Binding of benzo[a]pyrene by purified cytochrome P-450c

Benzo[a]pyrene (BP) fluorescence-emission intensities in phospholipid micelles are quantitatively described over a broad range of lipid and BP concentrations by excitation that is linearly dependent upon BP concentration and an offsetting excimer quenching that is dependent upon the square of the BP concentration. The fluorescence of BP is quenched by the presence of cytochrome P-450c in proportion to the concentration of the cytochrome in the micelles and in accord with stoichiometric complex formation. Parallel optical titrations indicate a change in spin state of P-450c to a predominantly high-spin state that correlates directly with the percentage fluorescence quenching of complexed BP. Neither change occurs with five other purified forms of rat liver P-450 that have low activity in BP metabolism. N-Octylamine, a ligand that binds to the heme of P-450, competitively inhibits both the spin-state changes and the fluorescence quenching in equal proportion. The Kd for the interaction of BP with P-450c is exceptionally low (10 nM) relative to the Km for monooxygenation (ca. 1 microM). Decreasing the concentration of either dilauroylphosphatidylcholine or dioleoylphosphatidylcholine concomitantly increases the high-spin state (from 30% to 80%) of fully complexed P-450c and the fluorescence quenching (50-100%) of the complexed BP (half-maximal at 80 micrograms of lipid/mL). It is concluded that spin state and fluorescence quenching both reflect the same changes in the interaction of the BP with the P-450 heme.(ABSTRACT TRUNCATED AT 250 WORDS)