CYTOCHROME-P450 CONFORMATION AND SUBSTRATE INTERACTIONS AS PROBED BY CO BINDING-KINETICS

The kinetics of CO binding to cytochrome P450, as measured by the flas h photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dy namics and are modulated by P450 interactions with substrates and othe r components of the microsomal membrane. Application of a difference m ethod to kinetic data analysis distinguishes the kinetic behavior of i ndividual P450 forms in the microsomal membrane. This approach shows t hat substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the bindi ng rate; and/or 2) steric effects that reduce the rate. Both mechanism s are observed, the relative contributions of each varying in a substr ate- and P450-dependent manner. In addition to microsomes, substrate i nteractions with individual P450s can be similarly probed using expres sed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognit ion of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fun damental P450-substrate interactions in a biological membrane environm ent.