Longitudinal (T1) relaxation studies were performed in order to examine the interaction of caffeine with the heme of human P450 1A2. Addition of caffeine to this P450 resulted in a small, incomplete conversion of the heme from high spin to low spin, as shown by changes in the optical spectrum. Determination of a relatively large dissociation constant (Ks = 2.6 mM) as well as the relative instability of the P450 after 2 h at room temperature necessitated the performance of these experiments at high concentrations (25 mM) of caffeine. The relaxation measurements on the three sets of methyl hydrogens led to the determination of the corresponding distances between the iron and the methyl groups on the bound caffeine as well as the position and orientation of caffeine within the active site of P450 1A2. The three methyl groups were found to be nearly equidistant from the iron (> or = 4.79-4.89 A), with slight preference for the N-3 position, and thus, the average position of caffeine was parallel to the heme. In vitro incubations with P450 1A2 and 5 mM caffeine led primarily to paraxanthine formation (N-3 demethylation), as expected. However, with 25 mM substrate, the overall extent of oxidation was doubled and there was more equivalent oxidation at each of the four potential sites on caffeine. This latter observation was consistent with the lack of selective positioning of the N-3 methyl group of caffeine relative to the heme.