Studies on the Redox Conformational Changes and Substrate Adaptability Changes in Cytochrome P-450_d Mutants

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The cytochrome P450(P450) is one of the superfamily of heme-containing monooxygenases which catalyze many types of biotransformation reactions of organic substrates in living organisms. Significant progress in understanding the P450 structure-function relationship has been made on the basis of the X-ray crystal structure of water-soluble P450_<cam> and alignments of amino acids of P450s. By combining this knowledge and site-directed mutagenesis technique, the structure of membrane-bound P450_d(CYP1A2) was elucidated with respect to the heme incorporation (Biochemistry, 27, b4138-4141(1988)) and the structure of putative distal site (Biochemistry, 28, 6848-6857 (1989) ; Biochemistry, 30, 1490-1496(1991) ; Biochbmistry, 30, 4659-4662 (1991)). In this project we found the important role of Glu318 at the putative distal site' of P450_d(CYP1A2) in the packing or the conformational stability of the putative distal site of the P450_d molecule (Biochemistry, 30, 11206-11211(1991)) and the important role of the Glu318 in the catalytic function of the P450_d (Biochbmistry, 31, 1528-1531(1992)). Furthermore, we found that Lys94, Lys99, LYS105, Lys440, Lys453, Arg455, Lys463, and the Arg cluster, Arg135-Arg136-Arg137, of P450_d(CYP1A2) participate in the inter-molecular electron transfer process by forming ionic bridges between the P450_d and NADPH-P450 reductase and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins (J. Biol. Chem., 266, 3372-3375(1991)). Well-conserved polar amino acids at position 318 of P450_d (Glu or Asp of P450s ; Asp251 of P450_<cam>) prior to the conserved Thr (Thr3l9 for P450_d ; Thr252 for P450_<cam>) significantly contribute to the activation of the oxygen molecule bound to P450.