not the result of a direct electrostatic change in the heme environment.
Mayuzumi
H
H
Institute for Chemical Reaction Science, Tohoku University Katahira, Sendia, Japan.
Shimizu
T
T
Sambongi
C
C
Hiroya
K
K
Hatano
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D006639:Histidine; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2