Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics

Biochimie. 1996;78(8-9):706-13. doi: 10.1016/s0300-9084(97)82528-x.


The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed 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 recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Carbon Monoxide / metabolism*
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Gene Expression
  • Humans
  • Kinetics
  • Male
  • Microsomes, Liver / metabolism
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism
  • Protein Conformation*
  • Rats
  • Rats, Sprague-Dawley
  • Structure-Activity Relationship
  • Substrate Specificity


  • Carbon Monoxide
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A