Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance

Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O 2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H 2 O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450 cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.