Structure-function relationships for the mammalian cytochromes P450 are examined. Specifically, we are elucidating the interactions between P450 and microsomal electron carrier proteins, substrates and ligands. We assessed the interaction of P450 with NADPH cytochrome P450 reductase to identify the specific amino acid residues involved in this protein- protein interaction. Molecular modeling in conjunction with P450 sequence alignments were used to predict the P450 binding site for reductase. Synthetic peptides corresponding to these regions on P450 2B1 were prepared and assessed for their ability to inhibit reductase-mediated P450 activities. A peptide corresponding to the C helix (residues 116- 134) inhibited benzphetamine demethylase, but had no effect when Arg-125 was replaced by Glu. In addition surface simulatory peptides were prepared which consist of sequences which are proximate in the tertiary structure but distant in primary sequence. A peptide consisting of elements of helices C and L was particularly effective in inhibiting several P450-mediated activities in both reconstituted and microsomal systems. These results thus identify both specific regions and a residue involved in P450 binding to reductase. The conformation and dynamics of P450 were examined with the CO flash photolysis technique. We evaluated the effect of a series of polycyclic aromatic hydrocarbons of varying sizes and shapes on the CO binding kinetics of both rat liver microsomal P450 1A1 and baculovirus expressed human P450 1A1. The results showed that substrates modulate CO binding by a dual mechanism involving both conformational and steric effects. Experiments with baculovirus expressed human P450 3A4, a form which metabolizes many important drugs with diverse structures, showed that this P450 consists of a population of conformers that differ in their substrate recognition properties. This finding helps explain the broad substrate specificity of this P450. These results demonstrate the utility of CO binding kinetics as a probe of P450 structure/dynamics in a native membrane environment.
