RECOMBINANT HUMAN CYTOCHROME-P450 1A2 AND AN N-TERMINAL-TRUNCATED FORM - CONSTRUCTION, PURIFICATION, AGGREGATION PROPERTIES, AND INTERACTIONS WITH FLAVODOXIN,FERREDOXIN, AND NADPH-CYTOCHROME P450 REDUCTASE

Previous work from this laboratory indicated that the N-terminus of re combinant human cytochrome P450 (P450) 1A2 expressed in Escherichia co li is blocked (P, Sandhu, Z, Guo, T, Baba, M, V, Martin, R, H, Tukey, and F, P, Guengerich, (1994) Arch, Biochem, Biophys, 30, 168-177), A m odification of this construct was done to insert an extra 12 residues containing a thrombin-sensitive site just beyond the most N-terminal h ydrophobic segment, and the protein was expressed, purified, and cut w ith thrombin. Treatment of E, coli membranes in which the P450 1A2 wit h 12 extra residues was present with thrombin did not release the trun cated form, suggesting that the added thrombin site may be imbedded in the membrane, The N-termini of the recombinant proteins were blocked but mild acid hydrolysis generated the expected Met residues as analyz ed by Edman degradation, Laser light scattering studies indicated that purified thrombin-cleaved P450 1A2 (devoid of the usual N-terminal 25 residues or the first 36 residues of the wild-type protein) was still aggregated in the absence of detergent and that some nondenaturing de tergents could reduce the apparent size to that of a tetramer, The N-t erminal truncated protein was as catalytically active as full-length P 450 1A2 but required a higher concentration of NADPH-P450 reductase, P 450 1A2 exhibited catalytic activity in E, coli cells, and activity of the purified enzyme could be supported by E, coli flavodoxin and NADP H-flavodoxin reductase, Spinach ferredoxin and NADPH-ferredoxin reduct ase could also substitute for NADPH-P450 reductase, These artificial e lectron donors did not require phospholipid for oxidation reactions; h owever, phospholipid was required for optimal activity when either P45 0 1A2 or the truncated form was used with NADPH-P450 reductase, Rates of oxidation of 7-ehoxyyresorufin were considerably higher for both P4 50 1A2 and the truncated form when NADPH-P450 reductase was replaced w ith the ''oxygen surrogate'' iodosylbenzene, indicating that P450 redu ction and oxygen activation are normally limiting in this P450 1A2 rea ction. (C) 1996 Academic Press, Inc.