Cytochrome P450s are a superfamily of metalloenzymes that are responsible for the monooxygenation of their hydrophobic substrates. P450’s retain the same general structural scaffold, however are able to bind promiscuously to substrates with distinct physico-chemical properties. It is believed that they possess considerable flexibility in the substrate binding regions as well as their active site to accomplish the diverse binding and catalytic chemistry with high regio- and stereo-specificity. In order to investigate the conformational dynamics inherent in these enzymes, especially in context of binding different ligands, we carried out amide proton exchange studies via NMR spectroscopy on a model P450 system of cytochrome P450cam (CYP101). These studies were performed in both camphor-bound and substrate-free forms of CYP101 and provide information on the motional properties of residues on slow timescales. Comparison of the exchange rates obtained from these studies for the two forms show that overall the substrate-free form is more dynamic than the camphor-bound form on the millisecond-second timescale due to its faster exchange rates, with regions being part of the substrate access site and active site in particular showing the largest differences in exchange rates. This study is the first of its kind looking at the residue-level changes in conformational dynamics upon ligand binding in a P450 and identifies specific regions where slow dynamic changes occur. Results from these studies help provide mechanistic insights into the process of differential ligand recognition by P450s
