ABSTRACT
Fluorescein isothiocyanate (FITC) has been shown to be selectively attached to the N-terminus of cytochrome P-450 LM2. The N-demethylase activity of cytochrome P-450 LM2 reconstituted systems modified in this way was inhibited by 25%. As revealed by CD measurements the overall conformation as well as the immediate heme environment of cytochrome P-450 LM2 remained unchanged after attachment of the FITC molecule. The binding affinity of modified cytochrome P-450 LM2 toward benzphetamine and aniline and the cumene hydroperoxide- or H2O2-supported N-demethylation of benzphetamine are maintained. However, the introduction of the electron via NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is impaired after modification of the alpha-amino group. The extent of reduced modified cytochrome P-450 LM2 in the cytochrome P-450 reductase-supported reduction reaction is diminished and the half-time of the reduction is increased. The diminished reducibility is ascribed to steric hindrance of groups directly involved in the interaction between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase or to blocking of the charge-pair interactions between the alpha-amino group of P-450 LM2 and the respective negatively charged group of NADPH-cytochrome P-450 reductase. By energy-transfer measurements distances between the heme and the alpha-amino group of 2.65 and 3.97 nm for the oligomeric and the monomeric forms of P-450 LM2, respectively, have been determined.
